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Structural and functional characterization of the DAXX N-terminal helix bundle and SUMO interaction motifs Escobar-Cabrera, Eric 2010

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STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE DAXX N-TERMINAL HELIX BUNDLE AND SUMO INTERACTION MOTIFS by  ERIC ESCOBAR-CABRERA  B.Sc., Trent University, 2002  A THESIS SUBMITTED 1N PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Chemistry)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2010 © Eric Escobar-Cabrera, 2010  Abstract The Fas death-domain associated (DAXX) protein was first discovered as an intermediary of a FADD-independent apoptosis signaling pathway. However, subsequent studies have established it as an important player in both transcription and cell cycle regulation. In this thesis, the first structural characterization of DAXX is presented. Sequence alignment and secondary structure prediction algorithms were used to define a number of constructs of DAXX. The C-terminal -l/3 of DAXX was found to be intrinsically disordered, whereas a welldefined folded domain was identified near its N-terminus. NMR spectroscopy was used to solve the three-dimensional structure of this domain, and to characterize its dynamic behavior. The calculated structural ensemble consists of five helices, and hence is named the DAXX Helix Bundle (DHB) domain. This domain has a very different topology to the Sin3 PAH domains, which until now have been used as a model for this region of DAXX. Rassfl C, an important tumor suppressor, was reported recently to interact with DAXX via the DHB domain. This interaction was linked to mitosis progression, and has potential implications in the treatment of cancer. The NMR-derived ensemble of the complex of the DHB domain with an N-terminal fragment of Rassf1 C revealed a short amphipathic a-helix filling the cleft between helices 2 and 5 of the DHB domain. Both hydrophobic and electrostatic interactions mediated complex formation. This structural characterization explains the observed in vivo interaction and provides clues as to how the binding might be regulated.  Additionally, two SUMO-interacting motifs at the termini of DAXX, SIM-N and SIM-C, were characterized. Their interactions with SUMO- 1 and SUMO-2 were examined structurally 11  and thermodynamically using NMR spectroscopy.  SIM-N was  also found to bind  intramolecularly to the DHB domain and SIM-C to mediate the interaction of DAXX with the sumoylated Ets- 1 transcription factor. Importantly, the latter did not involve any direct contacts between DAXX and Ets- 1, but rather derived from the non-covalent binding of DAXX SIM-C to SUMO- 1, which in turn was covalently linked in a “beads-on-a-string” fashion to Ets- 1. These results provide new insights into the binding mechanisms and biological roles of DAXX-SUMO interactions.  111  Table of Contents ABSTRACT  .  ii  TABLE OF CONTENTS  iv  LIST OF TABLES  x  LIST OF FIGURES  xi  LIST OF ABBREVIATIONS  xiv  LIST OF AMINO ACID ABBREVIATIONS  xvii  ACKNOWLEDGMENTS  xviii  CHAPTER 1- INTRODUCTION  1  PREAMBLE  2  1.1 1.1.1  The human genome  2  1 .1 .2  Signal transduction  2  1.1.3  Modularproteins  3  1.1.4  Intrinsically disordered proteins  3  1.1.5  DAXX, a multi-domain protein with intrinsically disordered regions  4  1.2  5  DAXX  1.2.1  What is DAXX  5  1.2.2  The mixed roles of DAXX in apoptosis  6  1.2.3  Where is DAXX9  7  1.2.3.1  DAXX in PML nuclear bodies  10  1.2.3.2  DAXX in other nuclear compartments  11  1.2.3.3  Cytoplasmic DAXX?  12  1.2.4  DAXX as a transcription regulator  13  1.2.5  The role of DAXX in mitosis  15  1.3  THE SMALL UBIQUITIN-LIKE MODIFIER PROTEIN SUMO  iv  16  .17  1.3.1  TheSUMOfamily  1.3.2  The mechanism of reversible sumoylation  17  1.3.3  Molecular consequences of sumoylation  20  1.3.4  Differences among SUMO paralogues  23  1.3.5  SUMO interacting motifs  24  1.3.6  DAXX and SUMO  25  THE RAS-ASSOCIATION DOMAIN FAMILY (RAssF)  1.4  27  1.4.1  Domain structure of Rassf  27  1 .4.2  Rassfl and cancer  31  1 .4.3  Rassfl and DAXX  32 35  THESIS OVERVIEW  1.5  CHAPTER 2- STRUCTURE OF THE N-TERMINAL DAXX HELIX BUNDLE 2.1  DAXX SEQUENCE AND SECONDARY STRUCTURE  2.2  CHARACTERIZATION OF  2.3  MAPPING OF THE  DAXX CONSTRUCTS  DAXX N-TERMINAL STRUCTURAL DOMAIN BOUNDARIES  37 38 42  46  2.3.1  Chemical shift assignments  47  2.3.2  Identification of the folded domain  49  2.3.2.1  ° secondary structure 4616 DAXX  49  2.3.2.2  N DAXX 5 ‘ ° relaxation analysis 4616  50  2.3.2.3  Comparison of various DAXX constructs  50  2.4  5544 STRUCTURE DETERMINATION DAXX  2.4.1  Assignments of side-chain signals  51 51  2.4.1.1  Assignment of signals from side-chain aliphatic nuclei  54  2.4.1.2  Assignment of signals from side-chain aromatic nuclei  57  2.4.2  55144 secondary structure DAXX  61  2.4.3  55144 tertiary structure DAXX  62  V  .68  55144 dynamics DAXX  2.4.4  STRUCTURAL COMPARISONS OF THE DHB DOMAIN  2.5  74  2.5.1  Comparison with Sin3  74  2.5.2  Comparison with other helix bundles  78 81  MATERIALS AND METHODS  2.6 2.6.1  Cloning  81  2.6.2  Protein expression tests  82  2.6.3  Protein purification  83  2.6.4  NMR spectroscopy  83  2.6.4.1  NMR relaxation measurements  84  2.6.4.2  Amide proton-deuterium exchange  84  2.6.5  Structure calculation  84  2.6.6  N Residual dipolar coupling measurements 5 lHNl  85  CHAPTER 3- STRUCTURE OF THE DAXXJRASSF1C COMPLEX  88  3.1  IDENTIFICATION OF RASSF 1 C AS AN INTERACTION PARTNER OF THE DHB DOMAIN  89  3.2  44 ’ 55 ° AND DAXX 5 NMR-M0NITORED TITRATION OF RASSF1C’  92  3.3  NMR-M0NITORED TITRATIONS OF TRUNCATION FRAGMENTS OF RASSF1C WITH 55144 DAXX  94  3.3.1  838 RassflC  94  3.3.2  ’ 82 RassflC  95  3.3.3  ’’ 2338 Rassf1C  95  3.4  SCREEN OF CONDITIONS FOR THE FORMATION OF A TIGHTER COMPLEX  103  3.5  DETERMINATION OF THE TERTIARY STRUCTURE OF THE COMPLEX  105  3.5.1  Changes in DAXX 55144 secondary structure upon binding  105  3.5.2  v secondary structure upon binding 2338 Changes in Rassf1C  106  3.5.3  ” complex 2338 / RassflC 55144 Tertiary structure of the DAXX  109  3.6  115  STRUCTURAL IMPLICATIONS  vi  3.6.1  Structural verification that the DAXX DHB and Sin PAH domains differ in binding 115 mechanisms  3.6.2  Other structural relatives share helix packing patterns  118  3.6.3  Other binding partners of DAXX  118  3.6.4  Possible new DIM-containing interacting partners of DAXX  122  3.7  BIoLoGIcAL IMPLICATIONS OF THE  3.8  MATERIALS AND METHODS  RASSF1C-DAXX INTERACTION  124 126  3.8.1  Cloning  126  3.8.2  Sample purification  126  3.8.2.1  ° 15 RassflC  127  3.8.2.2  N and RassflC 2344 Rassf1C ° 235  128  3.8.3  NMR-rnonitored titrations  128  3.8.4  Structure of the complex  129  3.8.4.1  Sample preparation  129  3.8.4.2  Resonance assignments  132  3.8.4.3  Structure calculation  133  CHAPTER 4- SUMO-INTERACTING MOTIFS (SIM) IN DAXX  134  DAXX SUMO-INTERACTING MOTIFS (SIM-N AND SIM-C)  135  4.1  4.1.1  Identification of SIM-N and SIM-C  135  4.1.2  Role of DAXX SIMs in its localization  136  4.1.3  Research objectives  136  NMR-M0NIT0RED TITRATIONS OF DAXX-SIM-N AND SIM-C WITH SUMO-1 AND  4.2  SUMO-2  138  4.2.1  Preamble. SIM binding by SUMO-1 and SUMO-2  138  4.2.2  NMR characterization of SIM-N, SIM-C, SUMO-1 and SUMO-2  142  4.2.3  NMR-monitored interactions  142  4.2.3.1  Mapping of the binding interface  vii  143  4.2.3.2  SIM-N binding to SUMO-1 and SUMO-2  .143  4.2.3.3  SIM-C binding to SUMO-1 and SUMO-2  146  4.3  COMPARISON WITH VALUES FROM THE LITERATURE  149  4.4  Two ORIENTATION BINDING OF  4.5  DETERMINATION OF THE BINDING MODE OF  4.6  INTRAMOLECULAR BINDING OF  4.7  CHARACTERIZATION OF THE INTERACTION BETWEEN SUMOYLATED ETs-1 AND  DAXX SIM-C TO SUMO-1  151  DAXX SIM-N WITH SUMO-1  155  SIM-N TO THE DHB DOMAIN  STM-C  160 DAXX 166  4.8  SUMMARY AND SIGNIFICANCE  170  4.9  MATERIALS AND METHODS  171  4.9.1  Constructs  171  4.9.2  Protein expression  172  4.9.3  Protein purification  172  4.9.4  NMR spectral assignments  174  4.9.5  NMRtitrations  174  4.9.6  Dissociation constants  176  4.9.7  Spin labeling  176  4.9.8  Paramagnetic relaxation enhancement measurements  176  CHAPTER 5- CONCLUDING REMARKS AND FUTURE EXPERIMENTS  178  5.1  STRUCTURE0FDAXX  5.2  SIGNIFICANCE FOR FUTURE TRUNCATION CONSTRUCTS OF  5.3  PROPOSED MECHANISM FOR ACTIVATION OF THE  5.4  FUTURE EXPERIMENTS IN DEFINING  5.5  DAXX SIMS  5.6  REMAINING QUESTIONS ABOUT THE  5.7  CONCLUDING REMARKS  179  DAXX  DHB DOMAIN  DHB DOMAIN INTERACTING MOTIFS (DIMS)  182 184 189 190  DAXX/RASSF1C INTERACTION  191 192  REFERENCES  194 Vlll  .214  APPENDICES APPENDIX 1  ° 34742 CIRCULAR DICHROISM (CD) SPECTRA OF DAXX  215  APPENDIX 2  EXPREssIoN TESTS OF DAXX CONSTRUCTS  216  APPENDIX 3  N HSQC SPECTRA ANNOTATED 1 H 15  221  APPENDIX 4  CALCULATION OF DISSOCIATION CONSTANTS IN NMR-M0NIT0RED TITRATIONS224  DISSOCIATION CONSTANT PLOTS FROM SIM/SUMO APPENDIX 5 NMR-.M0NITORED TITRATIONS  228  APPENDIX 6  EXCHANGE REGIMES  231  APPENDIX 7  ASSIGNMENT OF THE DHB-D0MAIN  238  APPENDIX 8  W COMPLEX 2338 ’’ IN THE DHB/RASSF1C 2338 ASSIGNMENT OF RASSF1C  245  APPENDIX 9  ’’ COMPLEX 2338 ASSIGNMENT OF THE DHB DOMAIN IN THE DHB/RAsSF1C  247  APPENDIX 10  TRIANGULATION OF THE SPIN LABEL  253  ix  List of Tables TABLE 2.1 NMR RESTRAINTS AND STATISTICS FOR THE ENSEMBLE OF THE TEN LOWEST ENERGY 55144 STRUCTURES CALCULATED FOR DAXX  65  TABLE 2.2 SUMMARY OF AMIDE THE DHB DOMAIN  70  -  -  RELAXATION DATA FOR DIFFERENT STRUCTURAL REGIONS OF  TABLE 2.3 INTERHELICAL ANGLE (Q)IN SIN3A CALCULATED WITH PROMOTIF -  TABLE 3.1 DISSOCIATION CONSTANTS OF UNDER DIFFERENT CONDITIONS -  PAH2 (IG1E.PDB) AND THE DHB DOMAIN 75  44 AND VARIOUS RASSF1C CONSTRUCTS ’ 55 DAXX 104  TABLE 3.2 NMR RESTRAINTS AND STATISTICS FOR THE ENSEMBLE OF THE TEN LOWEST ENERGY v COMPLEX 2338 /RASSF1C 5544 STRUCTURES CALCULATED FOR THE DAXX  110  TABLE 3.3 PROTEINS PREVIOUSLY REPORTED IN THE LITERATURE TO INTERACT WITH THE NTERMINUS OF DAXX  120  -  -  TABLE 3.4-  2338 BLAST SEARCH FOR SEQUENCES SIMILAR TO RASSF1C  TABLE 4.1  DISSOCIATION CONSTANTS BETWEEN  -  SIM-N” AND SIM-C” WITH SUMO-1’’ AND 149  SUMO-2’ TABLE 4.2 TABLE 4.3  -  -  SUMMARY OF REPORTED DISSOCIATION CONSTANTS OF INTERMOLECULAR NOES BETWEEN UNLABELED  SIMs/SUMO  -  150  SIM-N AND 13 N LABELED 5 C/’ 156  SUMO-I TABLE 4.4  123  SAMPLE CONDITIONS USED FOR TITRATIONS PRESENTED IN CHAPTER 3  x  175  List of Figures FuNcTIoN AND LOCALIZATION OF DAXX  .8  GENERIC MODELS FOR TRANSCRIPTIONAL REPRESSION IN EUKARYOTIC SYSTEMS  14  SUMOYLATION MECHANISM  19  MoLECuLAR CONSEQUENCES OF SUMOYLATION  22  THE RAS-ASSOCIATION DOMAIN FAMILY (RASSF) AND THEIR DOMAINS  29  FIGURE 1.6- PROPOSED MODELS FOR MECHANISMS CONTROLLING DAXX/RASSF INTERACTIONS  33  FIGuRE 1.1 FIGuRE 1.2 FIGURE 1.3 FIGURE 1.4 FIGURE 1.5  FIGURE 2.1 FIGURE 2.2 FIGURE 2.3 FIGURE 2.4  -  -  -  -  -  -  -  -  -  SUGGESTED “DOMAIN” ORGANIZATION OF DAXX  39  ALIGNMENT OF THE DAXX SEQUENCE  41  IDENTIFICATION OF STRUCTURED DOMAINS IN DAXX THROUGH DELETION ANALYSES  44  H-’ HSQC SPECTRA OF SOLUBLE CONSTRUCTS OF DAXX 1 N 5  45  FIGURE 2.5 MAGNETIZATION TRANSFER PATHWAYS FOR EXPERIMENTS USED FOR BACKBONE ASSIGNMENT  48  FIGuRE 2.6 IDENTIFICATION OF THE BOUNDARIES OF THE STRUCTURAL DOMAIN WITHIN ° 4616 DAXX  52  FIGURE 2.7 ANNOTATED ‘H-’ N HSQC SPECTRUM OF DAXX 5 55144  53  FIGURE 2.8 ExPERIMENTS USED FOR THE ASSIGNMENT OF NMR SIGNALS FROM THE SIDE-CHAIN ALIPHATIC ‘ C AND 1 3 H NUCLEI OF UNIFORMLY 15 C/ DAXX 3 ‘ N-LABELED 55144  55  FIGURE 2.9 STEPS INVOLVED IN THE ASSIGNMENT OF SIGNALS FROM THE AROMATIC SIDE-CHAINS OF TYR AND PHE RESIDUES IN UNIFORMLY 5 C 3 ‘ N -LABELED /’ DAXX 5544  59  FIGURE 2.10  ASSIGNMENT OF SIGNALS FROM HISTIDINE RESIDUES  60  NMR-DERIvED SECONDARY STRUCTURE OF DAXX 55144  63  STRUCTURE OF THE DHB DOMAIN  66  THE GLOBAL AND LOCAL BACKBONE DYNAMICS OF THE DHB DOMAIN  72  -  -  -  -  -  FIGuRE 2.11 FIGuRE 2.12 FIGURE 2.13  -  -  -  -  FIGURE 2.14- COMPARISON OF THE DHB DOMAIN WITH THE SIN3 PAH DOMAINS  76  FIGURE 2.15  80  -  STRUCTURAL COMPARISON OF THE DHB DOMAIN WITH SIMILAR HELIX BUNDLES  FIGURE 2.16 USE OF RDC VALUES IN THE STRUCTURAL CALCULATION OF THE DHB DOMAIN -  xi  87  FIGuRE 3.1 FIGURE 3.2  RAssF Ic 1  -  334,  , BOUND THE 576598 BUT NOT AxIN  91  DHB DOMAIN  ° THAT MEDIATED THE INTERACTION 5 DETERMINATION OF THE REGION OF RAssF1C’  -  93  5544 WITH DAXX FIGURE 3.3 FIGuRE 3.4 FIGURE 3.5  838 RESIDUES THAT INTERACTED WITH IDENTIFICATION OF THE RAssF1C  -  Sw, BUT NOT RASSF1C 233 ASSFIC , BOUND 821  -  44 ’ 55 DAXX  44 ’ 55 DAXX  100  5544 ’ BINDING INTERFACE ON DAXX 2338 3.6 IDENTIFICATION OF THE RAssF1C  FIGURE 3.7  -  107 RASSF1C UPON w CHANGES IN THE AMIDE CHEMICAL SHIFTS AND RELAXATION OF 2338  -  108  5544 BINDING DAXX FIGURE 3.9  102  55144 UPON ”’ AND DAXX 2338 CHANGES IN THE SECONDARY STRUCTURES OF RAssF1C  -  BINDING FIGURE 3.8  97  ’’ AND 2338 ELECTROSTATIC INTERACTIONS CONTRIBUTED TO THE BINDING OF RASSF1C  -  55144 DAXX FIGuRE  96  W COMPLEX 2338 DAXX RASSFIC / TERTIARY STRUCTURE OF THE 5544  -  111  55144 FIGURE 3.10 RESIDUES MEDIATING THE INTERMOLECULAR INTERACTION BETWEEN DAXX w 2338 RAS5F1C AND  113  FIGURE 3.11 DAXX AND SIN3 PAH DOMAINS USE DIFFERENT BINDING MODES. OTHER STRUCTURAL RELATIVES OF THE DHB/RAssF1C COMPLEX  117  2338 COMPLEXES WITH COMPLEMENTARY C /RASSF 1 ’’ 5544 FIGURE 3.12 PREPARATION OF DAXX LABELING FOR STRUCTURAL STUDIES  130  -  -  -  FIGURE 4.1 FIGuRE 4.2  -  -  D1FFERENCEs BETWEEN SUMO-1 AND SUMO-2  139  MAPPING OF THE BINDING INTERFACE OF SUMO-1 AND SUMO-2 WITH SIM-N AND  SIM-C FIGURE 4.3  144 -  NMR-M0NIT0RED TITRATIONS DEMONSTRATED THAT SIM-N’ BOUND BOTH  SUMO-I’” AND SUMO2w FIGURE 4.4  -  147  NMR-M0NIT0RED TITRATIONS DEMONSTRATED THAT  SIM-C’’ BOUND BOTH  SUMO1w AND SUMO-2” FIGURE 4.5  -  148  SIM-C BOUND SUMO-1 IN BOTH PARALLEL AND ANTI-PARALLEL ORIENTATIONS  FIGURE 4.6- SIM-N BOUND SUMO-1 IN A PARALLEL ORIENTATION FIGURE 4.7 FIGURE 4.8  -  -  152 157  44 SELF-ASSOCIATION WAS IONIC STRENGTH DEPENDENT DAXX  161  5544 THROUGH SIM-N 156 INTERACTED WITH DAXX DAXX  163  567739 VIA THE COVALENTLY FIGuRE 4.9 SUMOYLATED ETs-l BOUND SIM-C OF MDAXX ATTACHED SUMO -  xii  168  FIGURE 5.1  -  DOMAIN STRUCTURE OF  DAXX  .  FIGURE 52 PROPosED AUTO-REGULATORY MECHANISM OF THE DHB DOMAIN. THE COMPETITIVE/CO-OPERATIVE COUPLED EQUILIBRIA (CCCE) MODEL  180  -  Xli  186  List of Abbreviations 1D  one-dimensional  2D  two-dimensional  3D  three-dimensional  aa  amino acid  AR  androgen receptor  ASKI  apoptosis signal-regulating kinase 1  ATM  ataxia telagiectasia mutated kinase  ATP  adenosine 5’-triphosphate  ATRX  cL-thalassaemia syndrome protein  BLAST  basic local alignment search tool  CBP  CREB binding protein  CC  coiled-coils  CCCE  competitive/co-operative coupled equilibria  CD  circular dichroism  CK2  casein kinase 2  CREB  cAMP-response element-binding protein  CSI  chemical shift index  Da  Dalton  DAG  diacyiglycerol/phorbol ester  DAXX  Fas death-domain associated protein  DHB  DAXX-Helix-Bundle (DAXX ) 55144  DHD  putative DAXX helical domain  DIM  DHB-domain interacting motif  Dr  ” 2338 / RassflC 15 C 3 ‘ /unlabelecl 55144 N-DAXX  DSSP  define secondary structure of proteins  DTT  dithiothreitol  El  SUMO/ubiquitin-activating enzyme  E2  SUMO/ubiquitin-conj ugating enzyme  E3  SUMO/ubiquitin-protein ligase  EDTA  ethylenediaminetetraacetic acid  xiv  FADD  Fas-associated protein with Death Domain  FAT  focal adhesion targeting domain  GR  glucocorticoid receptor  GST  glutathione-s-transferase  HDAC  histone deacetylase  hDAXX  human DAXX  HEPES  4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid  HIPI R  huntingtin -interacting protein-i related  HIPK-i  homeodornain-interacting protein kinase 1  HMBC  heteronuclear multiple bond correlation  HPLC  high performance liquid chromatography  HSQC  heteronuclear single quantum coherence  IDPs  intrinsically disordered proteins  IPTG  isopropyl 13-D- 1 -thiogalactopyranoside  ITC  isothermal titration calorimetry  INK  Jun N-terminal kinase  KD  dissociation constant  kDa  kilodalton  MA LD 1-To F-MS  matrix-assisted laser desorption/ionization-Time of Flight-Mass Spectrometry  rnDAXX  murine DAXX  MEF  mouse embryonic fibroblast  MES  2-(N-morpholino)ethanesulfonic acid  MOPS  3-(N-morpholino)propanesulfonic acid  MSP58  nucleolar microspherule protein  MTSL  S-(2,2,5,5-tetramethyl-2,5-dihydro- I H-pyrrol-3 -yl)methyl methanesulfonothioate  MW  molecular weight  MWCO  molecular weight cutoff  N CB I  national center for biotechnology information (http://www.ncbi .nlm.nih.gov)  NDSM  negatively-charged amino-acid dependent sumoylation motif  NMR  nuclear magnetic resonance  NOE  nuclear Overhauser effect  NOESY  nuclear Overhauser effect spectroscopy  PAGE  polyacrylamide gel electrophoresis  xv  PAH  paired amphipathic helices  PCR  polymerase chain reaction  PDB  protein data bank (http://www.pdb.org)  PDSM  phosphorylation-dependent sumoylation motif  p1  isoelectric point  PIAS  protein inhibitors of activated STAT  PML-NB s  promyelocytic leukemia oncoprotein nuclear bodies  ppm  parts per million  PRE  paramagnetic relaxation enhancement  RA  Ras-association  Rassf  Ras-association domain family  Rd  N-Rassfl ”/unlabeled 2338 DAXX C 55144 sample C/ 5 3 ‘ 1  R[)C  residual dipolar coupling  RNAI  RNA interference  rmsd  root mean square deviation  SARAH  Sav/RASSF/HPO domain  SDS  sodium dodecyl sulphate  SENP  sentrin specific protease  SIM  SUMO-interacting motif  SIM-C” SlMNw  DAXX C-terminal SUMO-interacting motif. 718739 (mDAXX’ ) W) 9 DAXX N-terminal SUMO-interacting motif. (DAXXII  SPT  Ser-Pro-Thr rich region in DAXX.  SSP  secondary structure propensity  SUMO  small ubiquitin-like modifier protein  1 T  longitudinal relaxation time  2 T  transverse relaxation time  TEMPO  2,2,6,6-tetramethylpiperidine- 1 -oxyl  TF  transcription factor  TFA  trifluoroacetic acid  TGF-3  transforming growth factor f3  TNF-cL  tumor necrosis factor a.  TOCSY  total correlation spectroscopy  xvi  List of Amino Acid Abbreviations A  Ala  alanine  C  Cys  cysteine  D  Asp  aspartic acid  F  Gly  glutamic acid  F  Phe  phenylalanine  G  Gly  glycine  H  His  histidine  I  lie  isoleucine  K  Lys  lysine  L  Leu  leucine  M  Met  methionine  N  Asn  asparagines  P  Pro  proline  Q  Gin  giutamine  R  Arg  arginine  S  Ser  serine  T  Thr  threonine  V  Val  valine  W  Trp  tryptophan  Y  Tyr  tyrosine  xvii  Acknowledgments My most sincere gratitude goes to Dr. Lawrence P. McIntosh, who throughout the years has been an excellent supervisor and a good friend. He was always available to answer my questions, and was open to my suggestions and ideas. He criticized my work always constructively, and made me grow as a scientist. His “work hard, play hard” policy was inspirational. He motivated me to get results, but also to enjoy the great things that the mountains and the ocean around Vancouver have to offer. I cannot emphasize enough how much I enjoyed working in his laboratory. I am forever grateful to my wife Claire, whom I married at the beginning of my graduate work. The research process often takes you through depressions and frustrations, and without her support, my success would not have been possible. Our son Natiü was born when I was in the middle of the experiments described in Chapters 3 and 4, and I can say for sure that without Claire’s extra support, I would not have been able to complete my graduate studies. I dedicate this thesis to her and our son. Of course, I would not be here without the support of many people. My mother Patricia, and my father Gerardo always motivated me to succeed academically, and this led to my scholarship to the United World College of the Adriatic (Italy) when I was 17. There, my chemistry teacher Anne Brearley inspired me to become a scientist. At my undergraduate studies, Dr. Steven  Rafferty and Dr. Mark J. Parnis inspired me to go to graduate school. In Dr. McIntosh’s laboratory, I had the privilege to work with great people: Meena, Cameron, Mario, Matt, Gary, Manuela, Dave, Greg, Hyun-Seo, Martin, Markus, Chris, Shaheen, xviii  Mark, Patrick, Genevieve, Jerome, Desmond and Simon. In particular, Genevieve and Hyun-Seo became really good friends and rock climbing partners. I thank them for their friendship through the good and bad times. My special gratitude goes to our collaborator Dr. Alexander Ishov for bringing a much needed biological perspective to my project. I would also like to thank the following people for their help in some of the work presented 38 studied in section 4.7 was prepared by the summer student in this thesis. The sumoylated Ets’’ C/ labeled 3 ‘ N Christopher Dart. The intermolecular NOEs between unlabeled SIM-N” and 15 SUMO-1 (Table 4.3 and Figure 4.6) were assigned by Dr. Mark Okon. Finally, I want to thank once again Dr. McIntosh, Dr. Gary Yalloway and Genevieve Desjardins-Seguin for reading my thesis and suggesting me how to make it better.  xix  Chapter 1  Introduction The Fas death-domain associated (DAXX) protein was first discovered over a decade ago as an intermediary of a FADD-independent Fas signaling pathway. However, subsequent studies have established it as an important player in both transcription and apoptosis regulation. Recently, a role of DAXX as a cell cycle regulator is emerging as well. Despite these important biological functions, the structural and dynamic properties of DAXX remained unknown prior to this thesis research. In this chapter, an overview of the biological functions of DAXX is presented, with emphasis on its interacting partners Rassfl C and the small ubiquitin-like modifier SUMO. An explanation of how DAXX mediates numerous essential biological functions requires detailed structural and thermodynamic analyses of DAXX with these protein partners. These experimental goals will be addressed in the following chapters of this thesis.  1  Chapter 1 11  —  Introduction  Preamble  1.1.1 The human genome A milestone in molecular biology was reached in 2001 when it was announced that the human genome had finally been sequenced (Lander et a!., 2001; Venter et a!., 2001). Exciting, yes, but intriguing as well, as it raised as many questions as it provided answers. The biggest question perhaps being how this sequence dictates the formation of a human being? It is clear that RNA and proteins, not coding DNA, do the “work” in biological systems, so knowledge of a genome by itself is insufficient to understand what makes living beings. Proof of this is the observation that the latest estimate of the number of protein-encoding genes in the human genorne is around 25,000, a number close to that of C. elegans, a free-living one millimeter-long  transparent worm (Pennisi, 2003). Obviously it is not the mere number of genes that defines the complexity of a species, but rather the properties of the encoded proteins, the mechanisms of their regulation, and the complicated networks that arise from protein-protein interactions and post-translational modifications. 1.1.2 Signal transduction Such complicated protein-protein-interaction networks are crucial for cell communication in multi-cellular beings, where cells die, differentiate, divide, or survive for the good of the organism as a whole. In a general sense, cell communication involves a receptor protein at the cell surface, which is in charge of detecting a signal. The receptor protein reacts to the signal by activating an intracellular signaling protein, which initiates a signaling transduction pathway. This involves multiple enzymes and adaptor proteins (scaffolds) that work together to convey the message to the transcription machinery in the nucleus, where genes for each cellular process are expressed or repressed. 2  Chapter 1  Introduction  1.1.3 Modular proteins Interactions between signaling proteins are mediated by modular binding domains, which are typically defined as “structural, functional and evolutionary components of proteins, which can often be expressed as a single unit” (Han et al., 2007).  It is estimated that out of 34,000  proteins encoded by the human genome, between 65% and 80% of proteins consist of two or more domains (Han et al., 2007). The modular nature of proteins facilitates their study by a “divide-and-conquer approach, in which the structure and function of isolated domains are studied, and their contribution to the overall function of the full-length protein is subsequently derived. Such an approach has proved useful in elucidating the functions of a number of proteins (Vogel et al., 2004). However, in order to understand the integrated roles of protein modules fully, it is critical to complement such reductionist approach with a “re-constructionist” approach, and study the wild type protein in its native cellular context at a system level. 1.1.4 Intrinsically disordered proteins Captivatingly, about 25% of the predicted protein sequences from eukaryotic genomes cannot be attributed to any known domains (Moore et al., 2008). Some of these cases certainly reflect inadequacies in sequence-based “homology searching” as well as yet undiscovered “novel” domains. However, it is clear that a significant amount of these sequences form natively or intrinsically disordered proteins (IDPs) or segments of proteins, which at least in isolation lack any predominant secondary or tertiary structural elements. There is a growing recognition that IDP sequences play crucial roles ranging from flexible linkers between structured domains in modular proteins (“beads-on-strings”) to linear recognition epitopes (motifs) for forming protein protein complexes. IDPs, as well as misfolded or aggregated proteins, are also directly linked to 3  Chapter 1  —  Introduction  a number of human diseases, including cancer, cardiovascular diseases, neurodegenerative diseases and diabetes (Uversky, 2009). The intrinsically disordered regions of proteins represent a challenge to the above structurebased definition of a domain. That is, they are often evolutionally and functionally conserved, but they lack a well-defined three-dimensional structure. However, as their presence is crucial to the function of the protein, they need to be studied as well. Proteins often contain both: welldefined structural domains juxtaposed to intrinsically disordered regions that often play a role in regulating the function of the full-length protein. 1.1.5 DAXX, a multi-domain protein with intrinsically disordered regions  The subject of this study is DAXX, an intracellular signaling adaptor protein with multiple structural domains and intrinsically disordered regions. Since its discovery in 1997 as a “Death Associated” factor (Yang et al., 1997), DAXX has been found to be an essential player in multiple cellular mechanisms such as apoptosis (programmed cell death) and transcription regulation. This brings special meaning to its structural characterization, which is presented in this thesis. In other words, by learning where its domains and intrinsically disordered regions are, one can better understand how it comes to play such an important role in so many mechanisms. The role of DAXX in various cellular processes is described in more than 250 publications. These roles are both widely varied and at times seemingly controversial. Thus, these publications are often confusing in the big-picture perspective. Fortunately, there are five reviews on DAXX (Lindsay et al., 2008; Michaelson, 2000; Salomoni and Khelifi, 2006; Salomoni and Michod, 2009; Shih et a!., 2007), to which the reader is urged to go for a more extensive summary of the numerous facets of this fascinating protein. 4  Chapter 1  —  Introduction  In this introductory chapter, a shorter review of the functions and localization of DAXX is first presented (section 1.2). This is followed by an introduction to SUMO (section 1.3) and Rassfl C (section 1.4), two important partners of DAXX, which were part of this study. Finally, at the end of this chapter, the main goals and organization of this thesis are described (section 1.5).  1.2  DAXX  1.2.1 What is DAXX? DAXX, a 740 aa protein, was first discovered and characterized in 1997 by Yang et al. (Yang et a!., 1997), who found that DAXX expressed in all mouse organs ubiquitously, and associated with the Fas death-domain (DAXX stands for Fas death-domain ssociated protein). This association was thought to initiate the activation of the Jun N-terminal kinase (INK) pathway, which ultimately led to cell death (apoptosis) (Figure 1. 1A). At the time, this appeared as a breakthrough in the field of death receptors, providing an alternative pathway for Fas-controlled death that is independent of the adaptor FADD (F as-associated protein with Death Domain). The same group identified a year later the poptosis igna1 regulating inase 1 (ASK1) as the link between DAXX and the JNK-pathway, reinforcing their original model (Figure 1. 1A) (Chang et al., 1998). However, conflicting reports on the role of DAXX started appearing shortly after its discovery. First, mouse embryo fibroblasts deficient in FADD or caspase-8 were unable to produce apoptosis in response to Fas activation, suggesting that DAXX did not dictate a parallel signaling pathway (Juo et al., 1998; Zhang et al., 1998). Second, Toni et a!. failed to reproduce the DAXX-Fas direct interaction and found that DAXX was primarily located in the nucleus, 5  Chapter 1  —  Introduction  particularly in PML-nuclear bodies, and not in the cytoplasm (section 1.2.3.1). Additionally, they identified DAXX as a transcription repressor and speculated that it modulated the genes activated by Fas-induction from the nucleus (Toni et al., 1999). Overall, these initial reports highlighted the two primary functions attributed to DAXX: a mediator in apoptosis, and a transcription regulator. 1.2.2 The mixed roles of DAXX in apoptosis In addition to the initial studies which implicated DAXX in Fas-induced apoptosis, another group identified DAXX as a binding partner of the transforming growth factor-13 (TGF-f3) and as a regulator of TGF-f3-induced apoptosis again via JNK activation (Perlman et al., 2001). This study was important as it established for the first time the mechanism by which TGF-f3 controlled apoptosis (Figure l.1A). Based on the reports implicating DAXX as a pro-apoptotic protein, one would predict that a knock-out mouse for its gene would display proliferation abnormalities. Such abnormalities were observed with p53 tumor suppressor knock-out mice, which were able to live but developed some type of cancer within 6 months of birth (Donehower et al., 1992). Surprisingly, the opposite was observed with DAXX. That is, DAXX was an essential protein for embryo development, as embryos with a truncated form of DAXX or with a complete knock-out of the DAXX gene displayed extensive apoptosis and lethality at embryonic day 9.5-10.5 (Ishov et al., 2004; Michaelson et al., 1999). These studies therefore suggested that DAXX plays an essential anti-apoptotic role, at least in embryonic development. Further evidence of an anti-apoptotic role for DAXX was shown by two independent studies in which DAXX expression was silenced via RNA interference (RNAi) experiments. Both 6  Chapter 1  —  Introduction  studies found that DAXX silencing resulted in sensitization of the cell to apoptosis induced by Fas, tumor necrosis factor a. (TNF-ct), or even ultraviolet light irradiation (Chen and Chen, 2003; Michaelson and Leder, 2003). It is then conceivable that DAXX has both pro- or anti-apoptotic roles in different situations. It has been suggested that this duality may arise from the distribution of the protein in cell compartments (Michaelson, 2000).  1.2.3 Where is DAXX?  The function of DAXX might be dependent upon its cellular location. Obviously, to interact with the Fas-death domain, it needed to be present in the cytoplasm. It was then surprising that the first study reporting the cloning and sequencing of human DAXX predicted that it would be localized in the nucleus due to the presence of nuclear localization signals (Kiriakidou et aL, 1997). Soon thereafter, multiple reports confirmed that DAXX was indeed predominantly a nuclear protein (Everett et a!., 1999; Ishov et al., 1999; Toni et a!., 1999). The cell nucleus is a complicated organelle where many essential processes take place. This is where gene expression, as well as DNA replication, repair and recombination occur. It is organized in a number of dynamic compartments that, in contrast to their cytoplasmic counterparts, do not have delineating membranes. However, they can be distinguished by light and electron microscopy and each contains defining subsets of resident proteins (Dundr and Misteli, 2001; Shiels et a!., 2007). Nuclear compartments include the nucleolus, the splicing factor compartments, the Cajal bodies, and the prornyelocytic leukemia oncoprotein (PML) nuclear bodies (Figure 1.1 B). The latter are where DAXX has often been reported to reside.  7  Chapter 1  —  Introduction  Figure 1.1 Function and localization of DAXX. (A) Attributed pro-apoptotic roles of DAXX. DAXX was discovered as a binding partner of the death domain of the Fas receptor (DD), which activated the INK-signaling pathway via ASK1 and ultimately led to cell apoptosis (Chang et al., 1998; Yang et al., 1997). This pathway was the first proposal of an alternative to the wellestablished FADD-caspase signaling pathway activated by Fas (Strasser et al., 2009). The cytoplasmic TRII domain of the Transforming Growth Factor-13 was also shown to interact with DAXX and activate the same signaling pathway (Perlman et a!., 2001). (B) Schematic representation of the compartmental organization within a cell nucleus, showing chromosome territories (green), cajal bodies (purple), nucleoli (violet), splicing-factor compartments (red), PML-NBs (light green) and nucleoplasm (white). Within chromosome territories, dots symbolize the location of euchromatin where transcription occurs, and stars symbolize the location of heterochromatic regions. This figure was adapted from (Dundr and Misteli, 2001; Schmitz and Herrmann, 2008; Shiels et a!., 2007). (C) Compartmental localization of DAXX is believed to control its function. When DAXX is recruited into PML-NBs by the PML protein during interphase, it cannot act as a transcription repressor. However, different stimuli can result in its release from PML-NBs into the nucleoplasm, where it can repress sumoylated transcription factors (TF) via its SUMO interaction motif (SIM) in the Ser-Pro-Thr rich region (SPT), or it can localize into the nucleolus, centromeres or heterochromatic regions. The function of DAXX within these compartments has not been fully characterized, although it has been proposed that nucleolus sequestration also inactivates DAXX, and that interaction with the ATP-dependent chromatin remodeling protein ATRX might play a role in its transcription regulation role. See text for details. Figure adapted from (Shih et al., 2007). -  8  _y7 ________  Chapter 1  A  —  Introduction  Extra-cellular matrix Fas receptor  Cytoplasm  \/ ASK1  Caspase-8  JNK Apoptosis  Nucleoplasm  B  \  Splicing-Factor Compartments  PML-NBs  Apoptosis  Nucleoli  Chromosome tories Transcription Cajal sites I-Ieterochromatin bodies  C ATRXAXX  Or  Nucleoplasm  Ets-1  53 p Smad4, etc.  9  Chapter 1  —  Introduction  1.2.3.1 DAXX in PML nuclear bodies PML nuclear bodies (PML-NBs; also known as nuclear domains-lO (ND-lU), PML oncogenic domains (PODs), or Kremer bodies) are present in 1-30 copies in most mammalian cell nuclei and typically measure 0.2-1.0 p.m (Bernardi and Pandolfi, 2007). They regulate a number of different cellular functions, including gene expression, DNA repair, apoptosis and viral infection control (Bernardi et al., 2008; Dellaire and Bazett-Jones, 2004; Tavalai and Stamminger, 2008). PML-NBs are composed of numerous proteins, with nucleic acids at the periphery of the bodies. Although variable in exact composition, key to PML-NB formation is the PML protein. Knock-out mice without the PML protein failed to form PML-NBs, and consequently were more resistant to apoptosis after DNA damage and prone to carcinogenesis (Wang et a!., 1998). The first groups that detected DAXX in the nucleus also reported that it was located in PML NBs, and identified DAXX as a binding partner of PML itself (Ishov et a!., 1999; Li et al., 2000a). In these studies, sumoylation of PML was proposed to be necessary for the recruitment of DAXX. As will be discussed shortly, the ability of PML to be sumoylated and to bind other sumoylated proteins is also essential for the formation of PML-NBs (Shen et a!., 2006). An extensive recent study confirmed that a UMO-jnteracting motif (SIM) within DAXX was needed for its recruitment to PML-NB, thus explaining the above proposition (Figure 1.1 C) (Lin et al., 2006). DAXX recruitment into PML-NBs has been proposed to serve as a regulator of its function (Ishov et al., 2004; Li et a!., 2000a; Shih et al., 2007). In this model, PML-NBs serve as “storage depots”, sequestering DAXX in an inactive state. Indeed, studies have reported that overexpression of the PML protein reversed DAXX-controlled repression by recruiting it from transcription sites into PML-NBs (Lehembre et al., 2001; Li et a!., 2000a). Overall these studies 10  Chapter 1  —  Introduction  highlighted the functional importance of the localization of DAXX, and the role that sumoylation and SIMs play in mediating its localization to PML-NBs (section 1.3). 1.2.3.2 DAXX in other nuclear compartments  In a study by Ishov et al., DAXX mice did not survive beyond embryonic day 10.5, but PML’ mice, which lacked PML-NBs, were viable (Ishov et a!., 1999). Therefore, PML-NB recruitment of DAXX is not essential for its biological function. Interestingly, in PML’ cells, DAXX was mostly found in areas of condensed heterochromatin. The association of DAXX with heterochromatin was  also  indirectly implied by Hollenbach et al., who used co  immunoprecipitation and gel filtration co-fractionation to demonstrate that DAXX was part of a large complex that included core histones, chromatin associated protein Dek and histone deacetylase 2 (HDAC-2) (Hollenbach et al., 2002). Numerous groups have subsequently reported a direct or indirect association of DAXX with histone deacetylases (Ecsedy et a!., 2003; Greger et al., 2005; Huang et al., 2008; Kuo et a!., 2005; Morozov et a!., 2008; Poleshko et a!., 2008). Since HDACs play a role in remodeling chromatin to an inactive state, their association with DAXX is believed to be key to the function of DAXX as a transcription repressor (discussed in section 1.3.3). However, many details of this model remain unknown. For example, what are the mechanisms that make DAXX move from PML-NBs to chromatin regions? This is unclear at the moment, although interaction between DAXX and the homeodomain-interacting protein kinase 1 (HIPK- 1), and the ct-thalassaemia syndrome protein (ATRX) have been shown to play a role in this process. In a study by Ecsedy et a!., HIPK-1 physically interacted with DAXX, re-localizing it from PML-NBs to chromatin via a direct interaction (Ecsedy et a!., 2003). The kinase also 11  Chapter 1  —  Introduction  phosphorylated DAXX at Ser669, increasing its repression ability by an unknown mechanism. ATRX, an ATP-dependent chromatin remodeling protein, and DAXX have also been shown by independent groups to form a complex (Ishov et a!., 2004; Tang et al., 2004; Xue et a!., 2003). This complex was relevant in inactivating ATRX via recruitment into PML-NBs in a cell-cycle dependent manner (Ishov et a!., 2004). Apart from heterochromatic regions, DAXX has been shown to interact with the centromere protein CENP-C and thereby it associated with centromeres (Pluta et al., 1998). DAXX also bound the nucleolar microspherule protein MSP58 and accumulated at the nucleolus (Lin and Shih, 2002). The role of DAXX in centromeres is unknown, whereas the latter report suggested that DAXX was recruited in the nucleolus with a functional consequence similar to PML-NB sequestration. That is, nucleolar DAXX was inactive and unable to mediate transcription.  1.2.3.3 Cytoplasmic DAXX?  Although the overwhelming evidence points to a nuclear localization of DAXX, a number of studies have reported DAXX trafficking from the cell nucleus to the cytoplasm under stress conditions (Akterin et al., 2006; Charette and Landry, 2000; Jung et al., 2008; Jung et al., 2007; Junn et al., 2005; Karunakaran et al., 2007; Song and Lee, 2004). However, most of these studies relied on immunofluorescence staining of transiently over-expressed (and hence unnaturally abundant) proteins. Further doubt to these findings has arisen from a very recent report. Lindsay et al. applied multiple approaches, including subcellular fractionation by various methods, and time lapse visualization by immune-fluorescent microscopy, to unequivocally show that DAXX does not translocate to the cytoplasm under previously reported conditions of stress or cell line type (Lindsay et a!., 2009a).  12  Chapter 1  —  Introduction  Therefore, the cytosolic association of DAXX with Fas, Ask-i or other cytoplasmic proteins remains in question. The authors suggested that these associations may be cell-cycle dependent (Lindsay et a!., 2007). Indeed, the same group has shown recently that DAXX interacts with RassflC, a cytoplasmic protein, once the nuclear envelope has broken down during mitosis ((Lindsay et a!., 2009b), discussed in section 1.2.5). L24 DAXX as a transcription regulator  A role of DAXX as a transcription co-repressor was first proposed by Hollenbach et al., who observed transcriptional repression of Pax3 when cells were co-transfected with DAXX (Hollenbach et al., 1999). Ten years later, the list of transcription factors regulated by DAXX includes more than 25 examples (summarized by Lindsay et al., 2008). Although some of these transcription factors appeared to be activated by DAXX (Emelyanov et a!., 2002; Huang and Shih, 2009), in most cases DAXX played a role in transcriptional repression. DAXX associates with HDACs, and this association is believed to be the main route to repression of gene expression (Figure 1 .2E and 1 .4B). Hollenbach et a!. proposed that DAXX functions in a similar way as the Sin3 co-repressor. Sin3 is a large multi-domain protein, which serves as a scaffold, linking HDACs with other transcription regulators and transcription factors (Grzenda et al., 2009; Silverstein and Ekwall, 2005). Key to the formation of these large complexes is the presence of four domains within Sin3 named “paired amphipathic helices” (PAH).  13  __  Chapter 1  —  Introduction  —Activator  A  RNA Pol H Holoenzynw  Ix.  4 B  -  TFHD  Gene  t  Repressor  C  Gene  i  D  j  ’ 1 iw  Gene  Remodeled Chromatin remodeling Nucleosome complex  Nucleosome  E  rii  Gene  F  •  JIVj  1t  Deacetylated histone tails  Figure 1.2 Generic models for transcriptional repression in eukaryotic systems. (A) Transcription activation requires the recruitment of the RNA polymerase II holoenzyme complex, which contains a large number of proteins that are omitted for clarity (Roeder, 2005; Sikorski and Buratowski, 2009; Thomas and Chiang, 2006). Recruitment is normally achieved by activators, which are multi-domain proteins with a DNA binding module and one or several protein-protein interaction domains. Activators not only recruit RNA polymerase II, but they also recruit co-activators such as histone acetylases for chromatin remodeling. Activators can be modulated by post-translational modifications, such as phosphorylation and sumoylation (not shown). Transcriptional repression can be achieved by many different pathways, but always involve one or more repressors, which can interfere with the recruitment of RNA polymerase II holoenzyme (B), neutralize activators (C), competitively bind activator DNA-binding regions (D) or recruit other complexes such as histone deacetylases (E) or chromatin remodeling complexes (F). Figure adapted from (Alberts, 2002). -  14  Chapter 1  —  Introduction  Sin3 PAH domains mediate interactions with transcription factors (TF5) and other co repressors. The cell tightly regulates these interactions (and thereby controls gene expression) via post-translational modifications to the TFs and competitive binding of activators to the same interaction interface on the TF. Hollenbach et a!. proposed the existence of two PAR domains in DAXX that could control its function in a similar way as the Sin3 PAR domains (this model is revisited in Chapters 2 and 3, as the N-terminal domain of DAXX is compared with the known structures of Sin3 PAR domains). Finally, in contrast to Sin3, a common theme that has appeared in recent literature is that DAXX-mediated repression depends on its ability to interact with sumoylated transcription factors. This is discussed in section 1.3.  1.2.5 The role of DAXX in mitosis  As discussed so far, previous studies have demonstrated that DAXX functions as an apoptosis and transcription factor mediator. Rowever, a new and exciting role of DAXX as a mitosis regulator has lately emerged. The evidence that DAXX is involved in cell cycle controlling processes is manifold. First, Lindsay et al. noticed an abnormal number of chromosomes (aneuploidy) in DAXX  ‘  mouse  embryonic fibroblasts (MEF5), suggesting an important role of DAXX in cell division (Lindsay, 2009). Second, DAXX  cells showed different cell-cycle progression with respect to control  cells. Third, cells depleted of DAXX via siRNA underwent cell division at a different rate with respect to control cells (Lindsay et al., 2009b). Fourth, Lindsay et al. noticed that DAXX localized at the mitotic spindle during mitosis, specifically during the prometaphase stage (discussed in section 1.4.3 and 3.7).  15  Chapter 1  —  Introduction  To understand the role of DAXX in cell-cycle control, Lindsay et a!. set up a yeast-two hybrid screen for interactors of DAXX that could be directly involved in cell-cycle controlling processes. In this way, they identified Rassfl C, a member of the tumor suppressor Ras associated domain family, as a DAXX partner (section 1.4.3). Further studies revealed that the DAXX/Rassfl C interaction was indeed important in mediating mitosis, and more importantly, in determining how cancer cells respond to a common chemotherapy agent, taxol. The details of their findings are discussed in Chapter 3, as the structure of the DAXX/Rassfl C complex is presented.  t3 The small ubiquitin-like modifier protein SUMO The small ubiquitin-like modifier protein (SUMO) derives its name from its structural resemblance to ubiquitin (rmsd of backbone C 3  A), despite having only 18% sequence  identity with it. It was discovered in 1996 by three independent groups (Boddy et al., 1996; Okura et al., 1996; Shen et al., 1996). Since then, the research field of SUMO has grown immensely, with well over 1500 publications and with a growing number of publications per year (in 2008 alone there were 265). This is not surprising, as SUMO has been implicated in multiple fundamental biological processes such as transcription control, DNA repair, nucleocytoplasmic trafficking, mitochondrial fragmentation, and viral infection. There are over 100 reviews on various aspects of SUMO functions. Excellent recent reviews on SUMO include: (Bergink and Jentsch, 2009; Garcia-Dominguez and Reyes, 2009; Geiss-Friedlander and Melchior, 2007; Geoffroy and Hay, 2009; Hay, 2005; Johnson, 2004; Kerscher, 2007). Here, an overview of the general principles and mechanisms of sumoylation is provided, with an emphasis on those that are relevant to DAXX localization and function.  16  Chapter 1  —  Introduction  L3.1 The SUMO family Despite limited sequence similarity, SUMO family members have three-dimensional structures very similar to ubiquitin. However, SUMO proteins contain an N-terminal unstructured 10-25 amino-acid segment that is not found in ubiquitin. Until now, the only function known of this disordered region is formation of SUMO chains. The human genome encodes four different SUMO paralogues (SUMO-1 to SUMO-4). SUMO-2 and SUMO-3 are virtually identical, with 97% sequence identity, and are commonly referred as SUMO-2/3. SUMO- 1 and SUMO-2/3 are expressed in all cells, whereas SUMO-4 seems to be mainly expressed in lymph nodes and the spleen (Guo et al., 2004). SUMO-2/3 have 50% sequence identity with SUMO-1, and 87% sequence identity with SUMO-4. All SUMO paralogues are expressed in inactive forms, with an extended C-terminus that needs to be removed proteolytically so that mature SUMO can be covalently attached to its target via a Gly-Gly motif (Figure 1 .3A). Whether maturation by SUMO-specific proteases is regulated or constitutive is unknown at the moment. Once matured, SUMO paralogues can be covalently attached to substrates via a reversible mechanism. 1.3.2 The mechanism of reversible sumoylation The process of sumoylation is very similar to ubiquitylation. It involves an El activating enzyme (Figure 1 .3B), an E2 conjugating enzyme (Figure 1.3 C), and in some cases, an E3 ligating enzyme (Figure 1 .3D). The latter ultimately attaches the C-terminal Gly carboxyl of the SUMO moiety to the amine of a lysine residue in the substrate via an isopeptide linkage. This reaction is reversible, as a number of sentrin-specific proteases (SENP) can specifically remove SUMO from substrates (Kim and Baek, 2009; Yeh, 2009) (Figure 1 .3E). 17  Chapter 1  —  Introduction  Clear differences to ubiquitylation exist, however. For example, a consensus SUMO-acceptor site has been recognized as ‘PKxE (where P is a branched aliphatic amino acid and x is any amino acid) (Rodriguez et a!., 2001). Recently this acceptor site has been expanded to a phosphorylation-dependent sumoylation motif (PDSM), or a negatively-charged amino-acid dependent sumoylation motif (NDSM), both of which depend on negative charges that are either added by phosphorylation (PDSM) or are constitutive of the residues downstream of the sumoylation site (Hietakangas et al., 2006; Yang et a!., 2006). Ubiquitylation-consensus sites, on the other hand, have not been identified. Furthermore, sumoylation involves only one E2, and not always an E3, whereas ubiquitylation involves more than 20 different E2s and hundreds of E3s (Geiss-Friedlander and Meichior, 2007). Sumoylation of substrates occurs very rapidly. Golebiowski et al. reported maximal sumoylation of proteins only 5 minutes after heat shock (Golebiowski et a!., 2009). Interestingly, while most heat-shocked-sumoylated proteins were de-sumoylated 2 hours after heat shock, heat shock-desumoylated proteins were not re-sumoylated over the same period of time. This hints to a reversible mechanism that is tightly and quickly regulated.  18  Chapter 1  .. ..-..  —  Introduction  SUM()  SF\P  +  +  E3?  I  . --..  II  O)  a.  SUMO  SUMO SUMO +  GG-COO  I  SUMO  ATP  j  c=o  rf qt  Figure 1.3 Sumoylation mechanism. Before SUMO can be conjugated, it must be matured by sentrin specific proteases (SENPs; A). This leaves a C-terminal Gly-Gly that can be covalently attached to Cys 173 in the El activating enzyme (AOS 1 /UBA2 in humans) by an ATP-dependent reaction (B). Cys92 of the E2 conjugating enzyme (UBC9) receives the activated SUMO from El (C). Finally, a lysine side-chain of a substrate (a transcription factor (TF) in the figure) is covalently modified to form an isopeptide linkage (D). This step is usually aided by an E3 ligating enzyme. The mechanism is reversible as SENPs can remove SUMO from the substrate (E). Structures used for this figure are: SUMO-1 (la5r.pdb); Senp2 (ltgz.pdb); Aosl/Uba2 (ly8r.pdb); Ubc9 (lu9a.pdb); Ets-1 TF (2jv3.pdb). -  19  Chapter 1  —  Introduction  Different mechanisms for regulating sumoylation have been identified. Various research groups have noticed that the expression levels of the sumoylating enzymes (i.e. El, E2 and E3s) changed under different conditions, such as in hypoxia, calcium signaling, or progesterone signaling (Comerford et a!., 2003; Deyrieux et al., 2007; Jones et a!., 2006; Liu and Shuai, 2008). Apart from expression levels, sumoylating enzymes have been reported to be regulated by inactivation under certain conditions or by translocation to different cellular compartments. For example, reactive oxygen species caused the formation of a disulfide bond between the catalytic cysteine residues of El and E2 in HeLa cells (Bossis and Melchior, 2006).  Similarly, pro-  inflammatory stimuli such as TNF-a induced phosphorylation of PIAS 1 (an E3 enzyme) and controlled its translocation to the promoters of necrosis factor-kB (Liu et al., 2007).  1.3.3 Molecular consequences of sumoylation Sumoylation can affect substrates in a number of ways. In a general sense, sumoylation can protect a lysine side-chain from other post-translational modifications such as ubiquitylation or acetylation. Most often, however, it alters the intra- or inter-molecular interactions of the substrate. This can either alter the activity of the substrate (if it is an enzyme), or it can promote or inhibit the formation of large complexes that carry out a process that is important for the substrate (Figure 1 .4A). In principle, SUMO can either add or block a binding interface from the substrate, or it can induce a conformational change in the substrate that affects its activity. Examples of the first are RanGap 1 (which interacts with RanBP2 when sumoylated), and the transcription repressor ZNF76 (which binds to a TATA-binding protein only when it is SUMO-free) (Mahajan et a!., 1997; Zheng and Yang, 2004). Consistent with the model of SUMO serving as a structurally independent tag on a protein, Macauley et a!. showed that SUMO-RanGAP behave as two 20  Chapter 1  —  Introduction  independent domains linked by a flexible isopeptide bond (Macauley et al., 2004; Macauley et al., 2006). In contrast, only one example of a protein whose conformation changes significantly upon sumoylation has been reported: thymine DNA glycosylase. This DNA-repair enzyme requires sumoylation to undergo a structural change that allows it to release its substrate once repaired (Baba et a!., 2005; Hardeland et al., 2002). Ultimately, it is not easy to predict the effect that sumoylation will have on a particular substrate. However, the observation that the vast majority of sumoylated proteins are transcription factors suggests that some common themes exist. These include the recruitment of chromatin remodeling complexes and histone-modifying factors such as histone deacetylases (Garcia-Dominguez and Reyes, 2009). It is worth noting that such chromatin remodeling is long-lasting. That is, a small amount of sumoylated transcription factor can have a big effect by leaving permanent repressive changes once the transcription factor or its repressive complexes are removed by de-sumoylation. This is important as only 5% of the transcription factors are sumoylated in vivo at the “steady-state” (Geiss-Friedlander and Meichior, 2007; Hay, 2005). Recruitment of chromatin remodeling complexes does not occur via a direct interaction with transcription factors, but rather it is thought to be mediated by scaffold proteins such as DAXX (Figure 1 .4B, section 1.3.6).  21  Chapter 1  —  Introduction  •k  Surnoviation  A  -  Lstra  rSubst  I  (  (SIts)  Desurnoylation  tratZ  :Subs  II1LLraLIor A  B  Ac  Ac Sumoykition  Nucleosome  Ac  £TF) ‘  Ac  Figure 1.4 Molecular consequences of sumoylation. (A) Before sumoylation, a substrate can either be free (and unable to interact with interactor B) or interact with another protein (interactor A). Sumoylation can have a number of consequences, including: (1) disrupt the complex of the substrate with interactor A; (2) induce a conformational change on the substrate; (3) supply a new surface for recruiting a new protein (interactor B). In principle, this could occur either independently from the substrate or with cooperative binding from both the substrate and SUMO. (B) Proposed mechanism for DAXX-mediated transcription repression. DAXX is unable to bind a transcription factor (TF) when it is not sumoylated (in this example the TF is present in active chromatin with acetylated histone tails). Once the TF is sumoylated, it recruits a large repression complex via the binding of DAXX to a SUMO. The repression complex may contain histone deacetylases (HDACs) that deacetylate the histone tails and remodel chromatin to prevent gene transcription. -  22  Chapter 1  —  Introduction  1.3.4 Differences among SUMO paralogues Since SUMO-4 was discovered more recently, its function is still unclear. There is also a controversy as to whether or not SUMO-4 can even be covalently matured and attached to substrates. Thus, its biological function appears to differ from that of the other SUMO paralogues (Owerbach et al., 2005; Wei et al., 2008). Most studies to date have focused on SUMO-1 and SUMO-2/3 differences. Saitoh et al. were the first to address some of the functional differences between these SUMO paralogues (Saitoh and Hinchey, 2000). In their study, they observed the following differences: (1) SUMO-2/3 was about 4 times more abundant than SUMO- 1. (2) SUMO-1 was mostly in a conjugated form, whereas there was a large pooi of free SUMO-2/3 in the cell. Overall, there was about fifty times more free SUMO 2/3 than SUMO-l. (3) Some substrates were modified by specific SUMO paralogues. For example, RanGapi was modified by SUMO-1 and not by SUMO-2/3. Indeed, a recent proteomic study found SUMO paralogue specificity is quite common among SUMO substrates. Out of 53 identified sumoylated proteins in HeLa cells, only 9 could be modified by either SUMO-1 or SUMO-2/3, whereas 25 were preferentially modified by SUMO-l, and 19 by SUMO-2/3 (Vertegaal et al., 2006). (4) Stress signals such as heat shock depleted the pool of free SUMO-2/3. This suggested that a number of proteins are sumoylated in response to these stimuli. Indeed, a very recent study identified 766 SUMO-2/3 substrates, many of which underwent sumoylation changes upon treatment with heat shock (Golebiowski et al., 2009).  23  Chapter 1  —  Introduction  Additionally, SUMO paralogues have been shown to have different localization and mobility in vivo by Ayaydin and Dasso (Ayaydin and Dasso, 2004). In their experiments, they  demonstrated that only SUMO- 1 is located at the nuclear envelope and in the nucleolus, whereas all paralogues were found throughout the nucleoplasm.  Furthermore, photobleaching  experiments revealed that SUMO- 1 mobility was much slower than that of SUMO-2/3. Other differences among SUMO paralogues include their ability to form polymers and their specificity towards interactors via non-covalent bonds. For example, SUMO-2/3 can form chains through its flexible N-terminal segment, whereas SUMO-1 cannot. These chains have been shown to be important in signaling and promotion of macromolecular complexes (Ulrich, 2008). Finally, differences in the specificity towards binding SUMO interacting motifs from different paralogues have been discovered recently (Hecker et al., 2006; Meulmeester et al., 2008). These differences are discussed in detail in Chapter 4. 1.3.5 SUMO interacting motifs  The first group that reported a specific SUMO-interacting motif (SIM; also known as SUMO-binding motif, SBM) noticed that proteins interacting with sumoylated p’73, a member of the p53 family, contained a common Ser-X-Ser sequence juxtaposed with hydrophobic and acidic residues (Minty et al., 2000). Four years later, Song et al. identified an entirely different SIM by NMR spectroscopy, and isothermal titration calorimetry (ITC). This SIM consisted of Val/Ile-X-Val/Ile-Val-Ile (Song et al., 2004). A year later, a crystal structure of SUMO 1/RanGapl-UBC9-Nup358 complex revealed that SUMO-l and RanGapi indeed interacted through this motif and that the hydrophobic core is fundamental for the interaction (Reverter and Lima, 2005). This example highlights the importance of a structural characterization to unequivocally determine the basis for the interaction between molecules. 24  Chapter 1  —  Introduction  A number of SIM-containing proteins have been documented since then (reviewed in (Kerscher, 2007)). SIMs contain a hydrophobic core juxtaposed with a cluster of charges. Common characteristics among SIMs are: (1) The hydrophobic core is essential for the interaction, and a single amino-acid change can significantly reduce its affinity for SUMO. (2) A cluster of charges juxtaposed to the hydrophobic core is almost always present, and helps to dictate the binding orientation of the motif (Hecker et al., 2006; Song et al., 2005). (3) Charges within the hydrophobic core and the juxtaposing residues can also influence SUMO-paralogue binding specificity (Hecker et al., 2006; Meulmeester et al., 2008).  (4) SIMs have dissociation constants in the range of 2-3 jtM, whereas ubiquitin binding motifs are more in the range of 10-500 tM (Hicke et al., 2005).  (5) Some proteins use multiple SIMs to increase their affinity towards SUMO-chains. An example of this is RNF4, an E3 ubiquitin ligase that bound weakly to monomeric SUMO-2/3, but strongly to SUMO-2/3 chains. Consistently, RNF4 only ubiquitinated PML that had been modified by SUMO-2/3 chains in vitro (Tatham et al., 2008). 1.3.6 DAXX and SUMO DAXX and SUMO interact in two possible ways: DAXX itself can be sumoylated, or DAXX and SUMO can interact non-covalently via SIMs within DAXX. In the first case, DAXX sumoylation can occur in a number of sites. DAXX has 15 lysine amino acids, of which only one has the PKxE sumoylation motif: Lys6O (YKLE). However, Jang et a!. failed to detect any sumoylation effect on K6OA or K385A DAXX mutants. Instead, they identified Lys630 or 25  Chapter 1  —  Introduction  Lys63 1 as possible non-consensus sumoylation sites in BOSC23 cells (Jang et al., 2002). These sumoylation sites were corroborated by a later study, which also identified Lys6O as a sumoylation site. However, Lys6O was sumoylated only if expressed as a peptide comprising residues 46-75, and not if expressed as 1-282 (Chen et al., 2006). Thus, it is unclear if this site is functional in the wild type protein. The sumoylation sites within DAXX have been suggested to be cell-type dependent. For example, Lin et al. reported detectable sumoylation at all lysine residues in HeLa cells (Lin et al., 2006). More interestingly, Lin et al. also reported that DAXX sumoylation was dependent on one of its SIMs, as mutations of the SIM-binding cleft of SUMO, or of the DAXX C-terminal SIM, significantly reduced DAXX sumoylation. A simple model for this effect is that DAXX interacted non-covalently with E2-SUMO via its SIM-C, thereby increasing the effective concentration of its acceptor lysine(s) towards the conjugating enzyme complex. The biological significance of DAXX sumoylation remains to be established. A mutant lacking all Lys residues showed no functional difference to the wild type, localizing in PML NBs and retaining its transcription repression ability (Lin et al., 2006). However, recent reports suggested that sumoylation of DAXX was up-regulated in viral infections or in heat shock (Golebiowski et al., 2009; Hwang and Kalejta, 2009). Whether this is biologically relevant remains to be tested. On the other hand, non-covalent SUMO binding by DAXX has been demonstrated to be essential for its recruitment into PML-NBs and/or transcription regulation. Two SIMs within DAXX that mediated non-covalent SUMO-binding have been reported in the literature, one at its N-terminus (SIM-N), and one at its C-terminus (SIM-C) (Lin et al., 2006; Santiago et al., 2009).  26  Chapter 1  —  Introduction  In Chapter 4, a detailed analysis of how these SIMs interact with SUMO- 1 and SUMO-2 is presented. In Chapter 5, some other interesting models for DAXX SIMs that have arisen from the structural characterization of DAXX are discussed.  1.4 The Ras-association domain family (Rassf) Rassfl C is a member of the tumor suppressor Ras-association domain family (Rassf). Its founding member, Nore 1 (now known as Rassf5), was first characterized a decade ago, almost at the same time as SUMO and DAXX were first identified (Vavvas et al., 1998). Ten years later, this family is at the center stage for cancer research. It is now known that many members of the Ras-association domain family play important roles as tumor suppressors and their inactivation is a common characteristic of various types of tumors. The Rassf family has been the subject of a number of recent excellent reviews (Avruch et al., 2009; Hesson et a!., 2007a; Richter et a!., 2009; van der Weyden and Adams, 2007). In the next sections, a brief overview of the characteristics of this family and its association with DAXX is presented. 1.4.1 Domain structure of Rassf The defining characteristic of all Rassf members is the presence of a Ras-association (RA) domain (Figure 1.5). Ras GTPases are a superfamily of monomeric GTPases that broadcast signals from the cell surface to the nucleus. When activated indirectly via cell surface receptors, they interact with different effectors that in turn initiate signaling transduction pathways to ultimately regulate cell proliferation, differentiation, motility and apoptosis (Alberts, 2002). So far, only Rassf5 has been established as a true Ras-effector, and the remaining members of the family are thought to either exert functions independent of Ras, or mediate Ras-activated processes via heterodimerization with Rassf5 (Avruch et a!., 2009).  27  Chapter 1  —  Introduction  Most members of this family also contain a Sav/Rassf/Hpo (SARAH) domain, a 50 aminoacid stretch shown to form heterodimers with proteins involved in cell cycle control and apoptosis development. Some members contain a Cl-type zinc finger domain, also involved in protein-protein interactions controlling apoptosis. Finally, Rassfl A and Rassfl C have an ataxia telangiectasia mutated kinase (ATM) recognition motif, which is phosphorylated by the ATM, an enzyme of central importance in DNA-repair and apoptosis (Figure 1 .5A) (Richter et a!., 2009). At the structural level, the domains in the Rassf members are just beginning to be characterized. The structure of the Cl domain of Rassf5 was solved by NMR methods recently, and was found to bind intramolecularly with its RA domain (Harjes et a!., 2006). Interestingly, this intramolecular interaction was disrupted by Ras binding, freeing the Cl domain to presumably interact with other partners. The SARAH domain structure of Mstl was solved by NMR methods a year later, and it was found to mediate homo- and heterodimerzation through two short amphipathic helices (Hwang et al., 2007). Finally, the crystal structure of the RA domain of Rassf5 bound to Ras was reported last year (Stieglitz et a!., 2008), revealing a novel mode of Ras binding with very tight association (KD between 0.02-0.3 tiM, depending on the method used) (Wohlgemuth et al., 2005) (Figure 1 .5B). To our knowledge, the work presented in Chapter 3 is the first to structurally characterize the unique N-terminus of Rassfl C.  28  Chapter 1  —  Introduction  Figure 1.5 The Ras-Association Domain Family (RASSF) and their domains. (A) Schematic of the most prevalent members of the family. The defining characteristic of this family is the presence of a Ras-Association (RA; red) domain. Most of the members have this domain preceding the Sav/RASSF/HPO (SARAH) domain (green) at their C-terminus. A newly discovered sub-family (Rassf7-RassflO) has the RA domain at its N-terminus. RassflA and Rassf5A-B have a cysteine-rich diacyiglycerol/phorbol ester (DAG)-binding domain, also called protein kinase C conserved region 1 (blue; Cl). RassflA, C have an ataxia telagiectasia mutated (ATM) kinase phosphorylation motif (magenta). The NCBI accession number of each member is shown as reference. (B) Cartoon representation of the structural domains of Rassf5A. The structure of the apo C-i domain (blue, 1 rth.pdb) with Zn 2 atoms ; the RA domain (red; 3ddc.pdb) bound to RAS; and the SARAH domain as a homodimer (green/gray; 2jo8.pdb). The structures are not to scale. -  29  Chapter 1  —  Introduction  A RASSF1A  NP 009113  RASSF1C  NP 733831  I  .  341)  —  LZZZZZEZZZ  270  —  RASSF2  TZZ._  NP 739580  .  326  —  RASSF3  NP 835463 230  RASSF4  NP 114412  •  RASSF5A  NP 872604  -•  RASSF5B  NP 872605  ASSF5C  NP 872606  •1410  336 265  RASSF6A  NP 803876  RASSF6B  NP 958834  337  RASSF7  NP 003466  RASSF8  NP 009142  1  -  -  ZZZZZZ  IZ 369  ZZZZZ ZZZZZI  373  —  ZZ 392  —  RASSF9  NP 005438  RASSF1O  NP 001073990  --  435  ---  ------------—--  615  B  RAS  166  349  ‘4O3 357 RASSF5A 366  -------  122  170  --  274  30  413  _s-__ 364  418  Chapter 1  —  Introduction  1.4.2 Rassfl and cancer Rassfl A and Rassfl C are identical at their C-termini, as they are different splice forms of the same gene, located on chromosome 3p2l.3. Their transcription uses two different promoters (3.5kb apart), resulting in a unique 120 and 50 aa N-terminus for RassflA, and RassflC, respectively. Very interestingly, deletions of the 3p2l .3 region are a common and early event in the formation of lung, breast, kidney and other cancers. Rassfl A inactivation is one of the most frequent marks so far described in human cancer (Hesson et al., 2007a). Mutations in the RassflA gene are rare in cancer cells, but rather the whole gene is often silenced by DNA methylation of its promoter, occurring in up to 100% of liver cancer cells, -60% of breast cancer cells, 80% of testicular cancer cells, -50% of ovarian cancer cells, and so on (reviewed in (Agathanggelou et al., 2005)). Since RassflC uses a different promoter, its inactivation by DNA methylation is not as frequent as for the Rassfl A gene, although loss of expression of RassflC does occur in some cancer cells (Li et al., 2004; van der Weyden and Adams, 2007). These observations have led a number of groups to use the Rassfl A as a tumor biomarker, prognostic marker, and even as a candidate for treatment with inhibitors of DNA methyltransferases (Hesson et al., 2007b). Based on this evidence, it is obvious that Rassfl genes must play an important role in normal cell function. So far, members of this family, particularly RassflA, have been implicated in the regulation of Ras signaling, cell cycle control, microtubule stability, and apoptosis regulation (Richter et a!., 2009). It is evident that they perform this by regulating protein-protein interactions with various players, and therefore, like DAXX, appear to function mainly as scaffolding proteins.  31  Chapter 1  —  Introduction  1.4.3 Rassfl and DAXX  Apart from Lindsay’s work (discussed in section 1.2.5), two papers have already implicated a direct interaction of DAXX with Rassfl proteins. Kitagawa et al. reported that DAXX and Rassfl C interacted directly at the PML-NBs during interphase. Interestingly, stress stimuli that produced DNA damage also induced degradation of DAXX, and released Rassfl C from the PML-NBs. Rassfl C then translocated to the cytoplasm and activated the INK signaling pathway, thus ultimately activating apoptotic pathways (Kitagawa et al., 2006) (Figure 1 .6A). This suggested mechanism is simple and appealing. However, Lindsay et al. failed to reproduce these results, and found that Rassfl C and DAXX are totally separated during interphase, with DAXX being recruited in PML-NBs while RassflC remained in the cytoplasm. Once in mitosis, they then did interact and presumably mediated cellcycle checkpoints (Figure 1.6C, discussed in Chapter 3) (Lindsay et al., 2009b). It is unclear at the moment what conditions determined these contradictory results, but nevertheless the DAXX/Rassfl C interaction has (an) important biological function(s). Interestingly, both studies identified roughly the same interacting regions of both proteins: the N-terminus of DAXX and the N-terminus of Rassfl C. More recently, Song et al. reported a direct interaction between RassflA and DAXX (Song et al., 2008). This depended on the translocation of RassflA from the cytoplasm to the nucleus, which they speculated occurred upon DNA damage. They found that RassflA disrupted the interaction of DAXX with Mdm2 and Hausp, two regulators of p53. Such disruption resulted in the activation of p53 genes, which mediate apoptotic pathways (Figure 1.6B).  32  Chapter 1  —  Introduction  Figure 1.6 Proposed models for mechanisms controlling DAXX/RASSF interactions. (A) Kitagawa et a!. reported that DAXX and RASSF1C interact at PML-NBs during interphase, and induced DNA damage resulted in DAXX degradation (presumably by ubiquitylation) and freed Rassfl C from PML-NBs. Subsequently, Rassfl C translocated to the cytoplasm and mediated activation of the INK-signaling pathway (Kitagawa et a!., 2006). (B) Song et al. reported direct interaction between RassflA and DAXX. This disrupted the DAXX-Mdm2-Hausp complex (Tang et a!., 2006), which regulated the ubiquitylation of the transcription factor p53. Rassfl A promoted Mdm2 auto-ubiquitylation and the increased level of p53 resulted in transcription activation (Song et a!., 2008). (C) Lindsay et al. reported different locations between RassflA/C (microtubules at the cytoplasm), which formed a heterodimer, and DAXX (PML-NB5 at the nucleus) during interphase. Once in mitosis, DAXX translocated to the microtubules via Ras sf1 C, and they mediated a mitotic checkpoint important for the progression of cell division (Lindsay et a!., 2009b). -  33  Chapter 1  —  Introduction  A PML bodies DNA damage  (Rascflc  -  DAXX  &19  Cytoplasm translocation ,  4  iNK pathway  i  Ub  Apoptosis  DAXX  Proteasome degradation  B Proteosome degradation  DAXX) MDM2  bX  MDM2 (ssf:9  _—.--  ssf1A)  DAXX  DNA damage  p53 Pr:osorne degradation  -  :  ()  C  Interphase  Mitosis (prornetaphase)  34  Chapter 1  —  Introduction  Importantly, Song et al. found that the N-terminus of DAXX once again mediated the interaction, but identified the C-terminus of Rassf1 A (common to Rassfl C) as the region regulating the interaction. On the other hand, Lindsay et a!. identified the unique 50 aa Nterminus of Rassfl C as the DAXX interacting region. In Chapter 3, it is confirmed that indeed this region of Rassfl C contains a DAXX interacting motif (DIM). It is possible that a second DIM is present at the C-terminus of RassflAIC, which would explain Song’s results. Further experiments will help to clarify this.  t5 Thesis overview It is well established that DAXX performs a number of important functions in the cell. Most of the knowledge on how DAXX functions as a scaffold protein has arisen from the ability of DAXX truncation constructs to bind the many effectors that DAXX regulates. However, these constructs have been designed based solely on a very poor knowledge of the structural domains of DAXX, derived only from sequence alignments with homologues and extrapolation of their function. The overall goal of this thesis is to provide a clear structural characterization of the domain boundaries of DAXX and the roles of the domains in mediating protein-protein interactions. This will aid future biological experiments that will improve our understanding of the detailed mechanisms that mediate the function of this important protein.  Chapter 2 presents the first study of the domain composition of DAXX. Different constructs of DAXX that contained predicted domains based on a consensus of secondary structure and disordered region prediction algorithms were designed. This less biased approach led to the discovery of a well folded eighty-residue domain that is predicted to be separated from other structured domains by long intrinsically disordered regions. Using NMR spectroscopy, its three 35  Chapter 1  —  Introduction  dimensional structure was solved. Although this domain did form a helix bundle, its fold was clearly different to that of the reported Sin3 PAR domains. Thus it was named as the DAXX Helix-Bundle (DHB) domain. The fold of the DHB domain suggested that it would also mediate protein-protein interactions in a different mode to the one reported for Sin3 PAH domains. Chapter 3 presents the investigation of the interaction between the DHB domain and the unique 50 aa N-terminus of Rassfl C. Indeed, an interaction by NMR-monitored titrations was corroborated. After a long iterative process, the optimal conditions for the formation of a tight and soluble complex were derived, and thereby its three dimensional structure was solved by NMR methods. This structure will be valuable in understanding how DAXX mediates protein-protein interactions. Excitingly, it explains previously reported interactions and it might even predict future binding partners of DAXX. Finally, Chapter 4 presents the characterization of the SUMO interacting motifs (SIMs) within DAXX. These are localized in conserved disordered regions at both ends of the protein. The strength of the SUMO/DAXX-SIMs interactions under various conditions was measured and their binding mode was determined by NMR-methods. Moreover, how a sumoylated transcription factor, Ets- 1, is able to interact with DAXX was studied. Interestingly, this interaction is found to be completely dependent of the presence of SUMO, as unsumoylated Ets 1 cannot bind DAXX. Additionally, no cooperativity in the binding is observed, suggesting that the ability of DAXX to bind sumoylated transcription factors depends solely on SUMO, and not on the transcription factor. Finally, an exciting finding of an intramolecular binding of SIM-N with the DHB domain is shown. This could open the door to new mechanisms at how DAXX is regulated in the cell, discussed in Chapter 5. 36  Chapter 2  Structure of the N-terminal DAXX Helix Bundle The first structural characterization of DAXX is presented in this chapter. Sequence alignment and secondary structure prediction algorithms were used to define a number of constructs of DAXX. After cloning and expression, the C-terminal 1/3 of DAXX was found to be intrinsically disordered. In contrast, a well-defined folded domain was indentified near its N terminus. NMR spectroscopy was used to define the boundaries of this domain, to solve its three dimensional structure, and to determine its dynamic behavior. The calculated structure consists of five helices, and hence it is named the DAXX Helix Bundle (DHB) domain. This domain has a very different topology to the Sin3 PAH domains, which until now had been used as models for this region of DAXX. The differences in the structure between the DHB domain and Sin3 also suggest that each utilizes a very different mode of binding. In Chapter 3, this is confirmed by the structural analysis of a Rassfl Cl peptide with the DHB domain. This work provides the first clear domain boundaries in DAXX, which in turn will provide clues as to how this key regulatory protein operates at the molecular level in a number of diverse, important cellular processes.  37  Chapter 2  2.1  —  Structure of the DHB domain  DAXX sequence and secondary structure Due to the lack of experimentally-determined structural information, various researchers  used sequence comparisons to provide “best guesses” regarding the structure-function properties of DAXX (Figure 2.1). The first group to do so suggested the existence of two coiled-coils formed by residues 180-212 and 356-388 (Pluta et al., 1998). Coiled-coils are known to regulate oligomerization or protein-protein interactions (Grigoryan and Keating, 2008). A second group shortly thereafter predicted a “Pair of Amphipathic Helices” (PAH domains) between residues 64-108 and residues 192-240. This was based on weak sequence similarities with the transcription repressor Sin3 (Hollenbach et al., 1999). Researchers have also defined “domains” in the C-terminal portion of DAXX based on unusual amino acid content (Figure 2.1, top). The first one, between amino acids 434-485, contains 80% of GluJAsp and hence was denoted “acidic”. The second one is -48% Ser-Pro-Glu residues (SPE: residues 495-596). The third one (SPT: residues 665-740), which is 46% Ser-Pro Thr residues, was reported to bind the transcription factor Ets-1 (see section 4.6). These sequence analyses reflect very low resolution views of DAXX, and thus a more exhaustive bioinformatic approach was undertaken to better identify possible structural domains within this protein. It is clear that three-dimensional structural information is needed for a detailed understanding of the function of DAXX. However, unstructured and disordered regions often prevent successful crystallization of a protein, or make the analysis of its NMR spectra much more difficult. This is particularly true for large, multi-domain proteins, which often behave as “beads on-a-string”. Therefore, following a reductionist’s approach, it is often necessary to define minimal structural domains for bacterial expression and in vitro characterization.  38  Chapter 2  < 55  1  >  >s 144  CC  CC  180212  35__fi  I’AmI  []]  DAXX  CL DHB  17  64  —  Structure of the DHB domain  <-> idi4  108  192  240  200  250  434  485 495  SPE 5%  I  665  SPT[] 723 740  PHD  HNN SOPMA NNPREDICT JNET SCRATCH PORTER PSIPRED 0  50  100  150  300  350  400  450  500 550 600 650 700 750  Residue Figure 2.1 Suggested “domain” organization of DAXX. (Top) Two “Pair of Amphipathic Helices” domains (PAH1 and PAH2) have been proposed to be located in the N-terminal portion of the protein (Hollenbach et a!., 1999). A region between amino acids 434-485 contains 80% Glu/Asp residues and hence termed “Acidic”. The C-terminus contains regions rich in Ser/Pro/Glu residues (SPE; residues 495-596), and in Ser/Pro/Thr residues (SPT; residues 665740). Two segments were suggested to form coiled-coils (CC) (Pluta et a!., 1998), the first one within the putative PAH2 domain (residues 180-212), and the second one between PAH2 and the acidic region (residues 356-388). Based on the analysis of Figure 2.2 using more extensive database, DAXX has three regions of conserved sequence, identified as CL (green, 60-145), 3 (orange, 1 85-400) and y (blue, —430-470), as well as two SIMs (red) and the DHB domain characterized in this chapter (magenta). (Bottom) Secondary structure prediction of DAXX using the following sequence-based algorithms: PHD (Rost, 1996), Hierarchical Neural Network (HNN; Guermeur et a!., 1999), the improved self-optimized prediction method (SOPMA; Geourjon and Deleage, 1995), Neural Network Predict (NNPredict; (Kneller et a!., 1990), JNET (Cuff and Barton, 2000), SCRATCH (Cheng et al., 2005), PORTER (Pollastri and McLysaght, 2005), and PSIPRED (McGuffin et a!., 2000). Predicted cL-hehces are shown in black and predicted 13-strands in red. -  39  Chapter 2— Structure of the DHB domain Comparison of the sequences of DAXX from various vertebrate organisms shows proteins of similar size with a number of regions that are highly conserved (Figure 2.2). Remarkably, the Nterminal and C-terminal segments of the protein each contain an essentially invariant SUMO Interacting Motif (SIM), which forms the basis of Chapter 4. Two additional regions that show a high degree of conservation are located between residues 60-145 (termed region c) and residues 182-397 (region 13). A more variable-length conserved region rich in Asp and Glu amino acids (residues 434-485, region y) is also present in each organism. These conserved regions suggest the existence of functional domains in the protein. As a first step to define the boundaries of possible structural domains, the presence of secondary structure elements in the protein was searched, particularly in the conserved regions. Recent advances in bioinformatics have provided a number of sequence-based algorithms that predict the secondary structure of proteins with relatively high reliability (Rost, 2001). Since each algorithm has strengths and weaknesses, a consensus of the following web-based servers to predict the secondary structure of DAXX was used: HNN (Guermeur et al., 1999); NNPredict (Kneller et a!., 1990); INET (Cuff and Barton, 2000); SCRATCH (Cheng et al., 2005); PORTER (Pollastri and McLysaght, 2005); PSiPred (McGuffin et al., 2000); PHD (Rost, 1996); and SOPMA (Geourjon and Deleage, 1995).  40  Chapter 2— Structure of the DHB domain  E—> 000  -  REDS 6CR ATTLE MONKEY  3ICU.CE .IALMCCI DCCC  SIM-N  I LE1E;  LCAr.A C. CR31 LE 7. lIKES  5CC-IAN -VINE  -  1031,2  —  !!  Ljj  ‘1  SCLl.CEKRiD5E3I  _Jii!  I  F  /C4V.CI.SL  LHECs1AC.4 ER  H  S  CF  HzI I;  0.  116  DOG Dli ICEWAC ClAD 6150655. —is STYLE —ii V .6V7NEY -.  V ClAD 55514(5 DoG CCRWAY RAT V. 615351CR CATTLE V MCNCCSY  Y  HOC-IAN MOOSE SALMON 05CC NC 54155 HAl MEORCA CATTLE C. MONKS? -.  HOC-CAD ICE 053.-lOll 304 IV V.  ICC —. F F -i-IF T 11 1 EEE MI5NDOFL’ 456 EECE EENT 5L3ENG 467 1-CA: -I -5503-Es:-. C /EI1FEI N C 448 cE:T; I SI-LIE.Lo5: 5 6 445 KEs’IISO-CHILI 1 C C 415 --—-16” IA: rQs:-. -366551 LEEI6LE50LEEE_l 445 1 .1-1655) C 4-CF r FT ‘‘SLFDCFD -  ..-  -  -  -  -  -  -  .  556 553 041  .IELLVIEV’. .LLEP3CDL?\ SON: INFD’,COSPJ C— -  s-s4:oo  ,OICCC’KlCC FL 1105.0 1.C,sClCC’p:o’p,CI LSCCFPLAPIC.It ‘“6 IDEFEHAA .C’T)C!NS?N SEGNE— ———GCIESFD PVFFH2R—NSE?HEN’LFT?EC54OiCLTEP55Po.ADLDPIVDL LI .IV’1E-O’C EEDENAEVOIN-—:lA1SGN3PI:QSYT. —:.:NLvDE5510:E :—co:s:ssto ‘:61 Fpr,ISTE1:l;LEFSC:3l3R:MSIV.:. FTVF5-—:ECI LAFC::0 Cl s1CE 1’6 C CDI ,‘l’IIF 211141 ‘Cll(RCCDGl LENL.CLDS1ASiLN-C.0 GLFVL -—0 ICEs]C:CIFA-EE——Es:Tt,C,-po,:IE000, :-[-CASIFAHSE’;C -0 OIIiTARICLLFECEEC. Es-’.&.,DJFCCLSEEIFAFS .411. 1_E5RE___CDC C, ‘CC 5FCsHp]IA’,C’0 ‘C 5 QT1TEBFiLFlKo7H ‘VIE. .‘:——--.p C C,:lAEPLCP.I.CIF —.  -.  ..  -  HAS 67 Y 5 .’V.. F5.CSE5COCDTVl.Z .. -C FE:lCA:I’c: F DIV I ‘_AELEC-4153P5’ ECI ‘hyC’sED.:DCIJDL 5’ H\’SADHISTsC’-D5YAsp .:-.DLO-OS:vzINT410 DOT 1 :.,E j RSDC ‘LFEI.ERAL’1FPEFFDI ‘.11150 EERL”TVCF 010 .‘CCC L I ElF A C 51 Cl -  MEOAFS. 14115110  1EE  k  TF  1  .  IF  IL  T/  1  F  L  F  LE 71111  81465 -V-C 56 15515011 DOS N 41WAI i-IL V. ME-ASIA CADDLE 7. IKNREY  11l7:sGf P  árWCZADlL DrrE3TVAD :Ts, -il  ODE C5DD 233 .CNCEPNFCCCCIC 0 C 0.3,, LLGGCYIKEC I4A.,VJ .0 C KECOK: YiVL2s0: :020.1(3 AEPD0N-YUEP.C Cl 1B ‘ASs  <—>  SIMC  74 577 DNDA—— -.61 1lCCI 6115 sppsFLC’-:DLIVA:cAP::KrpCIs:FHDFGTI roSSPLA’.:,:_.. 1’ J L’C SC.I_-JCI’. ‘355 6011 .,‘53H COD F 078 ‘D’ A DIII’ IT 1 :C4:51 576 SPFSP:?IOD4 ‘41 T60:OSSOPGTI 1 3. 5664 32605 1, L_SC110_’, 16656531 4 CDA  743 737  -  74E 766  ‘•-  Figure 2.2 Alignment of the DAXX sequence. The following organisms were used for the alignment: (common name, accession numbers): Homo sapiens (Human, CAG33366), Mus musculus (Mouse, AAC53284), Salmo salar (Salmo salar ABQ59664), Canis familiaris (dog, CAl 11447), Rattus norvegicus (Norway rat, NP_543 167), Orzias latipes (Japanese Medaka, BAD93268), Bos taurus (Cattle, AA134603), and Cercopithecus aethiops (Vervet monkey, AAB66586). Positions with 90% conservation identity are highlighted in black, residues with 90% conservation similarity are highlighted in gray. There are two SIMs in all the forms of DAXX, one at its N-terminus (SIM-N) and one at its C-terminus (SIM-C), shown with red double-headed arrows. Three regions with high degree of conservation and predicted to have secondary structure elements are also identified with arrows and named in order as (green), f3 (orange) and ‘y (blue). The structured residues of the DAXX Helix Bundle (DHB) domain characterized in this chapter are marked (magenta box). -  41  Chapter 2— Structure of the DHB domain As summarized in Figure 2.1, the N-terminal 2/3 of DAXX is predicted to contain many c helices that cluster roughly into the three regions (cL,  i, and y)  of highest sequence conservation  (Figure 2.2). In contrast, the C-terminal —l/3 of DAXX is predicted to be predominantly unstructured  (“intrinsically  disordered”),  as  expected  from  the  abundance  of polar  Ser/Thr/Pro/Glu residues. Thus, secondary structure prediction algorithms and sequence conservation suggested the presence of clear domain boundaries.  2.2 Characterization of DAXX constructs With the aim of using NMR spectroscopy to characterize potential structural domains of DAXX, a set of constructs encoding various truncation fragments corresponding to the two most conserved regions, c. and f3, were cloned (Figure 2.3). The region  ‘  was not investigated, as it  contains the acidic “domain” with 31 Glu and 12 Asp residues and thus would not be expected to be well structured. The constructs were tested first for protein expression in E. coil BL21 (2DE3) cells. In the cases where protein of the expected size was indeed produced, cells were fractionated to differentiate soluble DAXX fragments from those forming insoluble inclusion bodies (see section 2.6.2 for details on the procedure). Of twenty three constructs tested, four expressed soluble proteins (Figure 2.3, blue boxes, Appendix 2): human , 60 DAXX’ ’ 46 DAXX , 61243 , and murine mDAXX 347420 DAXX . The first two contain the putative PAH1 and coiled 566739 coil/PAH2 domains respectively. The third construct covered another predicted coiled-coil (Figure 2.1). The fourth construct contained the C-terminal SPT region (including SIM-C). In the absence of any functional assays, the four soluble DAXX fragments were examined for structure by NMR spectroscopy.  By far the best construct was , 60 yielding a ’ 46 DAXX 42  Chapter 2— Structure of the DHB domain spectrum that contained over one hundred dispersed, sharp signals indicative of a likely monomeric, well structured protein (Figure 2.4). This region contains the putative PAH1 domain, and thus it was decided to solve its three-dimensional structure as a route towards understanding its function. DAXX’ -243 precipitated when dialyzed into NMR buffer (see section 2.6), and In contrast, 61 2 elution buffer which is high in ionic strength (500 mM NaC1) was found to be stable only in Ni and has a pH of 7.4. The spectrum of this construct under the latter conditions still showed only a limited number of broad signals, indicative of aggregation. Furthermore, mass spectrometry revealed that the sample was also degraded. This result was disappointing as DAXX’ 61243 contains the putative PAH2 domain. Further deletion or addition studies may be necessary to obtain fragments of this region that remain stable and soluble, as required for any structural analysis. ° was stable in NMR sample buffer and gave a good ‘H-’ 34742 DAXX N HSQC spectrum, 5 albeit with much less dispersion than exhibited by DAXX ° (Figure 2.4). Although such 6 ’ 46 limited  H’ 1  dispersion is often indicative of a predominantly unstructured polypeptide, coiled  coils often give poor dispersion despite having secondary structure (Kovacs et al., 2002). Indeed, the circular dichroism (CD) spectrum of this construct did show features diagnostic of cL-helix content (Appendix 1). It is interesting to note that this construct contains the region originally predicted to contain a coiled coil (Figure 2.2) (Pluta et al., 1998). However, an improved prediction algorithm for coiled coils gave a probability score of only l6% for residues 359-399 (McDonnell et al., 2006), so further structural characterization is needed to determine if this region is indeed involved in coiled coil formation.  43  Chapter 2— Structure of the DHB domain  <>4— cc 100  [J  I  17  I’I  64  100  cc  212  356  360  [idj  PAHZ 192  240  434  480 495  SPE  SPT 596  665  Expression Solubility  724 730  1-160  No  46-160  Yes  Yes  Yes  No  55-243  Yes  Partial  161-243  Yes  Yes  161-342  Yes  No  161-420  Yes  No  181-335  Yes  No  181-365  Yes  No  181-385  Yes  No  181-407  Yes  No  181-365  Yes  No  *181407  Yes  No  206-335  Yes  No  206-365  Yes  Partial  206-385  Yes  Partial  206-407  Yes  I’artial  Yes  No  Yes  No  <  46-420  —> ——.——>  246-342 261-420  -  261-342  No  279-342  No  [420  Yes  rn566-739  Yes  Yes  Yes  No  1  47-560  -  -  Figure 2.3 Identification of structured domains in DAXX through deletion analyses. All constructs were His -tagged except for two that were GST-tagged (*). Expression tests were 6 performed in BL21 (2DE3) cells grown at 30 °C for 5 hours. Blue frames highlight constructs that expressed soluble proteins. The last construct (566-739) was made using murine DAXX (mDAXX) instead of human DAXX. For reference, a schematic drawing of the proposed domain organization (described in Figure 2.1) of DAXX is shown on the top. -  44  Chapter 2  110  e  115  •  o 120  Structure of the DHB domain  61243 DAXX’  0  ° 466 D.A)cX  —  rO  0 0  C  0 C  C  0  •0,  125  110  ° 742 DAXX C  115  0  •  000  120 0  C  125  10  9  8  7  ‘H (ppm)  ‘H (ppm)  Figure 2.4 5 H-’ HSQC spectra of soluble constructs of DAXX. All spectra were recorded 1 N in NMR sample buffer at 25 C except for DAXX 161-243 , which was recorded in Ni.2+ binding buffer (50 mM 4 HPO buffer, 500 mM NaC1, pH 7.5) to minimize precipitation at lower ionic 2 K strength. The dispersion of the amide resonances in the DAXX 6 spectrum is indicative of ’ 46 stable secondary and tertiari structure elements. DAXX 161 243 gave a poor spectrum, probably due to aggregation. DAXX 47-420 showed slight IHN dispersion, which along with CD studies (Appendix 1), suggested that it may be partially structured. mDAXX 566739 had random coil IHN shifts, indicating that it was predominantly unstructured. -  45  Chapter 2— Structure of the DHB domain 566739 gave a typical spectrum of an intrinsically unstructured (or natively mDAXX disordered) random coil polypeptide, with relatively sharp amide narrow 7.5  -  11 1 N  resonances clustered in the  8.5 ppm range (Figure 2.4). Interestingly, this region has been reported to interact  with a number of proteins (discussed in Chapter 5, Figure 5.1C). It is hypothesized that these interactions may occur via “linear peptide recognition sequences,” such as the conserved SIM-C (Figure 2.1). Indeed, SIM-C was able to bind independently to a sumoylated transcription factor (section 4.6). It is very likely that the partially conserved region 675-699, which is very rich in Ser/Thr residues, has a yet uncharacterized biological role as well.  2.3 Mapping of the DAXX N-terminal structural domain boundaries Unstructured polymer segments typically present a challenge for the elucidation of the three dimensional structures of the ordered regions of biomolecules, including proteins, nucleic acids, and carbohydrates. In the case of X-ray crystallography, they both interfere with the crystallization of the molecule and fail to give detectable electron density in any resulting crystal. In the case of NMR spectroscopy, they often yield strong, poorly dispersed signals that overlap other resonances co-incidentally in the same spectral region. Furthermore, they are often susceptible to aggregation, chemical degradation or proteolytic cleavage, thus producing a heterogeneous sample that is difficult to analyze. Also, disordered regions typically do not yield useful long range NOEs that might otherwise provide possible restraints on their conformations. Thus, unless the unstructured region has a known biological function, it is usually prudent to first work with minimal-sized constructs containing only the folded domain of interest. Once structurally characterized via this reductionist approach, one can then undertake a constructionist approach to investigate the domain within its native context. This is particularly true given the more recent recognition that disordered segments of proteins indeed have numerous important 46  Chapter 2  —  Structure of the DHB domain  ° were 6 ’ 46 C, and ‘ 3 N resonances of DAXX 5 functions. With this goal, the main-chain ‘H, ‘ assigned, to enable a better definition of the boundaries of the structural domain within this N relaxation measurements. 5 truncation fragment via chemical shift analyses and ‘ 2.3.1 Chemical shift assignments NMR spectroscopists have a number of powerful multi-dimensional heteronuclear experiments at their disposal for assigning signals from the main-chain or backbone nuclei of proteins (a description and pulse sequences of these experiments are reviewed in (Sattler et al., 1999)). Two of these, namely the HNCACB and CBCA(CO)NH, were used to assign the 60 ’ 46 DAXX backbone nuclei of . The HNCACB pulse sequence transfers magnetization “out-and-back” from the amide proton to the directly bonded nitrogen ‘ N nucleus, then to both the ‘ 5 C° and 13 3 C of the same residue (i, intra-residue) and of the preceding residue (i-i, inter-residue) (for frequency labeling in ti), then back to the nitrogen (t ) and finally back to the amide proton for detection (t 2 ) (Figure 3 2.5A). This means that in the final three dimensional spectrum, at each amide 1 H” (wa) and ‘ N 5 (02),  there will be four  13 and ‘ cross-peaks: two from the intra-residue Cx C nuclei, and two 3  13 and ‘ from the preceding ‘ C’ and ‘ 3 C nuclei (Figure 2.5B). Conveniently, the Cu 3 C signals 3 have opposite signs due to a constant time delay used for the pulse sequence.  The CBCA(CO)NH pulse sequence transfers magnetization from the ‘H”/’H to the 13 C 3 C/’ (t,),  HN for then via the carbonyl to the nitrogen of the next amide (t ) and finally to the amide 1 2  detection (t ) (Figure 2.5A). The result is that each residue gives only two cross-peaks in the 3 C nucleus and one for the ‘ three dimensional spectrum: one for the 13 C nucleus of the residue 3 (i-i, w,) preceding the amide (i;  02,03)  in question (Figure 2.5B). 47  Chapter 2 HNCACB  A  H—,.©—H —  H  Structure of the DHB domain  CBCA(CO)NH  H—©çH  H—©—H  çII) C —C&•—©— C I I II II  N  —  H—Co—H  —  H  0  0  r  B  -  Cr  -  I 1 c  Cr  0.  -•+ C cr 1 c; oS  II  AIO3NH  SIO2NH  II  A1OINH 111  It  LIOONH  (ppm)  Figure 2.5 Magnetization transfer pathways for experiments used for backbone assignment. (A) Magnetization transfer pathway for the I-fl\TCACB (1) and CBCA(CO)NH (2) experiments. Arrows indicate the direction of the magnetization transfer, and circles define the atoms whose chemical shifts are recorded in the experiment. (B) Example of how the HNCACB and CBCA(CO)NH experiments were used to assign uniformly 5 C 3 ‘ N -labeled /’ . 60 ’ 46 DAXX C and ‘ Both experiments give cross peaks for 13 C resonances of the residue before (i-i), but 3 HNCACB also gives cross peaks for the intra-residue (1) ‘ C and ‘ 3 C. Additionally, in 3 I-LNCACB spectra, positive peaks arise from ‘ C nuclei (black), and negative from ‘ 3 Cr nuclei 3 (red). All this information can be used to connect sequential residues (dotted line). Shown are o (vertical; 13 c) (03 (horizontal; 1 H’) strips taken at the ‘ N chemical shift (o2) of the indicated 5 residues. -  -  48  Chapter 2— Structure of the DHB domain By using these two experiments, one “walks” along the backbone of a protein, linking Ca and ‘ 3 C correlations 3 crosspeaks from the intra-residue (i), as well as the inter-residue (i-i) l  with the  IHN  Cx 3 and 15 N signals of each amide (i). Ala, Gly, Ser and Thr have very diagnostic l  and 13 C chemical shifts, and thus are easily identified as starting points for this process. In the ideal case it is possible to unambiguously assign most, if not all, of signals from the backbone nuclei of a protein using this approach. In cases of ambiguity, due for example to extensive spectral overlap, complementary pulse sequences such as those that detect the carbonyl carbon 0 the H1TCACB and , 6 ’ 46 can also be used to resolve problem areas. However, for DAXX CBCA(CO)NH experiments were sufficient to assign most of the resonances from the backbone nuclei in this fragment. 2.3.2 Identification of the folded domain ° 6 ’ 46 With spectral assignments in hand, the boundaries of the folded domain within DAXX were easily refined. This was done by determining the secondary structure of each residue N (section 2.3.2.2), and 5 (section 2.3.2.1), by studying the relaxation dynamics of its backbone ‘ by making a number of constructs differing slightly in length to determine the boundaries of the ° fragment (section 2.3.2.3). 6 ’ 46 minimum folded domain within the DAXX ° secondary structure 4616 2.3.2.1 DAXX 13 3 The chemical shift of a ‘ C’ nucleus (as well as Ca, 3 ‘ C , ‘He) is extremely sensitive to its 3 of an amino acid shifts downfield with respect to the secondary structure. In particular, the C corresponding random coil value in x-helices and upfield in 13-sheets (Wishart and Sykes, 1994). Caobserved 3 Therefore a plot of “secondary chemical shifts” (l  —  Crandom coil) 13  is diagnostic of the  secondary structure of a protein, with contiguous stretches of residues having positive  49  Chapter 2— Structure of the DHB domain Cu secondary chemical shift values readily identifiable as being in helices, and negative values 13 indicating the presence of a f3-strand. Figure 2.6A shows that amino acids 60-136 in DAXX ° 6 ’ 46 contain a number of c-helices, and no n-strands. Remarkably, these boundaries agreed well with the secondary structure prediction algorithms (Figure 2.2) and with the conserved region a. in the alignment of DAXX sequences from various organisms (Figure 2.1). 2.3.2.2 15 N DAXX ° relaxation analysis 4616 A complementary way to determine the boundaries of the folded domain is to examine the NMR relaxation behavior of its backbone. ‘ N relaxation data reflect the local mobility of the 5 amide bonds in the polypeptide backbone on a residue-specific basis. In general, the heteronuclear NOE decreases from approximately +0.8 to -3.6 with increasing fast timescale mobility of the ‘ N-’H bond (Kay et al., 1989). Decreasing ‘ 5 N {‘H}-NOE values indicate 5 enhanced flexibility on a nsec-psec timescale. Folded regions tend to have elevated ‘ N {‘H} 5 NOE values (i.e.  >  0.6) due to the lack of fast timescale flexibility in the backbone, whereas  unfolded regions have much smaller or even negative ‘ N { 5 H}-NOE values. Figure 2.6B shows 1 that amino acids 60-140 of DAXX ° were well ordered, as expected based upon their 6 ’ 46 predicted cL-helical secondary structure, whereas the flanking residues were significantly more mobile and thus conformationally disordered. 2.3.2.3 Comparison of various DAXX constructs To refine the boundaries of the DAXX fragment containing a structural domain for minimal size and optimal expression and spectral quality, four additional constructs based on chemical shifts and relaxation analyses were made. DAXX 4 was first examined, which contained the ’ 59 minimum folded domain and does not have an oxidizable N-terminal Cys58. However, this  50  Chapter 2  —  Structure of the DHB domain  fragment was almost completely insoluble, and thus presumably too short to fold stably (Figure 58 C 5544 DAXX 2.6C). The N-terminus was then extended and Cys58 was replaced by Gly. G  proved to be more soluble, but still with a significant amount of protein in the insoluble fraction 4 retained the wild-type Cys residue, and exhibited ’ 55 of an E. coil extract. In contrast, DAXX 44 This ’ 55 DAXX improved solubility. Finally, the C-terminus was extended slightly to produce . construct was completely soluble when expressed in E. coil (Figure 2.6C). N HSQC spectra of DAXX 15 44 and DAXX ’ 55 ° showed that 6 ’ 46 A superimposition of the ‘Hthe optimized fragment indeed retained the structured elements from . 60 The only ’ 46 DAXX 1 N chemical shifts. significant spectral changes were the absence of signals with random coil H HNCACB and CBCA(CO)NH spectra of DAXX 44 were recorded to confirm previous ’ 55 assignments and assign signals from residues close to the new N- and C-termini, which expectedly differed slightly from . 60 Figure 2.7 shows the fully annotated ‘H-’ ’ 46 DAXX N 5 HSQC spectrum of the optimized construct . 44 ’ 55 DAXX 2.4 DAXX 144 structure determination 55 2.4.1 Assignments of side-chain signals The next step towards determining the three dimensional structure of DAXX 44 was to ’ 55 assign the signals from its side-chain ‘H, ‘ C, and ‘ 3 N nuclei. The successful calculation of a 5 protein structure by NMR methods generally requires the assignment of at least 90% of all its side-chain 1 H signals (Habeck et al., 2004). Therefore, a suite of three dimensional heteronuclear experiments was employed to assign the chemical shifts of nuclei in the aliphatic and aromatic side chains of DAXX 44 (Figures 2.8-2.10). ’ 55  51  _____  Chapter 2—Structure of the DHB domain  8  B  C  I  :a... •s --  •‘—  -e—— e=  SW—  -*1’  .  55-141  55-141C’50C  59-141  55-144  -2  -4  110  D eQ  C  z  e  Q 0 0  Is  .  O  •-  I  z  120  -0.5 0 125  • 9,0  e  0  0,5  0,0  ‘El 50  60  70  80  90  100  110  120  130  140  150  7,5  7.5  (ppm)  160  Residue Figure 2.6 Identification of the boundaries of the structural domain within . 60 ’ 46 DAXX C 3 l a (A) The difference between the observed and corresponding random coil chemical shifts. A stretch of residues whose ‘ C values shifted more than ‘—1 ppm downfield (dotted line) indicate 3 the presence of an x-helix, whereas a stretch of residues with ‘ Cc values that are ‘—1 ppm or 3 more upfield shifted (dotted line) indicate the presence of a 13-strand. According to this criterion, residues 60-136 contained at least 4 x-he1ices (double-headed arrow). (B) Heteronuclear 15 N {‘H}-NOE values of DAXX ° reveal that the fast timescale dynamics of residues 60-140 6 ’ 46 correspond to those of a well ordered domain. Missing points correspond to prolines or residues with overlapping or anomalously weak signals. (C) Expression tests of , 41 DAXX ’ 55 DAXX 55 141C58G 41 and . ’ 59 DAXX , 44 Lanes identified as M: molecular weight marker (see ’ 55 DAXX Appendix 2 for size marks); un: cell lysate of cells prior to induction; 5h: the total cell lysate 5 hours after induction; sol: soluble proteins in the cell lysate 5 hours after induction; ins: extracted proteins from the insoluble pellet 5 hours after induction. DAXX 44 showed the greatest ’ 55 solubility of all the constructs. (D) Superimposition of the ‘H-’ N HSQC spectra of DAXX 5 ° 6 ’ 46 (red) and DAXX 44 (black). The excellent overlap between the two spectra indicated that the ’ 55 55144 construct had the same secondary and tertiary structures as in DAXX DAXX . The 46160 spectra differed by the absence of signals within the random coil region. -  52  Chapter 2  —  Structure of the DHB domain  T78  110  G55 fJQ77NC  S114 S118  )110 Yi24  N107NS  4  -Ii  ‘1R117 S138 0  115  Q77  Q3  ‘Q93N  8137  S102 4C74  I  ‘C131  N63Ø 0E83N—HN  UK122  H96. 11271  120  )K91  N90 L61 E104 P132 NJ. Y89  4  N107 -H81  iDJ24fl128 t 67 F.  I  121 A —.1tKG  r  3F99 1  865  A136L123  L109 F/O..  71116 1  4  1k  I  ?L71  yKi35  -  E129 -  K75  KS ‘.1.  8147  0  L73114  ‘:)C106 3JALO1  v12S  P126  )RliJ .  .741,  ‘L98 1,97  KV84  1108  4L8s 11)E62  87  4V1 33  E64 7  4cs8  O8144 7119 ,,.  L143’O  125  N636  03 .3A139  9  8  7  ‘H (ppm) Figure 2.7 Annotated 15 H- HSQC spectrum of . 1 N 44 (1 mM, 10 mM phosphate, ’ 55 DAXX 100 mM KC1, pH 6.5, 25 °C). Signals from main-chain and AsnlGln side-chain amides are identified. The peaks corresponding to the side chain ‘ H of Asn63 were aliased from an 2 N 5 unusual chemical shift of 103.4 ppm in the ‘ N dimension, due to proximity to the aromatic ring 5 of PhelO5. -  53  Chapter 2— Structure of the DHB domain 2.4.1.1 Assignment of signals from side-chain aliphatic nuclei The side-chain assignment strategy naturally uses the assigned main-chain or backbone nuclei as starting points. Figure 2.8 shows how this is achieved for A1a95 and Arg94. In the first C nuclei in residue (i-i) 3 experiment, the (H)CC(CO)NH-TOCSY, signals from all of the ‘ preceding the amide group of residue i are detected. It is often possible to identify these signals 13 based on expected chemical shift ranges. in case of 13 3 l C o or Cc ‘ C , ‘ Cr, 3 3 as arising from Ca, ambiguity due to overlap or to anomalous shifts (i.e. resulting from tertiary structural effects such as the close proximity to an aromatic ring), additional experiments that detect only specific C 3 signals can be used. For example, the previously introduced CBCA(CO)NH only detects ‘ and  The assignment of signals from aliphatic hydrogen nuclei follows a similar strategy. The H(CC)(CO)NH-TOCSY experiment detects the side-chain ‘H of the residue (i-i) preceding a specific amide (i). The possible degeneracy of ‘Hp, ‘H and ‘H chemical shifts in some residues, such as lysine, is worse than with their carbon counter-parts, so the HBHA(CO)NH is very useful, since it displays only the ‘H and ‘H chemical shifts. A complementary solution is to use the HCCH-TOCSY experiment, which both correlates signals from all the aliphatic carbon and hydrogen nuclei in a residue and identifies the directly bonded ‘ C-’H pairs. One problem with 3 the amide-directed experiments is that they fail to provide assignments for residues that are followed by a proline. For those residues, a ‘ N-TOCSY-HSQC experiment was used, which 5 correlates signals from intra-residue side-chain protons with that of the main-chain  54  HN 1  Chapter 2— Structure of the DHB domain  Figure 2.8 Experiments used for the assignment of NMR signals from the side-chain 44 (Top) Magnetization ’ 55 DAXX /’ . C 3 ‘ N -labeled aliphatic 13 C and ‘H nuclei of uniformly 5 transfer pathways for (H)CC(CO)NH-TOCSY (1) and CBCA(CO)NH (2), H(CC)(CO)NH N-TOCSY-HSQC (6) experiments. 5 TOCSY (3), HBHA(CO)NH (4), HCCH-TOCSY (5), and ‘ Arrows indicate the direction of the magnetization transfer (open arrows for TOCSY steps), and circles define the atoms whose chemical shifts are recorded in the experiment. (Bottom) Representative example of the assignment of the side-chain atoms of A1a95 and Arg94 using the HN) strips taken at the ‘ N 5 C or ‘H) o (horizontal; 1 3 above experiments, using o (vertical; ‘ chemical shift (02) of the indicated residues. -  -  55  Chapter 2  —  Structure of the DHB domain  H-H —N——C—()  —N— 1I  (H)CC(CO)NH-TOCSY  ;) i  CBCA(CO)NH  cD  I  I.)  Fl  HBHA(CO)NH  I’) II © 0  1)  ©  N  I’) II  I) N  H(CC)(CO)NH-TOCSY  ©  —N—C  I  I  H®O®  H  HCCH-TOCSY  -®-C—C— N\  C  JW  TOCSY-HSQC  ©  0 20  20  R94-C’ 30  O  A95-I-1  o. R94-C  )  R94-C  R94-H R94-H’  2  2  22  3  3  4  4  R94-H 40  p40.  ‘0  R94-C’  -3  -3  50  50  R94-H O  R94-C’  ê4  R94CA  60  60  RM1T  0 A95-H”  70  70  A95-NH  A95-NH  A95—NH  A95-NH  56  A95-NH  Chapter 2— Structure of the DHB domain 2.4.1.1.1 Stereospecific assignments Va! and Leu methyl groups were stereospecifically assigned using the method described by 44 was expressed in M9 media containing 10% ’ 55 Senn et a!. (Senn et al., 1989). In short, DAXX C-labeied 3 -giucose. Stereospecific biosynthesis makes the pro-S ‘ 6 C 2 -giucose and 90% ‘ 6 C 3 ‘ C-labeied 3 C-labeied neighbor, whereas the pro-R ‘ 2 62 have a ‘ C methyl groups (Val C 2 or Leu ) C-labeled neighbor. These can be distinguished by 3 61 have a ‘ C methyl groups (Val C’ or Leu ) C CT 13 C HSQC spectrum or by the sign in a ‘H3 C couplings in a high resolution ‘H-’ 3 C-’ 13 HSQC spectrum. Stereospecific assignments of the side-chain amides of Gin and Asn were carried out using the EZ-HMQC experiment (McIntosh et ai., 1997). This approach exploits the difference in the JC-H 3  coupling between ‘ C (y for Gin, 3  13  for Asn) and its Z- and E- amide proton (7 Hz for the  former, negligible for the latter). 2.4.1.2 Assignment of signals from side-chain aromatic nuclei The assignment of aromatic side-chain signals follows a different strategy, since Trp, His, and Phe/Tyr all have distinctly different spin systems (and ring nuclei do not show correlations in standard main-chain-detected experiments). Figure 2.9 shows the procedure used for assigning signals from the aromatic rings of Tyr89 and Phe67. The first step is to link the previously assigned ‘ C shift with the ‘H 3 6 shifts of aromatic resides using the (HB)CB(CGCD)HD experiment. The corresponding ‘ 6 shift can then be identified in a ‘H-’ C 3 C CT-HSQC spectrum. 3 The IHe shift can be obtained either from the HCCH-TOCSY spectrum using these ‘ 6 and ‘H C 3 6 ‘ C C and ‘H shifts, or from the (HB)CB(CGCDCE)HE experiment. Finally, for Phe residues, the 3 shift can be obtained from the HCCH-TOCSY spectrum. 57  Chapter 2— Structure of the DHB domain Figure 2.10 shows the steps in assigning signals from histidine imidazole rings using His96 62 and C 3 as example. The first steps are as above, assigning the ‘  11162  using the  N HMBC experiment was 5 (HB)CB(CGCD)HD and CT-HSQC experiments. Then, the ‘H-’ used, which exploits the small  JNH 2  and  JNH 3  15 ‘W’, ‘ , 2 couplings to assign the NE ’ and 6 N 5  11162  shifts. Note that the labile nitrogen-bonded protons of histidines are usually not observed due to rapid exchange with water, and that the chemical shifts of an imidazole ring are highly diagnostic of its charge state and, if neutral, its tautomeric form (Pelton et al., 1993). His96, and His53 were HE position, and His8l, and His137 were found to be found to be neutral with the proton on the 2 positively-charged. 2338 peptide discussed C 44 does not contain any Trp residues, the Rassfl ” ’ 55 Although DAXX in Chapter 3 contains one. The steps in assigning Trp residues are similar, using the HE shifts / 1 NE (HB)CB(CGCD)HD experiment to obtain the ‘H ’ shift as starting point, and the 15 6 N-TOCSY-HSQC and ‘ 5 N-NOESY-HSQC spectra. The rest of the 5 are easily derived from a ‘ C-NOESY-HSQC experiments. 3 aromatic ring can be assigned using HCCH-TOCSY and ‘ Combining all of these strategies, it was possible to assign signals from 98% of all non-labile ‘H, ‘ C, and ‘ 3 N nuclei in DAXX 5 44 (Appendix 7). ’ 55  58  _________ _______  Chapter 2— Structure of the DHB domain  I H  I H  -  ::  II C)  I H  (HB)CB(CGCL))HD  38  V  C\-  H% /  I H  II 0  I H  (HB)CB(CGCDCE)HE  t  II 0  I  H  CT-HSQC  (HB)CB(CGCD)HD  ()  I  H  II  0  HCCH-TOCSV (HB)CB(CGCDCE)HE  38  .j  \9Y89-C1_H  I H  Y89-C-H 1 F67-CH  I F7_C_Hb  : 40  —  I. F679 F67Sc4  \%/  V  133.0  131.0  1315  1190  HCCH.TOCSY  -  7.1  7.1  q.N  7,1  72  6.8  6.6  6.8  6.6  F67_C_HiHh F67-C -H —H  .  -  72  7.3  7.3  ..  Y89-C -  —  118.5  ‘\\  Y89-C-H’ 1335  I  :  40  CT-HSQC  H -H  ..  F67-C -H -H  -H-H 6 Y89-c  7A  7.4  7.2  7.0  6.8  6.6  7.4  ‘H (ppm)  7.2  7.0  ‘H (ppm)  Figure 2.9 Steps involved in the assignment of signals from the aromatic side-chains of Tyr and Phe residues in uniformly 5 C 3 ‘ N -labeled /’ . 44 Magnetization transfer ’ 55 DAXX pathway for a Phe residue in the (HB)CB(CGCD)HD, (HB)CBCGCDCE)HE, CT-HSQC and HCCH-TOCSY experiments. Arrows indicate the direction of the magnetization transfer (open arrows for TOCSY steps), and circles define the atoms whose chemical shifts are recorded in the experiment (see text for details). -  59  Chapter 2— Structure of the DHB domain  Ic  (HB)CB(CGCD)HD  //“.\  31.5  -  32.0 -H t H96C  9  32.5  Ic  CT.-HSQC 1175  -  118.0 H96C  /(,.\  1T96 N  II  9  II  118.5  01196 N  P168 :172  1-IMBC  II  F  2 €  CT-HSQC  -  138.5 139.0  I I96-C”-1-1  9  139.5 II—  —II  (I)  u—  /I\  Q H96-N-H SAl  HMBC  H96.N’-H’  7.5  7.0 H (ppm)  6.5  L244 -248  2 9  6.0  Figure 2.10 Assignment of signals from histidine residues. The aromatic ring of His96 in uniformly 5 C 3 ‘ N -labeled /’ DAXX 44 was assigned using (HB)CB(CGCD)HD, CT-HSQC, and ’ 55 HMBC experiment. The sign of the peaks in the CT-HSQC experiment depends on the number of neighboring ‘ C nuclei. Positive peaks (black) have either zero or two ‘ 3 C nuclei directly 3 connected, whereas negative peaks (red) have one. The circles indicate the atoms detected in each particular spectrum. -  60  Chapter 2  —  Structure of the DHB domain  55144 secondary structure 2.4.2 DAXX As discussed above in section 2.3.2.1, the “secondary chemical shifts”  (sobs  -  random coji)  of  13 3 C’ nuclei of a given residue are dependent upon secondary 3 ‘ C , and ‘ main-chain ‘He, Ca,  structure within a protein. Wishart et al. were the first to formulate a simple algorithm, termed the “Chemical Shift Index” (CSI), to predict the secondary structure of a protein from these main-chain chemical shifts (Wishart and Sykes, 1994; Wishart et al., 1992). In this algorithm, residues are given scores of + 1, 0, or -1 depending upon the “secondary chemical shifts” of each of their main chain nuclei, followed by averaging over all nuclei. A more sophisticated “Secondary Structure Propensity” (SSP) algorithm includes additional weighing and scaling terms to yield a score ranging from -1(100% 3-strand) to +1(100% x-helix) that indicates the propensity of a residue to adopt a given secondary structure (Marsh et al., 2006). Alternatively, the program TALOS uses a database of known high resolution structures and chemical shifts to estimate the backbone dihedral  p  and  w angles for each residue, which in turn reflect secondary  structure (Cornilescu et al., 1999). Complementing chemical shift analyses, the secondary structure of a protein can also be determined from patterns of near-neighbor ‘H-’H NOE interactions. For example, c-helices 1 NOEs (historically denoted as a short dNN(i,i+]) distance), as typically show strong lHNlHN+ well as the presence of NOE reflecting intermediate daN(I, 1+3), d(1, i+3) and dc,N(i, 1+4) distances. Finally, secondary structure elements can be confirmed by determining which amide groups are protected from rapid hydrogen exchange (HX) when the sample is transferred to a D 0 2 solvent. This protection is usually interpreted to result from inter-residue amide-carbonyl  61  Chapter 2  —  Structure of the DHB domain  hydrogen bonding, as present between residues (I, i+4) along a helix or between paired 13-strands of a 13-sheet.  As summarized in Figure 2.11, a combination of the above methods clearly demonstrates five ct—helices in . 44 ’ 55 DAXX  2.4.3 DAXX 55144 tertiary structure  The three dimensional structure of DAXX 44 was determined using distance restraints ’ 55 derived from inter-proton NOEs, dihedral angle restraints from chemical shifts, and orientational restraints from residual dipolar couplings. The NOEs were measured using simultaneous aliphatic ‘ N-NOESY-HSQC-, aromatic ‘ 5 C-NOESY-HSQC, and CT-methyl-methyl 3 C- and ‘ 3 and amide-methyl-NOESY spectra. The tertiary structure of DAXX 44 was calculated from these NMR-derived restraints using ’ 55 ARIA 1.2 (Linge et al., 2001). Manual assignments of unambiguous peaks were incorporated in the initial calculation, although most NOEs were automatically assigned by ARIA, followed by a manual correction of violated restraints (details in the Materials and Methods section). A final ensemble often water-refined structures was obtained with an RMSD value of 0.54  A  for backbone heavy atoms in structured regions of the protein (Table 2.1). Based on the algorithms DSSP, PROMOTIF and PROCHECK, the boundaries for each helix are: x1 (residues 60-77); a2 (85-93); c3 (97-100); ct4 (103-118); and c5 (123-136) (Figure 2.12A) (Hutchinson and Thornton, 1996; Kabsch and Sander, 1983; Laskowski et al., 1996). This is in good agreement with the secondary structure analysis described in the previous section.  62  Chapter 2  —  Structure of the DHB domain  Figure 2.11 NMR-derived secondary structure of DAXX . A combination of HX, CSI, 55144 SSP, TALOS and NOE-derived parameters defined the helical regions of . 44 For the ’ 55 DAXX HX experiment, amide protons that were detectable 45 minutes after transferring the sample to 0 are indicated with a For the CSI analysis, only residues predicted to be c-helica1 using a 2 D combination of C’ and ‘H chemical shifts are shown as ‘H’. For the SSP, only residues 3 ‘ with a helical propensity score greater than 0.6 are shown as ‘H’. Similarly, residues with TALOS-predicted p and w angles of -57 ± 20° and 47 ± 20°, respectively, in nine out of the ten closest database entries are labeled as H. Bars connect atoms for which the indicated NOE crosspeak (denoted as a short distance between two nuclei) has been identified. The thickness of the bars indicates the intensity of the NOE. The presence of strong dNN(i, 1+1), as well as intermediate daN(I, i+3), d(i, 1+3) and dcN(1, 1+4) distances, is indicative of a-helices (Wüthrich, 1986). For comparison, the cylinders above the sequence show the secondary structure elements identified by PROCHECK and DSSP using the three dimensional structural ensemble of 44 derived in section 2.4.4. ’ 55 DAXX -  .  63  I -  r  (1  .  I:I4  i::i  Qz U-K  I1  Z  U  C) Cl)  C!)  x  xx  x  xx  XX x  —  I  I  —  c-  c  c  -  1-  I,,  :  .  IeI  i1 —  II  z ztt  xx  X  IH XZ  >  Cl)  P1 P1 (l)  P1  U-K i::i C/)  I’-4 ‘-  P1  (Y P1 —  z  —  ro--’---  --  r  --  t’  -  t  z  U —  z  —  o  o---H  1)U)  ---  -  Chapter 2— Structure of the DHB domain Table 2.1 NMR restraints and statistics for the ensemble of the ten lowest energy . 55144 structures calculated for DAXX -  Summary of restraints. NOESa Intra-residue Sequential Medium range (i-j 2-4) Long range (i-j 5) Total Dihedral angles N’ 4) ‘H-’N Residual Dipolar Coupling (RDC) Deviation from restraints NOE(A) Dihedral restraints (degrees) RDC(Hz) Deviation from idealized geometry Bonds (A) Angles (degrees) Improper angles (degrees) Residues in generously allowed regions of the Ramachandran plot Mean energies” (kcal moF’) Evdw d 0 Eb Eangies Eimproper  514 (69) 468 (151) 463 (330) 369 (278) 1814 (828) 70 70 87 0.05 ± 0.02 1.8 ± 0.8 1.1±0.1  (%)  ENQE  EdIh  Esani Residual dipolar couplings Qvalue RMSD from average structure’ Residues in c-helices Backbone atoms Heavy atoms All residues Backbone atoms Heavy atoms  0.005 ± 0.000 0.71 ± 0.07 1.95 ± 0.25 97.5 205±61 36±8 210 ± 40 170 ± 60 190±90 30± 30 110±20  0.047  (A) 0.54 ± 0.07 1.28 ± 0.05 0.73 1.41  ± ±  0.15 0.11  a number of unambiguous restraints, with ambiguous restraints in parentheses. b  final ARIA/CNS energies for van der Waals (vdw), bonds, angles, NOE restraints (NOE), dihedral restraints (cidh) and residual dipolar coupling restraints (sani). C  Residues in a-helices: 60-77, 85-93, 97-100, 103-118, 123-136. All residues: 60-136. 65  Chapter 2— Structure of the DHB domain  Figure 2.12 Structure of the DHB domain. (A) Ribbon diagram of one low-energy NMR 44 with c-helices shown in red. The ’ 55 derived structure of the DHB domain contained in DAXX (B) Superimposed backbone atoms from the for reference. labeled are and C-termini Nstructural ensemble of the DHB domain. (C) Surface of one low-energy NMR-derived structure of the DHB domain colored according to an electrostatic potential (blue positive; red negative; white neutral; calculated using MOLMOL with the “simplecharge” method and 100 mM salt). Selected residues are labeled for reference. A cluster of positively-charged residues corresponding to Arg9l, Arg94, Lys135, His137, Lysl4O, Lysl4l and Lys142 stands out on one side of the molecule, whereas a cluster of negatively-charged residues corresponding to G1u62, G1u64, G1u68, G1u69 and G1u72 is present on the opposite side of the molecule. (D) Exposed hydrophobic residues (green) on the surface of the DHB domain (polar residues in gray). A cluster of hydrophobic residues comprising Pro86, Phe87, Va184, Tyr124, Va1125, 11e127 dominates one side of the molecule. -  66  Chapter 2— Structure of the DHB domain  B  A  055  900  C  H-’ 180°  Ri 15—  R115  11 K141  1  V. -i K56 K57 K142  D  180°  67  Chapter 2— Structure of the DHB domain The ioop regions connecting cd—a2, cL2—x3, c3—a4, and c4— cL5 are also well defined in the N relaxation measurements 5 structural ensemble. This is consistent with their low mobility by ‘ (next section) and likely reflects a number of potential hydrogen bonds. DSSP and PROMOTIF (but not PROCHECK) identified residues 120-122 (linker a4— c5) as a 3io helix. Due to its helical content, this structure will be named the DAXX N-terminal Helix Bundle (DHB) domain. 44 ensemble reveals a number of ’ 55 An examination of the surface the DAXX physicochemical features that might be relevant to the function of the DHB domain. First of all, the molecule is predominantly positively-charged, in agreement with its theoretical p1 value of 9.45 (Wilkins et al., 1999). More so, a cluster of positively-charged residues (Arg9l, Arg94,  Lysl35, Hisl37, Lysl4O, Lysl4l and Lys142) stands out on one side of the molecule. In contrast, several negatively-charged residues (Glu62, Glu64, G1u68, Glu69 and G1u72) are located on the opposite side of the molecule (Figure 2.12C), thus giving the DHB a very asymmetric electrostatic surface. Exposed hydrophobic residues (Figure 2.1 2D, green) on the surface of the DHB domain also show a clear clustering. In particular, Pro86, Phe87, Val84, Tyr124, Val125, and 1le127 are all located on one side of the molecule, surrounding the positively-charged residues. In the next chapter, it will be shown that this feature is key to the function of the DHB domain as a protein protein interaction module. 2.4.4 DAXX 55144 dynamics The dynamic properties of the DHB domain in DAXX 44 were investigated using ‘ ’ 55 N 5 relaxation methods (Figure 2.13A-C). From the measured amide ‘ N T 5 1 and T 2 values, the effective correlation time for the global isotropic tumbling of DAXX 44 was determined to be ’ 55 68  Chapter 2— Structure of the DHB domain 7.5 ns (Tensor2 (Dosset et al., 2000)). This value is within the range expected for a monomeric species having the molecular weight of the DHB domain (l1 kDa) (Daragan and Mayo, 1997). N 5 In addition to providing a measure of the global rotational diffusion of a protein, ‘ relaxation data reflect the local mobility of its polypeptide backbone on a residue-specific basis. 2 lifetimes indicate enhanced flexibility N { ‘H } -NOE values and elevated T In general, reduced 15 on a nsec-psec timescale, whereas short  12  lifetimes arise from conformational exchange  N {‘H}-NOE, T,, and 5 broadening on a msec-jisec timescale. The ‘  2 values of the DHB T  domain, summarized in Table 2.2, are typical of a well ordered domain, with an average value of 0.75 ±0.07 (NOE), 0.48  ±  0.02 s (Ti), and 0.10  ±  0.01 s (T ) for residues 60-136, which include 2  the loops. Often ioops tend to be more flexible than helical or strand regions. However, in the case of the DHB domain, there is very little difference in the relaxation parameters of the loops versus a-helical regions. This can be explained by the likely presence of a number of hydrogen bonds in the short turns of the loops. For example loop a4-a5 adopts a  helical conformation, due to  the inferred hydrogen bonding between the carbonyls of residues Arg 119, Pro 120, and Lys 122 and the amides of Lys122, Leu 123, and Val125, respectively. This inferred hydrogen bonding network likely contributes to the low RMSD value of the ioops in the NMR structure ensemble (Figure 2.12B). In contrast to the core residues 60-136, the N- and C-terminal residues of 44 are clearly conformationally disordered, based on both their ‘ ’ 55 DAXX N relaxation 5 properties and high RMSD deviations in the structural ensemble.  69  Chapter 2— Structure of the DHB domain N relaxation data for different structural regions of the Table 2.2 Summary of amide 15 DHB domain. The residues comprising each region, as determined by PROMOTIF, PROCHECK and DSSP, are indicated in parenthesis. Standard deviations are also shown. -  Region  H}-NOE 1 N{ 15  1 (s) T  2 (s) T  Helixi (60-77)  0.75 ± 0.06  0.49 ± 0.02  0.10 ± 0.01  Helix2 (85-93)  0.77 ± 0.05  0.48 ± 0.02  0.09 ± 0.00  HeIix3 (97-100)  0.68 ± 0.04  0.46 ± 0.03  0.10 ± 0.01  Helix4 (103-118)  0.79 ± 0.04  0.48  0.10 ± 0.00  Helix5 (123-136)  0.76 ± 0.06  0.48 ± 0.01  0.10  Loop cd—c2 (78—84)  0.69 ± 0.05  0.49  ± 0.02  0.08 ± 0.01  Loop a2—a3 (94—96)  0.77 ± 0.06  0.48 ± 0.02  0.10 ± 0.01  Loop ct3—a4 (101—102)  0.79 ± 0.01  0.48 ± 0.02  0.10  ±  0.00  Loop a4—cL5 (119—122)  0.77 ± 0.04  0.48  ±  0.02  0.10  ±  0.01  0.75  0.48  ±  0.02  0.10  ±  0.01  All residues 60-136  ±  0.07  ± 0.01  ±  0.00  Amide proton-deuterium exchange (HX) rates were also measured to probe the local stability and dynamics of the DHB domain (Figure 2.1 3D). In addition to the known effects of sequence, the exchange rate of specific amide hydrogen atoms in a protein is dependent upon its structural environment, including hydrogen bonding and solvent accessibility, as well as local and global fluctuations leading to water contact (Hoofnagle et al., 2003). Under EX2 conditions, protection factors can be interpreted as the inverse of an equilibrium constant for fluctuations between a closed non-exchangeable state and a transiently open exchange-competent state. Thus protection factors provide a measure of residue-specific free energy changes, 70  1 AG  RTln(krc/kex),  Chapter 2— Structure of the DHB domain governing local and global conformation dynamics allowing exchange. Based on the largest protection factor (Leu7 1, 1.1 x 1 Os), the minimum estimate of the free energy changes for global unfolding of the DHB domain is 6.8 kcal/mol. The measured exchange lifetimes (1/kex) of the DHB domain reveal a striking difference in the protection of each helix to exchange with the buffer. Residues in helix 1 and helix 4 had the largest protection factors, residues in helix 3 and helix 5 had intermediate protection, and residues in helix 2 underwent facile HX (Figure 2.13D). It is interesting to note that functionally important residues are often more dynamic, which enables them to accommodate conformational changes needed to accommodate ligands, substrates or other proteins (Elcock, 2001). Indeed helices 2 and 5, the least stable of the bundle, are involved in substrate binding (Chapter 3).  71  Chapter 2— Structure of the DHB domain  The global and local backbone dynamics of the DHB domain. (A) Figure 2.13 1 (B) and ‘ NT 5 2 (C) relaxation data measured at 25 NT 5 H}-NOE. (B-C) ‘ N {1 5 Heteronuclear ‘ Missing data points corresponded to prolines or °C, pH 6.5 using a 500 MHz spectrometer. residues with overlapping signals. (D) HX lifetimes for the DHB domain at pH 6.5 and 25 °C. Missing data were for prolines, residues with no detectable signal in the first spectra recorded (i.e. helix 2), or residues with overlapping signals. Overall, with the exception of the terminal residues, the DHB domain was well ordered on the nsec-psec timescale. -  72  Chapter 2— Structure of the DHB domain  1.0•  IJ.  C  iizi A  I•1  0.5  z z  0-  J,0 . 1 -  I  I  I  I  I  I  I  I  I  I  I  I  I  I  I  I  1.0-  I  0.8 0.6 ——  I  0.4 0.2 0 0.5  ———————— ——— ——— ——— —  10cI_j5 10  i0 3 i0 102  60  70  80  90  I  100  1i bi  Residue 73  110  120  Hii  130  140  Chapter 2  —  Structure of the DHB domain  2.5 Structural comparisons of the DHB domain 2.5.1 Comparison with Sin3 Previous to this study, the region encompassing the DHB domain was predicted to contain a ‘Pair of Amphipathic Helices” (PAH) domain (Hollenbach et al., 1999). Historically, the name PAH domain comes from an early sequence analysis of the yeast Sin3 gene (Wang et al., 1990). The encoded Sin3 transcriptional repressor was simply predicted to contain two adjacent amphipathic helices. The first structural analysis of a Sin3 fragment demonstrated that the so-called PAH domain is in fact a four helix bundle that binds partners (such as a short helical segment of Mad 1) using a hydrophobic patch between all helices (Figure 2.14A, see also Figure 3.11C) (Brubaker et a!., 2000; Spronk et a!., 2000). Sponk et a!. suggested the name of “wedged helical bundle” for the domain, which may be more appropriate in a descriptive sense. However, the somewhat erroneous PAH nomenclature has prevailed in the literature of Sin3. Since these original studies, five additional structures of Sin3-fami!y PAH domains have been reported (Nomura et al., 2005; Sahu et al., 2008; Swanson et a!., 2004; van Ingen et al., 2006). Figure 2.14A summarizes the structure of a!! the PAH domains known to date. It is clear that all of these structures are very similar, including the utilization of the same cleft between helix 1 and helix 2 to bind their targets. At first sight the DHB domain indeed looks like a four-helix bundle similar to the PAH domains. However, closer inspection reveals a number of very significant differences. First of all, the DHB domain contains an additional helix (helix 3 in Figure 2.14). Although short, this helix is well defined and traverses diagonally from helix 2 to helix 4. As a result of this cross 74  Chapter 2  Structure of the DHB domain  —  over, the Sin3 PAH domains and the DHB domain have a different topological arrangement of their core 4 helix bundles (see next section). Second, helix 3 of the DHB blocks the cleft used by Sin3 PAR domains to bind their targets. This immediately suggests that the binding mode of the DHB domain is different to the one used by Sin3 PAH domains. Third, the interhelical angles (Q) between helices are quite different (Table 2.3). In all of the structures of Sin3 PAR domains helices 1 and 2 have an angle approximately of 1400 to accommodate substrate binding (Figure 2.1 4A), whereas in the DHB domain that angle is only l 65°, providing much less “room” to accommodate a binding partner. Fourth, the isolated DHB domain is very well structured in isolation, whereas the two available structures of ligand-free Sin3 PAR domains show a substantial increase in the RMSD of helices 1 and 4 (Figure 2.14A), suggesting that the unbound domain is relatively dynamic.  Table 2.3 Interhelical angle calculated with PROMOTIF.  (Q)a  -  DHB Sin3B  1  1 3  in Sin3A PAH2 (1G1E.pdb) and the DHB domain  2 -164  2  137  )b 4 (  35  172  -159  -23  )b 5 ( 4  )b 4 (  )b 5 ( 4  27  -140  -169  24  3  167  165  a  Angles are in degrees. The sign convention is explained in (Drohat et al., 1996). In short, to obtain the interhelical angle, it is necessary to (1) orient the two helices of interest (if) such that they are in a plane parallel to the screen; (2) align the first helix (i) vertically (0°) with its N —* C vector pointing upwards. (3) transpose the vector of the second helix (I) such that its tail is at the same location as the tail for the vector of the first helix (i) (4) rotate an imaginary vector aligned vertically (0°) with its tail at the N-terminus of both helices until it aligns with the N —k C vector of the second helix (f). A clockwise rotation gives a positive interhelical angle, and a counterclockwise rotation gives a negative value.  b  Helix numbering in Sin3 is used as reference, and numbering in DAXX is shown in parenthesis for comparison.  75  Chapter 2— Structure of the DHB domain  Figure 2.14 Comparison of the DHB domain with the Sin3 PAH domains. (A) Structure comparisons of the PAR domain with the DHB domain. There are seven available NMR-derived structures of Sin3 PAR domains: Sin3B PAH2 with a target peptide from Madi (lE9lpdb); Sin3A PAH2 with Madi (1G1E.pdb); Sin3B PAH2 with HBP1 (1S5Q.pdb); Sin3B PAH1 with NSRF/REST (2CZY.pdb); apo-Sin3B PAH2 (2F05.pdb); apo-Sin3A PAHI (2RMR.pdb); and Sin3A PAH1 with SAP25 (2RMS.pdb). Helices 1, 2, 3 and 4 of the four-helix bundle are shown in red, orange, blue and purple, respectively. The DHB domain contains a different fold in which a fifth helix blocks the position where the Sin3 PAH domains bind their targets (green). This suggests a distinct mechanism of ligand binding for the DHB domain. (B) Topographical representation of the left-handed DHB and Sin3B PAH2 and, as an example, right-handed cytochrome b562 helical bundles. To determine the handedness of the bundle, it is necessary to place the N-terminus of helix 1 at the bottom of the page and its C-terminus to the top, then place a hand with the thumb aligned with helix 1. The handedness is determined by which hand follows helices 2 and 3 with its fingers. Although differing in handedness, Sin3B PAH2 and cytochrome b562 both adopt the more common all anti-parallel configuration. In contrast, the DHB domain has helices 1 and 4 and helices 2 and 5 parallel to each other. In addition, cytochrome b562 and Sin3B PAH2 have their N- and C-termini next to each other, whereas the DHB domain has them on opposite sides of (i.e. diagonally across) the molecule. -  76  Chapter 2  —  Structure of the DHB domain  A  SIN3B PAH2 Mad 1  SIN3A PAH2 Mad 1  SIN3B PAH2 HBP 1  SIN3B PAH1 NRSF/REST  C  SIN3B PAH2  SIN3A PAll SAP25  SIN3APAH1  N DHB  B Lefl hand  Left hand  Right hand  b562  DHB  77  Chapter 2  —  Structure of the DHB domain  2.5.2 Comparison with other helix bundles The DHB domain can be described as having a core four-helix bundle (with an additional short helix between the 2’ and 3’ helices of the bundle). Four-helix bundles, which are found frequently both as independent structural units and as parts of larger domains, can be categorized by a number of descriptors, including (i) connectivity, (ii) handedness, and (iii) helical orientation (Kamtekar and Hecht, 1995). The polypeptide connectivity of the core helical bundle (i.e. helices 1, 2, 4, and 5 in Figure 2.14B) of the DHB domain can be described as up-down-up-down, which is similar to that of Sin3 PAH (Figure 2.14B). Furthermore, the DHB domain and the Sin3 PAH domains are also both left-handed (Figure 2. 14B), although this is by no means uncommon (Kamtekar and Hecht, 1995). Importantly, however, the unit direction vectors of the individual helices in the DHB domain are not all anti-parallel to each other, as seen in most helical bundles including Sin3 (Kamtekar and Hecht, 1995; Presnell and Cohen, 1989). This is due to the crossover of helix 3, making helices 1 and 4 and helices 2 and 5 parallel to each other. Based on these factors, the DHB domain can be classified in the same topological family as that of the domain one of the transcription elongation factor TFIIS (Figure 2.1 5A). However, DHB and TFIIS only share 12% sequence identity and 26% sequence similarity (Figure 2.15B) (Booth et al., 2000), and the C backbone atoms of their helices align with an rms deviation of 4.7  A. According to the “hierarchic classification of protein domain structures” database CATH  v3.2.0 (Orengo et al., 1997), the ubiquitin-protein ligase CBL (CATH code 1.20.930.20) (Gay et al., 2008; Zheng et al., 2000), also contains a helical bundle within its phosphotyrosine-binding domain with a similar topology to that of TFIIS and the DHB domain. Despite the low sequence  78  Chapter 2— Structure of the DHB domain identity (18%) and similarity (27%) with the DHB domain, the backbone Ca atoms of the CBL helical bundle align with the DHB domain at a modest level (2.9  A rmsd) (Figure 2.15B-  C). Indeed, the DALI server, which searches for proteins sharing similar tertiary structures, returned the CBL helical bundle domain as being most similar to the DHB domains (a high Z score of 6.2) (Holm and Sander, 1995) (Figure 2.15B-C). As noted above, the presence of the cross-over helix 3 almost certainly prevents the DHB domain from binding any partner proteins via the “wedged helix” mechanism exhibited by Sin3 PAH domains. Although sharing similar topological features, both TFIIS (an RNA polymerase II elongation factor) and CBL (a ubiquitin ligase) have low sequence similarity to the DHB domain and thus provide no functional clues as to its role with DAXX. In the next chapter, it will be examined how the DHB recognizes a motif from Rassfl C, which provides insight on the function of this important domain.  79  Chapter 2— Structure of the DHB domain  A  TFIIS  CBL  DHB  B  C I  TFII S DHB  --  2  j----------3  1 55 2---tZJ--  TFIIS 0KB  4 I 1 50 VNFK3TNOK VKKIS— 115 PAS?AKLY I N CTVKAF  PA KFI  77 144  I_--[—1-CBL mip  47 P 55  --  ‘VDKKMVEKCW KC--YKLENE  KV’ FEFLE  -ZZZZ.LJZZ IZI-CBL DK3  NPKLALKNSPPY MQTADHP-----E  2  .  DT YNR  ----  LRTI SRYEGKME -All  LA  R----  4 —--[Z] KTKQTI LFKEGKERMYEENSQPRRN TKLSLIFSH NI SVLSRA AK YVYINELCT P -Ej-  108 TLGENEYFR 102 SA  3  -  -  E  ELKGI KAHS-  Figure 2.15 Structural comparison of the DHB domain with similar helix bundles. (A) The establishing member of the family, TFIIS (1EOO.pdb), CBL (black) in complex with UBCH7 (gray) and a ZAP7O peptide (red circle) (1FBV.pdb) and the DHB domain have the same fold. Helices 1 (red), 2 (orange), 3 (blue) and 4 (purple) and short helices in the loops (green) of the four-helix bundle are highlighted. (B) Sequence comparisons based on structural alignments between CBL and the DHB domain, as well as between TFIIS and the DHB domain, show little sequence identity (black, 12-18%) or sequence similarity (gray, 27%). The numbering of the helices is based on an ideal four-helix bundle. (C) Structural alignment between CBL and the DHB domain show that helices in CBL are much longer, although the orientation of the four principal helices is remarkably similar, with an RMSD of 2.9A. -  80  Chapter 2  Structure of the DHB domain  2.6 Materials and Methods 2.6.1 Cloning Figures 2.3 and 2.6C outline most of the constructs utilized in this chapter. All of the genes  encoding His -tagged DAXX constructs were cloned from a human cDNA library (accession 6 CAG3 3366) from Invitrogen, kindly provided by Dr. Graves (University of Utah) (except for , which was originally cloned from a mouse cDNA library by Cameron Mackareth). 566739 DAXX Briefly, the desired DNA was amplified by PCR methods, purified using a PCR purification kit (Qiagen), and subjected to restriction enzyme digestion (NdeI/XhoI). After incubation overnight at 37 °C, the DNA was re-purified and used for ligation mixtures with previously digested and dephosphorylated pET28a (Novagen) vector. Ligation reactions (20 jiL) were carried out at room temperature for a minimum of 8 hours. The salt in the ligation buffer was removed by dialysis (10 LL on a “V” series Millipore membrane in Petri dish with 10% glycerol for at least 1 hour) prior to transformation by electroporation into E. coil BL21QDE3). Colonies were grown for less than 16 hours after transformation, and then picked for overnight growth in 2 mL of LB media at 37 °C. The cultures were transferred to 25 mL of fresh LB and grown at 37 °C until the 600 was 0.6-0.8. Then a glycerol stock (1 mL, 15% glycerol, frozen at -70 °C) was created for 0D each colony, and the rest of the mixture was induced (1 mM IPTG) and screened for expression (next section). Glycerol stocks from colonies expressing protein of the correct size on SDS PAGE were used to extract the cloned plasmid (Qiagen kit) which was sent for DNA sequencing (NAPS, UBC). GST-tagged DAXX (constructs 181-365 and 181-407) were cloned using the same procedure into pGEX-2T expression vector (GE Healthcare) using BamHI and EcoRI restriction enzyme sites.  81  Chapter 2  —  Structure of the DHB domain  2.6.2 Protein expression tests To verify the viability of a construct for expression (summarized in Figure 2.3), 1 mL solution from the 25 mL culture (described in previous section) was extracted prior to induction (uninduced sample), then induced bacteria were grown at 30 °C (or lower temperature for some 600 was recorded, and 2 mL solution were constructs, see Appendix 2) for 5 hours, their 0D extracted: 1 mL for total expression analysis, and 1 mL for a solubility test (see below). The extracted 1 mL solutions were immediately centrifuged at 6000 rpm for 1 minute, then the 600 supernatant media was discarded, and the pellet was re-suspended proportionally to the 0D 600 (Reference volume: for 0D  =  0.6 use 100 pL of cracking buffer [0.125 M Tris, p1-I 6.8, 2%  SDS, 20% glycerol, 0.002% BPB]). To test the solubility of the construct, 1 mL was extracted from a culture that had been induced for 5 hours. It was then centrifuged, the supernatant discarded, and the pellet re suspended in Bugbuster (Novagen, with 500 mM NaC1) instead of cracking buffer. The volume used for this was half of the volume used with cracking buffer. It was then incubated under constant mixing at 4 °C for at least 30 minutes. After that, sample was centrifuged at 14,000 rpm for 5 minutes. The supernatant (which contained the soluble fraction of proteins) was extracted and diluted 1:1 with cracking buffer, whereas the pellet was re-suspended in 1 mL of water (washing), re-centrifuged, and then finally re-suspended with the same volume of cracking buffer as the total expression sample. All samples were placed in boiling water immediately for 5 minutes and kept at -20 °C until the next step.  82  Chapter 2— Structure of the DHB domain SDS-PAGE was used to compare the intensity of the bands of the expressed protein in the uninduced sample, total expression sample, soluble fraction sample, and insoluble fraction sample (Figure 2.6C, Appendix 2).  2.6.3 Protein purification  ° is the same as will be 6 ’ 46 The overall procedure for the expression and purification of DAXX 44 had the ’ 55 described in sections 4.7.2 and 4.7.3. The protocol for the expression of DAXX following modifications: (1) The sample bound to the Ni 2 column was washed with 125 mM imidazole and eluted with 250 mM imidazole. (2) DTT was added to all buffers except for the thrombin cleavage buffer. (3) After thrombin cleavage, the sample was diluted 1 Ox into 50 mM phosphate buffer, ph 6.7, 10 mM DTT, and applied to an ion exchange column (SP Sepharose, 44 eluted at approximately 350 mM ’ 55 GE Healthcare) and eluted with a salt gradient. DAXX NaCl. 2.6.4 NMR spectroscopy  Unless stated otherwise, all proteins were in NMR sample buffer (5% D 0, 10 mM 2 HPO 0.1 mM EDTA, 10 mM DTT, pH 6.5 buffer with 100 mM KC1) with 5% DO added 2 K , 4 for the lock signal. DAXX ° was concentrated to 2OO iM in ‘—280 1 6 ’ 46 iL for assignment of the backbone signals. Due to the low concentration and partial degradation of the sample, long HNCACB and CBCA(CO)NH experiments were recorded for 1 day each. DAXX 44 used for ’ 55 structural calculation was concentrated to 1.2 mM in ‘—280 1 iL. Non-randomly fractional 10% ‘ C 3 44 used for stereospecific assignments was 0.8 mM. ’ 55 DAXX All spectra were recorded at 25 °C using a 500 MHz Varian Unity and 600 MHz Varian Inova NMR spectrometers. The latter is equipped with a triple resonance cryoprobe. All pulse 83  Chapter 2— Structure of the DHB domain sequences were provided by Dr. Lewis Kay (University of Toronto). NMR data were processed using NMRpipe (Delaglio et al., 1995) and analyzed by SPARKY (Goddard and Kneller, 2004). 2.6.4.1 NMR relaxation measurements Amide ‘ N relaxation parameters were acquired at a proton frequency of 500 MHz as 5 H}-NOE 1 N { 5 described by Farrow et al. (Farrow et al., 1994). Steady-state heteronuclear ‘ spectra were recorded with and without 3 seconds of ‘H saturation and a total recycle delay of 5.017 seconds. Relaxation rates and correlation times were calculated using Sparky and Tensor2 (Dosset et al., 2000), respectively. 2.6.4.2 Amide proton-deuterium exchange After recording reference ‘H-’ N HSQC spectra, ‘ 5 N-labeled 55 5 DAXX 4 ’ 4 in 500 iiL protonated NMR buffer was lyophilized and re-dissolved in an equivalent volume of D 0. 2 Amide exchange rates were measured by recording a series of sensitivity-enhanced ‘H-’ N 5 HSQC (Kay et al., 1992) spectra at 25 °C, starting 5 mm after the addition of D 0. Initial 2 spectra were obtained with a small number of scans to detect the exchange of the least protected residues, while later spectra were recorded with a greater number of scans for improved signalto-noise. Spectra were scaled according to the acquisition time and residue-specific exchange rates, kex, determined by fitting peak heights, I(t), to the equation  = ‘(t)  Ioet  +  I, where I is  the baseline value, close to zero, due to residual protonated water. 2.6.5 Structure calculation The program ARIA vl.2 (Linge et al., 2001; Nilges, 1995; Nilges and O’Donoghue, 1998) was used for structural calculations. A collection of manually picked and partially assigned peaks from 3D simultaneous ‘ C- and ‘ 3 N-NOESY-HSQC, simultaneous constant time methyl-methyl 5 84  Chapter 2  —  Structure of the DHB domain  and amide-methyl NOESY, and aromatic-NOESY spectra were provided for the generation of distance restraints (all with a 100 ms mixing time). Dihedral angle restraints, obtained from TALOS for residues with agreement with at least nine database entries, were also used for the calculation. After a complete ARIA run, violations were manually corrected by consideration of the resulting structure, and a new run initiated. The ensemble in Figure 2.1 6C, left was obtained after convergence with no violations greater than 0.5  A. This structure was then used as a  seeding structure for a final structure calculation that incorporated RDC data (next section). The quality of the ensemble of structures is summarized within the text (Table 2.1). 2.6.6 1HN..15N Residual dipolar coupling measurements 4 was partially aligned using acrylamide gel stretching, as the more common DAXX 4 ’ 55 method using filamentous phage led to aggregation (Chou et a!., 2001). In short, a control IPAP 55144 was first recorded (Figure 2.16A) (Ottiger et a!., 1998). Then, a 5% HSQC on the DAXX (29:1 acrylamide:bis-acrylamide) polyacrylamide pellet was prepared, and dialyzed into NMR buffer. The protein (300 jtL, 0.8 mM) was dialyzed into the polyacrylamide pellet (2 cm, 4.2 mm diameter) in an Eppendorf tube for 24 hours. The gel was stretched by using a Teflon funnel to inject it into an open-bottom NMR tube (New Era #NE-370). The IPAP-HSQC of the aligned protein was then recorded, and compared with the control experiment (Figure 2.1 6A). 0 splitting produced immediately after loading the gel was 6.7 Hz, which decreased to 2 The D 5.4 Hz over the course of five days. The resulting average RDC in the amides was 20 Hz (Figure 2.1 6A). The program RDCA (Lewis Kay, University of Toronto) was used to estimate the alignment tensor Da and R values (0.53 and -13.8 Hz, respectively) required for the SANI module in ARIA vi .2. Figure 2.1 6C shows the DHB domain structural ensemble calculated with and without  N 5 lHNl  RDCs. Without RDC data, the DHB domain showed an average RMSD 85  Chapter 2— Structure of the DHB domain value of 0.61  ±  0.13  A and 1.21  ±  0.23  A for backbone and heavy chains, respectively (residues  60-136) of a? member ensemble. In comparison, an ensemble of the 7 lowest energy structures calculated using RDC data had an improved RMSD value of 0.49  ±  0.11  A and 1.14 ± 0.21 A for  backbone and heavy chains, respectively. One notable difference is that RDC data allowed the ARIA program to orient the loops between helices 1 and 2, and between helices 4 and 5, parallel with the helices, in contrast to the almost perpendicular orientation that they had in the structure ensemble without RDCs.  86  Chapter 2 A  I  —  1i EI I  I  ‘  Structure of the DHB domain  I I  ‘‘  “  I  Y89  30 1l0.0. Y89  1196  —-1  1(96  20 N  110.5.  10  942Hz  E  0  74.1Hz ((9.0.  z  -  93.7Hz  117.4Hz  —20  ll9.5  60  120.0.  ((.05  11.110  ((.75  0.70  8.55  8.80  8.75  70  D  0.7(1  80  90  100 110 120 130 140 Residue  25 0  ‘H(ppm)  N  01  c  20  0  0  0 >0  150 0  o  11:  -  0 0  Co  o  5  0 10 0  .2-15 CO  5(7  Q-factor=O.402  >  .  25 20 0) 15-  -10 C) ( -15  -  0  -20  Q-Iusctor=O.047 -10  0  10  20  30  Expt. dipolar coupling (Hz)  Figure 2.16 Use of RDC values in the structural calculation of the DuB domain. (A) Measurement of the RDC values was carried out by comparing the nitrogen splitting in the IPAP-HSQC spectrum before (left) and after (right) aligning the molecule in a stretched 5% polyacrylamide gel. (B) Histogram showing the RDC values for each amide group in the molecule. Remarkably, residues in helices 1, 2, 4 and 5 showed very similar RDC values, consistent with the parallel alignment that they have in the three dimensional structure. (C) Comparison of the 7 lowest energy structures calculated before (left) and after (right) RDC data was incorporated in the calculation. (D) Plot of calculated vs. experimental dipolar coupling shows a poor correlation for a structure calculated without RDC data, and a remarkable improvement in the correlation for a structure calculated using RDC data. The Q factor (rms(RDC0b5_RDC1D)/rms(RDC0T)) was determined with RDCA -  87  Chapter 3  Structure of the DAXX/RassflC complex In this chapter the first structural characterization of a DAXX complex is presented. Through an extensive screening of truncation mutants, the residues 23-3 8 of RassflC were identified as responsible for mediating the interaction of this tumor suppressor with the DHB domain. By systematically screening a selection of buffers, conditions were found for the formation of a tight complex suitable for structural calculations (KD  20 jiM, 10 mM MOPS buffer, 0.1 mM EDTA,  2338 formed a short pH 6.5). The NMR-derived ensemble of the complex reveals that RassflC amphipathic a-helix with hydrophobic residues filling the cleft between helices 2 and 5 of the DHB domain. A negatively-charged surface of the Rassfl C peptide also faced the positively charged surface of the DHB domain. Thus both hydrophobic and electrostatic interactions mediated complex formation. Consistent with this structural observation, the affinity of 2338 for the DHB domain significantly decreased with increasing ionic strength. This RassflC work provides important information on the function of the DHB domain, and could prove key in elucidating the mechanism by which DAXX is regulated and controls cellular functions through protein-protein interactions.  88  Chapter 3  —  Structure of the DAXX!RASSF1C complex  3.1 Identification of RassflC as an interaction partner of the DHB domain With the goal of identifying the function of the DHB domain, proteins in the literature reported to interact with DAXX were examined. Many of these studies used truncation constructs in an attempt to identify the regions of DAXX and its partners responsible for the interactions. Given that there is little structural information on DAXX and most of its partners, these mapping studies at best provided very coarse “guesses” of domain boundaries. Disappointingly, a possible common motif among all of these partners could not be identified through sequence alignments (not shown). Notable among these studies, Li et al. investigated a large number of truncation constructs of 97 (Li et al., 2007). As documented in Chapter 2, this Axin, a protein that interacts with DAXX’’ fragment includes the DHB domain. All of the Axin constructs that showed a positive interaction with DAXX’’ 97 by co-immunoprecipitation shared residues 576-598 (Figure 3.1). To test whether this region was indeed responsible for mediating the interaction, NMR was used to 576598 with 55 monitor the titration of purified unlabeled GST-Axin N-DAXX 5 ‘ 4 ’ 4 (Figure 3.1 B D). Disappointingly, the lack of any spectral perturbations indicated that these two constructs do not interact in vitro. Indeed, it is a common observation that reported protein-protein interactions, deduced from co-immunoprecipitation or other such pull-down assays using “crude” cell extracts or cell-free transcriptionltranslation systems, do not stand the test of in vitro biophysical analyses using purified proteins. In the summer of 2008, a fruitful collaboration was started with Dr. Ishov, whose laboratory specializes on studying the function of DAXX. As mentioned in section 1.4.3, Dr Ishov’s group recently identified Rassfl C as a binding partner of DAXX, and found that the interaction mediated a mitotic checkpoint (Lindsay et a!., 2009b). Kitagawa et al. also recognized this  89  Chapter 3  —  Structure of the DAXX/RASSF1C complex  interaction (Kitagawa et al., 2006), albeit they found the interaction to mediate activation of the INK-signaling pathway (see sections 1.4.3 and 3.7). Nevertheless, it is clear that the DAXX/Rassfl C interaction is biologically important. Importantly, Dr. Ishov’s group mapped the region of each protein that was responsible for the interaction to residues 1-142 of DAXX and 1-50 of Rassfl C. Since an intramolecular interaction 4 had already been identified (section 4.5), a similar motif in DAXX 4 ’ between DAXX’ ° and 55 2 ° was searched. Sequence alignment revealed residues 13-34 of RassflC as a likely 5 RassflC’ DAXX-binding sequence (Figure 3.1A). Similar to the SIM-N sequence, this region contains a number of negatively-charged residues juxtaposed by hydrophobic residues. 334 was To test if residues 13-34 were the DAXX binding sequence, unlabeled GST-RassflC’ 4 (Figure 3.1E-G). In 55 N 5 ‘ 6 4 DAXX -His expressed and purified for titration studies with ’ 334 produced profound changes in the ‘H , unlabeled GST-Rassfl C’ 576598 contrast to GST-Axin 4 (Figure 3.1F). That is, most peaks from the DHB 55 N 5 ‘ 4 -DAXX N HSQC spectrum of ’ 5 ‘ domain were broadened beyond detection due to the large size of the resulting complex, which also included the GST-tag (27kDa). Such broadening could of course result from an interaction between the GST-tag and the DHB domain. However, this is unlikely as such effects with GST 576598 were not observed. Furthermore peak broadening was alleviated by incubating the Axin sample with thrombin to remove the GST-tag (Figure 3.1G).  90  Chapter 3  —  Structure of the DAXX/RASSF1C complex  A 576 KPRSYSENAGTTLSAGDLAFGGKTS  576598 Axin  ° 12 DAXX RASSF1C’°  20 1 MAN2IIVLDDDAAA 1 [‘4GEAEAPSFEMTWSSITSGYCSQSSLEQYFTARTSLARRPRRDQD 50  44 + GST-bait DAXX”  4 DAXX’ 110  B  C  0  0  115  .:  0  0  °  0  0  0 *°  0  0  o  C • •00 o%o 0 00 0 • ° 000°0. 0 0  0 00  o 0  q 0  >  :  000 0  00  00 0  0  000  0  0  0  0  110  00  0  C) ci, H  0  0  0  0  0  ‘0 00 0 00  0  00  00  , 125  0  ,$  0  ‘Of  • 0  •  S  •o0o  0  0 0  00 OOQ  0  0  0  0  0  00 00 0  0  0  * C  —  0  44 + GST-bait+thrombin ’ 55 DAXX  C 00°o  0  0  120  598  E  0  0  F 0  JG 0  0  0  0000  .o’°  0  115  0  0000  0  0.  oo0*  0  120  o 0  00  e  0  •  •  0  )  0  ci) ci)  00 0  o  0  C) (/2 H  0  • •  0  0  000  0  0  Oe 0°  00 j°o0 O 0 0 0  0  0°  0  00  0.  0  0  eo 0  00  :  00 0  CD 0  12S °  0  o  9.5  9.0  8.5  8.0  7.5  7.0  9.5  9.0  8.5  8.0  7.5  7.0  9.5  9.0  0  0  8.5  8.0  7.5  7.0  H (ppm) 0  Figure 3.1 RassflC , but not Axin 1334 , bound the DHB domain. (A) Residues 13-34 of 576598 RassflC align well with DAXX’ , suggesting that, like this N-terminal segment of DAXX 20 (section 4.5), this portion of Rassfl C will also bind the DHB domain. In contrast, Axin 576598 does not share any sequence similarity. (B-G) The ‘H-’ N HSQC spectrum of ‘ 5 N-labeled His 5 6 44 (177 iM B, 155 .iM E) was not affected when unlabeled GST-Axin ’ 55 DAXX 576598 was added in excess to a 3:1 molar ratio (C), but was profoundly perturbed when unlabeled GST-Rassfl C’ 3 was added (4:1; F). The loss of signal was due to formation of a high molecular mass GST 334 / 55 RassflC’ -DAXX complex. Removal of the GST-tag (and His 6 His ”’ -tag) by addition of 6 thrombin dramatically sharpened the signals of the resulting smaller DAXX 44 / Rassfl C’ ’ 55 44 in the presence of Axin ’ 55 complex (G). In contrast, the spectrum of labeled DAXX 576598 was not significantly altered (D). -  91  Chapter 3  —  Structure of the DAXX/RASSF1C complex  Importantly, this study confirmed that DAXX and RassflC interact in vitro. However, it was ° flanking 13-34 were also involved. 5 still possible that other additional residues in RassflC’ ° was purified and NMR methods to determine the exact region that 5 Therefore RassflC’ 4 were employed. DAXX 4 ’ mediates its interaction with 55  144 55 ° and DAXX 15 3.2 NMR-monitored titration of RassflC ° was challenging, as it contained an internal 5 Expression and purification of Rassfl C’ pseudo-thrombin cleavage site at Arg38. The sample was also insoluble when expressed with a N-labeled and a 5 -tag. After optimizing the conditions for its purification, homogenous ‘ 6 His C/’ 3 ‘ N ) 5 38 partially proteolyzed (i.e. heterogeneous, with a significant amount of RassflC’ labeled samples were obtained (see section 3.7.2.1 for details). By combining the information N-labeled peptide, 5 N-NOESY-HSQC spectra recorded with the ‘ 5 from ‘ N-TOCSY-HSQC and ‘ 5 /’ 5 C 3 ‘ -labeled along with HNCACB and (HB)CBCA(CO)NH spectra recorded with the N ° were assigned (Figure 3.2A). 5 sample, the signals from all of the amide residues in RassflC’ C chemical shift values are typical of random coils (not shown), it was Since its lHa and 3 ° is predominantly unstructured in isolation. 5 concluded that Rassfl C’ N HSQC experiments were used to monitor the titration of 5 With assignments in hand, ‘H-’ 4 Suspecting an electrostatic contribution to . 4 ’ 55 ° with unlabeled DAXX 5 N-labeled RassflC’ 15 the interaction of these two species, the titration was performed using a low ionic strength buffer. N HSQC spectrum of 5 Profound peak broadening and chemical shift changes in the ‘H-’ 44 ’ 55 Rassfl , 150 as well as the formation of precipitate, were observed upon addition of DAXX C (Figure  3.2B).  At  the  stoichiometric  point,  92  a number  of RassflC’ ° 5  peaks  had  Chapter 3  —  Structure of the DAXX/RASSF1C complex  A  2 T39  115  J136 ‘  S8 29  S27  ‘H (ppm)  SN r-S4O  S19  s15  —  Sis  2  R38 S23  Z  0.12  —Q49 .-R43 —F35 -F5 —Y21 .‘E32 Q33—  120  D2 E25  —R46  [)25  —L31  MU r11o)  -  0.10  -  E 0.08  --  —E3O  C22  -R47 Q24  R44  --  < 0.06  A42 TAI  -  125  ...WI3  -A3  0.04 A37 0.02  .D5O 10 8.4  8:2  8.0  20  30  40  50  RassflC residue number  7:8  ‘H (ppm)  Figure 3.2 Determination of the region of RassflC ° that mediated the interaction with 15 44 (A) Fully annotated N ’ 55 DAXX . 15 HSQC spectrum of RassflC H 1 . (B) Superimposed 50 -50 spectra of ‘ N-Rassfl C’ (red, 115 tM) upon titration with 0.5 (cyan) and 1 (black) equivalents 5 of unlabeled DAXX 44 (initially, 1.9 mM) in 10 mM phosphate buffer, pH 6.5. Full/open ’ 55 arrows show peaks that shifted and were detectable/undetectable at the stoichiometric ratio. Most peaks were significantly broadened during the titration, but still observable at a DAXX 55 44 C ‘ :Rassfl 150 ratio of 0.5:1 (cyan). (C) Chemical shift perturbations resulting from addition of 44 to ‘ ’ 55 DAXX N-labeled RassflC’ 5 50 at a DAXX:RassflC ratio of 0.5, calculated as (H)2 +(0.2o,N) 2 reveal that residues 28-38, and to a lesser extent, 9-18, are perturbed upon -  ,  binding.  93  Chapter 3  —  Structure of the DAXX/RASSF1C complex  already broadened beyond detection (Figure 3.2B, open arrows). Fortunately, when the unlabeled ° 5 44 concentration was at half the stoichiometric point, most signals from RassflC’ ’ 55 DAXX were still visible (Figure 3 .2B, cyan), thus allowing the delineation of the region of Rassfl C that interacts with the DHB domain. Significant chemical shift changes were observed for residues 28-3 8, and to a lesser extent, 334 construct was 9-18 (Figure 3.2C). This showed that the originally tested GST-RassflC’ indeed too short. With this information at hand, a number of intermediate-sized constructs were designed to determine the optimum Rassfl C peptide for structural studies.  3.3  NMR-monitored titrations of truncation fragments of RassflC with 144 55 DAXX  3.3.1 RassflC 838 The first investigated construct was RassflC , which contained all the residues perturbed 838 in the RassflC’ ° titration. When unlabeled RassflC 5 838 was titrated into a sample of 5 -’ 6 His N 44 substantial irreversible precipitation occurred if the NMR buffer did not contain any ’ 55 DAXX , added KC1. However, in the presence of 100 mM KC1, precipitation was completely absent (not shown). Under the latter conditions, the  H” 1  and ‘ N chemical shifts of ‘ 5 N-labeled RassflC 5  changed in the fast exchange regime upon addition of unlabeled DAXX 44 (Figure 3.3A). This ’ 55 titration confirmed that residues 3 1-36 of RassflC were most affected upon binding (Figure 3.3C). The KD for this interaction was found to be 210  ±  30 iM based on least-squares fitting of  chemical shift changes for 6 RassflC amides as a function of total ligand ) 44 ’ 55 (DAXX concentration (Figure 3.3B).  94  Chapter 3  —  Structure of the DAXX/RASSF1C complex  3.3.2 RassflC 21 8  ° showed modest chemical shift changes for 5 The previous experiments with RassflC’ 44 (Figure 3.2C). To examine further the role of these ’ 55 residues 9-18 upon binding DAXX N HSQC 5 , was expressed and purified. The ‘H-’ 821 residues, an unlabeled construct, RassflC 6 His N 5 4 ’ -DAXX 4 ’ in NMR sample buffer lacking KC1 remained unperturbed spectrum of 55 upon addition of a stoichiometric amount of this peptide, indicating no measureable binding (Figure 3.4A). Note however, since spectral changes can only be observed when the KD values do not exceed approximately the protein concentration times five (i.e. KD < 5 [P1), the lower limit 44 must be greater ’ 55 for the KD value for any interaction between this isolated region and DAXX than 500 ElM.  3.3.3 ’’ 38 23 Rassf1C  v 2338 A complementary construct, Rassf1C  ,  was produced. This construct includes those  residues in RassflC’ ° and RassflC 5 838 displaying the greatest chemical shift perturbations upon addition of DAXX ’. In contrast to the case of RassflC 55 , stoichiometric amounts of 821 ” produced significant changes of selective amide signals in the ‘H-’ 2338 RassflC N-HSQC 5 spectrum of 55 -DAXX (Figure 3.4B). These changes occurred in the fast exchange regime 6 His ”” (Figure 3.5A), allowing the determination of the dissociation constant for the binding of ” by DAXX 2338 RassflC 44 (KD ’ 55  =  65 ± 16 1 iM, Figure 3.5E). The reverse titration, adding  unlabeled DAXX ” to ‘ 55 N-labeled RassflC 5 ”, produced much larger chemical shift 2338  () Constructs identified with a ‘w’ contain non-native C-terminal Trp residue, which was added when a native Trp residue was not present in the sequence. This facilitated quantification via UV-spectroscopy, and in the case of v, extended the C-terminus from the interacting Arg38. 2338 Rassf1C  95  __  __  Chapter 3  —  Structure of the DAXX/RASSF1C complex  [;i3:1  B  •  w 0.24  120  I  E  E  V • •  E32 Q33 Y34 F35 T36  I I V  LQ  z  a  0.16 a 0.08 B  KD=210±30  jiM  1 2 3 DAXX:RassflC ratio  4  I •1  10  15  20 25 30 RassflC residue number  35  Figure 3.3 Identification of the RassflC 838 residues that interacted with . 44 (A) ’ 55 DAXX Superimposed regions of the ‘H-’ N HSQC spectra of 15 5 N-labeled RassflC 838 with increasing amounts of unlabeled . 44 Arrows indicate chemical shift changes from the free to the ’ 55 DAXX bound state. (B) The chemical shifts of residues 31-36 of Rassfl C (initially 90 iM in 500 pL) as a function of added DAXX 55144 from a 1.3 mM stock. Fitting the titration curve (solid line) yielded a KD value of 210 ± 30 .tM. (C) Chemical shift perturbations resulting from addition of 4:1 DAXX 44 to ‘ ’ 55 N-labeled RassflC 5 , calculated as .J(zx6H)2 +(O.2MSN) 838 2 Although -  .  residues 3 1-36 showed the greatest changes upon binding, residues 11-29 were also slightly perturbed.  96  Chapter 3  110  A  —  Structure of the DAXX/RASSF1C complex B  0 0 0  0  •0 0 0 o  115  I  .  fl  0  0°  0  000 0  0  0 0  0  0  0  0  0  0  0  ,  0  0  0%  V  0o0 0  0  $  0  0,  00  0  0 0  0  000 ‘  00  0 S  0  0  125  0  0 ç,  0  0  120  V  0  0  0  • 0  0  0  9.5  90  8.5  8.0  7.5  7.0  9.5  9:0  8.5  ‘H (ppm)  8.0  7.5  7.0  ‘H (ppm)  E  z  8.2  8.1  8.0  7.9  7.8  •  2  DAXX:RassflC ratio  ‘H (ppm)  w, but not RassflC 2338 RassflC Figure 3.4 , bound . 821 44 (A) ‘H-’ ’ 55 DAXX N HSQC 5 spectrum of 55 NAXX 5 ‘ D 6 4 ’ -His 4 in the absence (red) and presence of a stoichiometric amount of unlabeled RassflC ’ (black). The lack of chemical shift changes indicated that Rassf1C 82 ’ 82 did not bind DAXX 44 measurably. (B) ‘H-’ ’ 55 N HSQC spectrum of 55 5 -DAXX 6 His 4 ’ 4 in the absence (red) and presence of a stoichiometric amount of unlabeled Rassfl ” 2338 (black). C Significant shift changes were observed in a number of resonances, thus confirming binding. (C) A section of the complementary ‘H-’ N HSQC s 5 pectrum of 15 5 ’” (125 jiM, red) 2338 N-Rassf1C with progressive increments in unlabeled DAXX 5 144 (from a 2 mM stock, cyan to black). The binding event was in the fast exchange regime. (D) The chemical shift of residues 31-37 of ” as a function of added DAXX 2338 RassflC 44 was used to fit the titration curve (solid line), ’ 55 yielding a KD value of 55 ± 5 jiM. -  97  Chapter 3  —  Structure of the DAXX/RASSF1C complex  perturbations, also in the fast exchange regime (Figure 3.4C). The dissociation constant fit for the reverse titration is consistent with this initial result  (KD  =  55  ±  5 M, Figure 3.4D). These  838 since these titrations values are about four times lower than those observed with RassflC were performed under lower ionic strength conditions. N-labeled DAXX was used. To test 5 -tagged ‘ 6 Throughout screening experiments, His whether binding was biased by the presence of this tag, thrombin was added to the solution from Rassf1C Beyond the expected v. N-labeled DAXX ’ with 2338 55 the last titration point of 15 disappearance of  ”-’ 1 ‘H N 5  changes were observed.  signals corresponding to the His -tag, no other significant spectral 6 Thus, the His -tag did not measurably affect the DAXXIRassf1C 6  interaction. Once the His -tag was removed, it was investigated whether there was indeed an important 6 electrostatic contribution to the DAXX/Rassfl C interaction. Addition of increasing amounts of KC1 produced progressive changes in the ‘H-’ N HSQC spectrum of ‘ 5 N-labeled DAXX 5 44 in ’ 55 the reverse direction, as observed during the initial titration. This clearly demonstrates a decrease in the population of the complex with increasing ionic strength, thereby verifying that electrostatic interactions indeed play a major role in complex formation (Compare Figure 3.5A with Figure 3.5C). Subsequently, it was investigated which residues were involved in the interaction by mapping chemical shift perturbations onto the structure of apo-DAXX ’. Figure 3.6 shows 551 that residues most affected by binding of Rassf1C ’’ form two clusters. 2338 The first cluster covered N-terminal residues, including Cys58 and Lys6O. Noteworthy, in lHNl signal of Lys6O N previous experiments, it was observed that the downfield shifted amide 5 98  Chapter 3  —  Structure of the DAXX/RASSF1C complex  is exquisitely sensitive to the salt concentration of the sample, i.e. changing from 9.3 ppm to 8.6 ppm with small increases in ionic strength (not shown). Such changes were attributed to the possible disruption of a hydrogen bond between the Lys6O amide and the Leu 100 carbonyl oxygen, and were not investigated further. Regardless, the chemical shift changes of Cys58 and ’’. In support of 2338 Lys6O likely reflect subtle sample changes during titrations with of Rassf1C this conclusion, the fitted KD values of these two residues are —10 fold higher than those 44 residues in the same titration experiment (Figure 3.5E-F; 700 .iM ’ 55 observed for other DAXX versus 65 !IM). Furthermore, the observation that the chemical shift changes of Lys6O and Cys58 did not reverse as salt was added to disrupt the complex, as observed with other residues (Figure v by these residues. 2338 3.5B-D), argues against a secondary, weak binding site for Rassf1C Svdependent chemical shift 233 The most predominant cluster of residues showing Rassf1C changes (residues 84-85, and 124-130) formed a cleft between helices 2 and 5 (Figure 3.6B,C). Most of these residues are hydrophobic, and importantly are not buried within the core of the DHE domain (Figure 3.6D, green). It is also worth mentioning that these residues have been  highly conserved during evolution, as the sequence alignment of DAXX from various organisms shows some variability in the DHB domain sequence, but very little variation in residues 84-85, and 124-135 is observed (Figure 2.1). These data strongly indicate that this region is the binding site for RassflC.  99  Chapter 3  —  Structure of the DAXX/RASSF1C complex  ” and 2338 Figure 3.5 Electrostatic interactions contributed to the binding of Rassf1C 4 (110 tiM, N 5 ‘ 6 4 ’ DAXX -His N HSQC spectrum of 55 5 44 (A-B) Sections of the ‘H-’ ’ 55 DAXX . 4 mM stock, cyan to black). 2 C ” 338 (from a red) with progressive increments in unlabeled Rassfl N-DAXX 5 ‘ N HSQC spectrum of thrombin treated 55 5 (C-D) The same sections of the ‘H-’ (blue). The resonances (black) to 450 mM from mM 0 with increments of NaC1 44 ” ‘ /Rassfl 2338 c from residues Asn128 and Va185 reversed direction towards the shifts from the free protein, indicating that the complex population decreased with increasing ionic strength. On the other hand, Lys6O continued to shift upfield with added salt, presumably by disrupting a hydrogen bond between its amide and the Leul 00 carbonyl oxygen. (E-F) The chemical shifts of residues ’’ were fit to obtain 2338 44 as a function of added Rassf1C ’ 55 58, 60, 85, and 125-128 of DAXX residue-specific dissociation constants. The anomalously high dissociation constant of residues 58 and 60 also suggested that these changes reflect a secondary local event independent of RassflC binding. -  100  Chapter 3  —  Structure of the DAXX/RASSF1C complex  122.5  z  123.5  124.5  D 119.0  z  122.5  123.5  119.5  V85 124.5  I  120.0  9.4 8.10  8.15  0  CQ  9.2  9.0  8.8  ‘H (ppm)  8.05  ‘H (ppm)  E 0.16 ---.  --  0.12  -  -  A  -  .  0.08  I .  S.  0.04  •  SI  .  1  KL= 65 2  3  4  ±  •  V125 127  •  Y126  16 tM 5  6  7  8  RassflC:DAXX ratio  RassflC:DAXX ratio  101  8.6  Chapter 3  —  Structure of the DAXX/RASSF1C complex  A 0.30  E 0.20  -  0.10  II I I  60  II.iI.i 1 . 111  I tIIIIIIIiIIIIiIIhIiIIIIIIIIII.IIiIII 70  80  90  100  110  120  130  140  DAXX residue number  Y124  B  D  ciA  C  Figure 3.6 Identification of the Rassf1C ’’ binding interface on DAXX 2338 . (A) 55144 Chemical shift perturbations resulting from addition of RassflC ” to ‘ 2338 N-labeled DAXX 5 , 5544 l()2 calculated as (B) Cartoon representation of DAXX 44 with its 5 helices ’ 55 2 +(O.2Aö,N) -  .  (al- c5). Residues Va184, Va185, Tyr124, Va1125, 11e127, Asn128, Glu129 and Leul3O, which showed significant spectral perturbations (A > 0.085 ppm), are highlighted in red. Also in red are Cys58 and Lys6O, which experienced spectral changes during titrations that were independent of the binding of Rassf1C ’” (see text). (C) Surface of DAXX 2338 44 showing how ’ 55 the side chains of the affected hydrophobic residues are solvent exposed, and presumably involved in substrate binding. (D) Cartoon representation of DAXX 55144 with non-polar residues shown in green. The amphipathic character of the helices is evident except for a few residues that are solvent exposed. These same residues were the most affected by addition of Rassf1C ’’. 2338 102  Chapter 3  —  Structure of the DAXX/RASSF1C complex  3.4 Screen of conditions for the formation of a tighter complex In order to obtain a high resolution three-dimensional structure of a biomolecular complex by NMR spectroscopy it is desirable that the components interact strongly (i.e. a 1:1 complex in slow exchange). Low affinity binding precludes saturation at the stoichiometric ratio, thus making intermolecular NOEs weaker and hence structural calculations more difficult (Nietlispach et al., 2004). As summarized in the previous section, it was found that residues 31-36 of Rassfl C are key ’’, bound DAXX 2338 55144 with a to binding with DAXX . The optimized construct, RassflC 55144 KD of 65  ±  16 .iM in 10 mM phosphate buffer, pH 6.5 and 25 °C. Thus, under the conditions  used for the titration experiments (100 jaM protein in the NMR tube, 5 mM peptide being added), only 45% saturation was reached at the stoichiometric point (i.e. equimolar amounts of each species). Based on mass action, this would improve to only 75% saturation by increasing the concentration of the protein to 1 mM. Therefore, with the aim of optimizing the conditions for the formation of a tighter 1:1 complex, the dissociation constant of the complex was measured as a function of temperature, pH, and buffer type, as well as examining slightly longer RassflC constructs (Table 3.1). From Table 3.1, one can conclude the following points. (1) Longer constructs, such as 838 or RassflC RassflC ° produced irreversible precipitation at low ionic strength, or showed 235  much weaker binding at high ionic strength, and hence were not suitable for structural studies. (2) A small extension of the C-terminus in Rassfl C 2344 did not produce significantly tighter  binding, and the non-specific proteolysis that the construct underwent makes it not suitable for  103  Chapter 3  —  Structure of the DAXX/RASSF1C complex  44 and various RassflC constructs under ’ 55 Dissociation constants of DAXX Table 3.1 different conditions. -  ‘ N 5 -labeled construct / concentration  Unlabeled construct / concentration  Buffer a  p11  Temperature  0 (.tM) K  Rassfl C 38  DAXX 55144 1.3mM  4 10 mM KPO  6.5  25 °C  Irreversible precipitation  DAXX 4 ’ 55 4 1.3mM  , 4 10 mM KPO 100rnMKC1  6.5  25 °C  210 ± 30  110 j.tM  RassflC ’ 82 2.3mM  4 10 mM KPO  6.5  25 °C  No binding  -DAXX 6 His 5144  v 2338 Rassf1C  110pM  4.1mM  10 mM KPO 4  6.5  25 °C  65  -DAXX 6 His 5144  Rassfl ’’ 2338 c  110J.LM  3.3mM  4 10 mM KPO  6.5  15 °C  69 ± 14  -DAXX 6 His 5144  Sw 233 RassflC 10 mM MES  5.5  25 °C  48 ± 13  25 °C  28±7  90j.tM  838 RassflC 90p.M 4 5 6 His 4 ’ DAXX  100 -DAXX 6 His 5144 100  0.8mM  2338 c Rassf w 1.8mM  4 5 6 His 4 ’ DAXX  wc 2344 RassflC  100 iM  0.65-3 mM ‘  4 55 6 His 4 ’ DAXX  Rassfl ° 235 C C  100  2.4rnMd  b  10 mM HEPES  *  16  10 mM KPO 4  6.5  25 °C  20-290  10 mM KPO 4  6.5  25 °C  Irreversible precipitation  a  All buffers also included 0.1 mM EDTA.  b  Resonances from amides from the termini (55, 57, 58, 140, 141, 142) and loops (83, 97, 121) were not detectable at pH 7.5 due to fast hydrogen exchange with the solvent.  C  Constructs 23-44w and 23-50 were purified using a different plasmid that contained a PreScission cleavage site instead of the thrombin cleavage site like the other constructs. This ° contains an internal thrombin cleavage site at Arg38. 5 was needed as Rassfl C’  d  MALDI-ToF-MS showed that construct 23-44w was a mixture of the intact peptide, and a shorter non-specifically proteolysed peptide 23-42 without a C-terminal Trp. The difference in extinction coefficient between both constructs introduced a large error in the calculated 235 also showed the presence of a smaller fragment. C concentration of the peptide. Rassfl °  e  The KD was calculated using either the concentration from an intact 23-44w (20 !IM) or a proteolysed 23-42 (290 tM). 104  Chapter 3  —  Structure of the DAXX/RASSF1C complex  structural studies (see Materials and Methods for details). (3) Temperature did not seem to play a major role in the interaction, as the dissociation constant of the complex at 15 °C and 25 °C were essentially identical. (4) Moderately tighter binding was observed at different pH values, although this is most likely due to the zwitterionic buffers used, which decreased the ionic strength of the sample with respect to the phosphate buffer. (5) The tightest complex observed was with 10 mM HEPES, pH 7.5, although a number of amide resonances in the termini and loops were not detectable due to fast exchange with the buffer at this alkaline pH value. One interesting observation is that amide signals in this buffer had a comparable signal-to, pH 6.5, despite obviously undergoing faster 4 noise ratio as their counterparts in 10 mM KPO hydrogen exchange. This is consistent with recent reports that demonstrate that cryogenic probes perform much better with low conductivity buffers relative to phosphate (Horiuchi et al., 2005; Kelly et al., 2002).  With this information at hand, a stable relatively tight complex  (KD  20 1 iM) suitable for  structural studies was prepared (10 mM MOPS, 0.1 mM EDTA, pH 6.5; see section 3.7.4 for details on how the final samples were prepared and how the structure was calculated).  3.5 Determination of the tertiary structure of the complex 4 secondary structure upon binding DAXX 4 ’ 3.5.1 Changes in 55 44 displayed only modest perturbations ’ 55 2338 the spectra of DAXX C Upon binding Rassfl ”, of a limited number of backbone amide groups  HN 1  and ‘ Cc 3 N (Figures 3.4B and 3.6A), and of ‘ 5  and ‘Hi’ values (Figure 3 .7C,D). This indicated that the tertiary structure of the protein was not significantly affected. Rather, small spectral changes for residues in a localized region of the  105  Chapter 3  —  Structure of the DAXX!RASSF1C complex  2338 binding. However, C protein likely reflect minor structural changes accompanying Rassfl ” 2338 C one residue stands out in these shift perturbation plots is Va184. In the presence of Rassfl ”, C and ‘H chemical shift values become more typical of residues in an tx—helix. its 13 Interestingly, Va184 lies at the N-terminus of Helix-2 (residues 85-93) in the apo-protein, and is 233 binding interface (Figure 4.6C). As it will be discussed in the next integral to the Rassfl C section, Va184 becomes part of a slightly elongated Helix 2  upon  binding RassflC.  38 secondary structure upon binding . 23 RassflC 3.52 Changes in w 2338 is predominantly unstructured, as evidenced by its random coil C The apo-Rassfl ”’ 23 44 however, RassflC ’ 55 DAXX chemical shifts and low SSP scores (not shown). Upon binding , 38w  N chemical 5 exhibits dramatic spectral changes. First, almost all of its amides show IHN and ‘  44 (Figure 3.8A). Second, its ’ 55 N-HSQC spectra when titrated with DAXX 5 shifts changes in ‘H-’ C chemical shift values move upfield and downfield, respectively (Figure 3.7A-B. ‘H and 13 N {‘H}-NOE values of most amides 5 This is diagnostic of a coil to u-helix transition. Third, the ‘ (0.45) are typical of a moderate degree of order on the nsec-psec time scale. In contrast, the negative NOE values of its N-terminal amides are typical of a peptide that is fully disordered on this timescale (Figure 3.8B). In all of these plots, residues 30-38 undergo the greatest changes 4 consistent with the original findings from the RassflC’ , 4 ’ 55 when binding DAXX ° titration 5 (Figure 2C).  106  Chapter 3  1.0  —  Structure of the DAXX/RASSF1C complex  C  0.8  ,—  E  0.6  Uk  04  c-) <0.2 II  0  I  Ii  .j  —  I  III  I  I  1.11111  1il lb I  11.111 III I ....  I  -  .0.2  0.05  p  -0.10  i  -0.15 -0.20  25  30  35  60  80  Residue  100  120  140  Residue  Figure 3.7 Changes in the secondary structures of RassflC ’’ and DAXX 2338 44 upon ’ 55 binding. (A-B) Changes in the corresponding ‘ Cc and ‘H° chemical shift values of RassflC 3 23 38w C 3 l a in its bound versus free states. The upfield and downfield changes in the and ‘H chemical shift values, respectively, are consistent with the formation of an a-helix (Wishart and Sykes, 1994; Wishart et a!., 1992). (C-D) Changes in the C and H chemical shift values of 55144 in its bound versus free states. Only Va!84, which is in the binding interface, showed DAXX a significant change due to a slight elongation of helix 2. This demonstrates that the protein did not undergo significant structural changes upon binding Rassft C. -  107  Chapter 3  —  Structure of the DAXX/RASSF1C complex  dl  A  Q24 :  115  S27 S29  I 7  120 —E25 QQZ4  L31  1)28 E39  125 A37  8.5  W39  8:0  ‘‘  7.5  7.0  II (ppm)  :: -0.2 -0.4  -  24  2  32  36  Residue w upon 2338 Figure 3.8 Changes in the amide chemical shifts and relaxation of RassflC DAXX5514. binding (A) Annotated ‘H-’ N HSQC spectrum of Rassf1C 5 ” in its free and 2338 44 form. Peaks corresponding to the region 3 1-38 underwent major spectral ’ 55 DAXX -bound perturbations, indicating significant changes in its secondary and tertiary structure. (B) Heteronuclear 15 N {‘H}-NOE values of -bound 44 Rassf1C ’ 55 DAXX ”’. The low/negative and 2338 low NOE values in residues 22-26 indicate a flexible N-terminus. The moderate values of residues 27-3 8 indicate a more ordered, structured region. The reduced NOE value of Trp39 indicates somewhat increased flexibility at its C-terminus. -  108  Chapter 3  —  Structure of the DAXX/RASSF1C complex  ’’ complex 2338 / Rassf1C 551 3.5.3 Tertiary structure of the DAXX 2338 complex was determined from an C 4 / Rassfl ’ DAXX 4 ’ The structural ensemble of the 55 extensive set of NMR-derived inter and intramolecular distance restraints (Table 3.2; Figure 4 / RassflC DAXX 4 ’ ” complex 2338 3.9B; details in section 3.7.4). A ribbon diagram of the 55 55144 retained the secondary and tertiary (Figure 3 .9A) demonstrates that peptide-bound DAXX structure of the free DHB domain (Chapter 2), based upon 5 x-helices (PROMOTIF boundaries: 60-77 red, 84-93 orange, 99-101 green, 103-118 blue, 122-137 purple; Figure 3.9A). This is reflected quantitatively in the low RIvISD between the helices in the lowest energy members of 4 structural ensembles (1.08 DAXX 4 ’ free and bound 55  A [backbone C/N/O atoms only] and 1.75  A [all C/N/U atoms in the helices]; Figure 3.100). Indeed, the most significant change in the backbone conformation occurred with the small elongation of helix 2 to include Va184. This resulted from the side-chain of Va184 being “pushed” back by the RassflC peptide (Figure 3.100). Additionally the packing of aromatic ” in the 2338 rings of Tyr124 and Phe87 changed significantly to accommodate RassflC hydrophobic binding cleft of the DHB domain (Figure 3.1 OD,F). Ras sf1 C 23  38W  lies in the cleft between helices 2 and 5 of the DHB domain (Figure 3.9 A-D).  The peptide forms a short amphipathic helix with Leu3 1, Tyr34, Phe35 and Thr36 making most of the intermolecular contacts with Val84, Phe87, Tyr124, and 1le127 of the DAXX DHB (Figure 3.1 OA-E). On the outer side of the amphipathic helix, the hydrophilic 5er29, G1u30, Glu32 and G1n33 face the solvent.  109  Chapter 3  —  Structure of the DAXX/RASSF1C complex  NMR restraints and statistics for the ensemble of the ten lowest energy Table 3.2 44 complex. i w 2338 55 /RassflC structures calculated for the DAXX -  Summary of restraints. NOESa Intra-residue Sequential Medium range (i-j 2-4) Long range (i-j 5) Intermolecular Total Dihedral angles:  849 (106) 385 (99) 256 (152) 217 (124) 33 (65) 1740 (546) 81 81  Deviation from restraints NOE(A) Dihedral restraints (degrees) Deviation from idealized geometry Bonds (A) Angles (degrees) Improper angles (degrees) Residues in allowed region of the Ramachandran plot Mean energiesb (kcal mor’)  0.052 ± 0.000 0.80 ± 0.13  (%)  -310±20 48 ± 2 204 ± 7 97 ± 8 280+20 6±2  Ed  Eangies Eimproper ENOE Edh  RMSD from average structureC Residues in a-helices Backbone atoms d Heavy atoms All residues Backbone atoms Heavy atoms  0.005 + 0.000 0.63 ± 0.01 1.61 + 0.08 98.1  (A) 0.35 0.92  ± ±  0.07 0.15  0.57 ± 0.22 1.13 ± 0.26  a Number of unambiguous restraints, with ambiguous restraints in parentheses. b  Final ARIA/CNS energies for van der Waals (vdw), bonds, angles, NOE restraints (NOE), and dihedral restraints (cidh). Residues in a-helices: DAXX (60-77, 84-93, 99-101, 103-118, 123-137), RassflC (29-33). All residues: DAXX(60- 137), Rassfl C (27-39).  d  “heavy” C/N/O atoms. 110  Chapter 3  —  Structure of the DAXXIRASSF1C complex  w complex. (A) Ribbon 2338 55144 / RassflC Figure 3.9 Tertiary structure of the DAXX diagram of one low-energy NMR-derived structure of the complex. Helices 1-5 of the DHB domain are shown in red, orange, green, blue and purple, respectively. Rassfl C is shown in old. 55 (B) Superimposed backbone atoms from seven low-energy members of the DAXX 2 C w 338 structural ensemble. (C) Surface of one low-energy NMR-derived structure of the Rassfl DHB domain in the complex, colored according to an electrostatic potential (blue positive, red negative, white neutral). A Rassfl C ribbon diagram with selected residues involved in the binding is shown for reference, and the negatively-charged residues 25, 26 and 28 in red. (B) ”’ in the complex, colored 2338 Surface of one low-energy NMR-derived structure of the RassflC 44 ribbon diagram with selected residues ’ 55 according to an electrostatic potential. A DAXX involved in the binding is shown for reference. The view on the left shows a “slab” picture from the inside to out of the DHB domain, highlighting helices 2 and 5, which mediate the binding process. The end of helices 1, 3 and 4 are in transparent gray. The view on the right shows the position of all helices in the complex. All electrostatic potentials were calculated using PDB2PQR with the AMBER forcefield (Dolinsky et al., 2007; Dolinsky et a!., 2004), and plotted using the APBS plug-in from Pymol (DeLano Scientific), with an electrostatic potential gradient from -8.0 to 8.0 kT/e. -  -  111  Chapter 3  Ra,’F1(  —  Structure of the DAXX/RASSF1C complex  B  Hi  R38  N4’ H2  90° +0 Q23  N144  G55  450  *  180w  45°  P  *  D 180°  1127  H’ 90°  F87’  * 112  Chapter 3  —  Structure of the DAXXIRASSF1C complex  55144 and Figure 3.10 Residues mediating the intermolecular interaction between DAXX Sw. Residues Leu3l, Phe35 and Thr36 in RassflC (A) and residues Va184, Phe87, 233 RassflC 44 (B) have the largest number of intermolecu]ar NOEs. ’ 55 Tyr124, 11e127 and Asn128 in DAXX (C) Diagram representing the intermolecular NOE connections between residues in helices 2 and 2338 peptide (gold). (D-E) Interaction surfaces C 5 from the DHB domain (cyan) and the Rassfl ’ of one low energy NMR-derived structure of the DHB/Rassfl C complex. The two molecules are separated and colored by surface physicochemical properties (hydrophobic green (D), red negative, blue positive, and white polar neutral (E). The arrow shows the hydrophobic cleft from the DHB domain occupied by the hydrophobic cluster from Rassfl C. The default values for calculating the electrostatic potential (at zero ionic strength) from MOLMOL were used. (F) Ribbon diagram of a low-energy NMR structure of the DHB (cyan) Rassfl C (gold) complex. The side-chain atoms of interacting residues are highlighted. Oxygen atoms are shown in red, nitrogen atoms in blue. (G) Ribbon diagram of superimposed low-energy structures of apo- and Rassfl C-bound . 44 Changes in the structure of free DAXX ’ 55 DAXX 44 (brown) and Rassfl C ’ 55 bound DHB (cyan) are located in the binding surface. The aromatic rings of Y124 and F87 move to accommodate the incoming Rassfl C peptide. Similarly, Va184 is “pushed” back by Leu3 1 in the peptide. -  -  113  Chapter 3  o  —  Structure of the DAXX/RASSF1C complex  C 30  20j  z 26  I  I  30  34  Residue  II 85  120  Residue  [  I)AXX HeIi’ 5  1132 S27  I)AXX.” HeIi 2  N120 1127 )2  V124 F32 Q3.i  35 [31,  5,55, v05  5  l(2’  57 R35 39  -,  130  D  E  026  DHB  RassflC  DHB  F  G  114  RassflC  Chapter 3  —  Structure of the DAXX/RASSF1C complex  44 ’ 55 Juxtaposed to the hydrophobic cleft, a cluster of positively-charged residues in DAXX (Arg9l, Arg94, Lys135, His137, Lysl4O, Lysl4l and Lys142) faces a cluster of negatively2338 (G1u25, Asp26, and Asp28) (Figure 3.1 OE). This electrostatic C charged residues in Rassfl ” interaction is consistent with the observed salt-dependence of binding (Figure 3.5). The final structure completely explains the preliminary experiments (sections 3.2-3.5.2). The peptide occupies the hydrophobic cleft predicted qualitatively from chemical shift perturbation mapping of titration data (Figure 3.6C) (Figure 3.1OD). PROMOTIF and DSSP identified ”’ helical 2338 residues 29-33 as the common helix boundaries (with residues 28 and 34 of Rassf1C in some members of the ensemble). This is consistent with chemical shift analyses and NOE data, which predicted that these residues formed an x-helix (Figures 3.7-3.8). The interactive ’’ construct. This resulted in 2338 region is close to the C-terminal non-native Trp39 of the RassflC Trp39 giving some intermolecular NOEs to Tyr124 (Figure 3.1OC), intramolecular NOEs to Thr36 (not shown), and a small positive ‘H-’ N heteronuclear NOE value (Figure 3.8B). 5 Attempts to extend the C-terminus with other constructs did not result in any improvement in complex formation, and in some cases led to aggregation (Table 3.1). These results suggest that Trp39 did not contribute significantly to the affinity of the DAXX 44 / Rassf1C ’ 55 ”’ complex. 2338  3.6 Structural implications 3.6.1 Structural verification that the DAXX DHB and Sin PAll domains differ in binding mechanisms With the structure of the DHB domainlRassfl C complex in hand, one can now conclude definitely that its binding mode is completely different to that of the Sin3 PAll domains (Figure 3.11). Sin3 PAll domains (discussed in Chapter 2) use a large hydrophobic cleft between helices 1 and 2 for binding (Figure 3.1 1B). The “opening” of the interhelical angle between helix 1 and 115  Chapter 3  —  Structure of the DAXX/RASSF1C complex  2 exposes hydrophobic residues from helices 3 and 4 to form a binding interface. For example, in the Sin3B PAH2/Mad complex (Spronk et al., 2000), this large 2100  2 hydrophobic cleft A  includes residues Phe7, AlalO, hell, 11e17, Tyr28, Phe3l, Leu32, Leu35, Phe59, Leu65, Phe66, Leu72, Leu73, Phe76, Phe79 and Leu8O (Figure 3.11C). In contrast, DAXX DHB domain uses a much smaller cleft along helices 2 and 5 to bind RassflC. The cleft is only 570  2 in A  hydrophobic surface area and lined by Val84, Phe87, Tyr124, 11e127 and Cysl3l (Figure 3.1OD). The Mad peptide forms a longer amphipathic helix (11 amino acids) when bound to Sin3B PAH2, whereas the Rassfl C peptide adopts a shorter helix (5 amino acids) in the resulting DHB domain complex. In the Sin3B/Mad complex, the hydrophilic residues of the Mad peptide are negatively-charged and complement the positively-charged residues of helix 2 from Sin3 (Figure 3.11 B), although they do not face each other directly as in the case of the DAXX/Ras sf1 C complex (Figure 3.1OE). 55 Additionally, Sin3B/Mad complex produces much tighter binding than the DAXX 44 complex. For example, a KD of 15-29 nM for Sin3A/Mad complex was ‘ ” 2338 /RassflC measured by ITC (Brubaker et al., 2000), and a KD of 1.4 jtM for the Sin3B/Mad complex was measured by surface plasmon resonance (van Ingen et al., 2004). These values are one or two orders of magnitude smaller than the best KD -‘20 tM (at low ionic strength) for DAXX 55 44 ” ‘ /Rassfl 2338 However, in the context of the full-length proteins, other domains could also C .  interact and thereby co-operatively enhance the specificity and affinity. Furthermore, post translational modifications on either protein could enhance the interaction. Interestingly, the Ser residues on Rassfl C 2338 are within ideal recognition sites of the CK2 kinase (discussed in Chapter 5). Further experiments are required to clarify these hypotheses.  116  Chapter 3  DAXX  —  Structure of the DAXX/RASSF1C complex  SIN3B  A  B  C  MAD  F  E  N C  DAXX  +  RassflC  H1PIR  FAT + Paxillin  Figure 3.11 DAXX and Sin3 PAH domains use different binding modes. Ribbon diagram of a low energy structure of the DAXX 55 2338 /RassflC complex (A) and the Sin3B PAH2/Mad 4 ‘ ” complex (B; 1 E9 1 .pdb). The helices numbering is shown as reference. (C) Surface of the Sin3B PAH2 in one low-energy structure of the Sin3B PAH2/Mad complex, showing the large hydrophobic cavity used for binding. The Mad peptide is shown in transparent cyan. Hydrophobic, negatively-charged and positively-charged residues of the Sin3B PAH2 domain are colored in green, red and blue, respectively. -  Other structural relatives of the DHB/RassflC complex. Ribbon diagram of a low energy structure of the /Rassfl 44 ’’ ’ 55 DAXX 2338 complex (D), the Huntingtin-interacting protein-i C (H1P1R) related (E) and Focal adhesion targeting (FAT) domain/Paxillin complex (F). The FAT/Paxillin complex displays a Paxillin peptide on each side of the protein on the same crystal structure. Helices 1 (red), 2 (orange), 3 (blue), 4 (purple), 5 or intermolecular helix (gold) and short helices in the loops (green) of the helix bundles are highlighted.  117  Chapter 3  —  Structure of the DAXX/RASSF1C complex  3.6.2 Other structural relatives share helix packing patterns 2338 complex were searched through C Additional structural relatives of the DHB/Rassfl ” DALI (Holm and Sander, 1995). The best hit was the five-helix bundle THATCH domain from the Huntingtin-interacting protein-i related (HIP 1 R), a protein-trafficking control multi-domain protein (Brett et al., 2006). Although not a complex, the first four helices of the helix bundle follow the same topology as the DHB domain, and the fifth helix packs on the groove of helices two and four, in a very similar way to how the Rassfi C helix packs along the equivalent groove in the DHB domain (Figure 3.11E). A more distant structural relative is the Focal Adhesion Targeting (FAT) domain in complex with Paxillin LD domains (Hoellerer et al., 2003). The FAT domain forms an all-antiparallel right-handed four-helix bundle that binds Paxillin LD peptides. In the structure of the complex, two LD peptides form amphipathic helices that pack on two sides of the FAT domain, each time using a cleft between two helices similar to the Rassf1 C peptide. Therefore the packing of a parallel helix (Rassfl C) along the groove of two other helices of a helical bundle (helices 2 and 5 of the DHB domain) is not uncommon. 3.6.3 Other binding partners of DAXX This work provides the first description of how DAXX interacts at the molecular level with other proteins. The structural characterization of the DHB domain and its interaction with a segment of Rassfl C also provide clues on how DAXX interacts with other proteins. The “dipolar” nature of the DHB domain, in which opposite faces of the protein have opposite electrostatic charges (Figure 2.1 2C), could prove important in mediating its interactions. The work presented in this chapter shows that the positively-charged face of the DHB domain 118  Chapter 3  —  Structure of the DAXX/RASSF1C complex  2338 peptide. This electrostatic interaction C interacts with the negatively-charged Rassfl ” 2338 for the formation of a C presumably helps to position the hydrophobic residues of Rassfl v stable complex. Indeed, recent studies have shown that formation of protein complexes involves an “encounter” complex, in which partner proteins display few specific interactions and assume multiple orientations (Ubbink, 2009). Electrostatic interactions dominate the formation of the encounter complexes. Subsequently, the complexes become “productive” with specific interactions leading to a single predominant conformation. It is worth mentioning that the opposite face of the DHB domain is negatively-charged and also contains a hydrophobic cluster formed primarily by residues in the ioops between helices 1 and 2, and 4 and 5 (Figure 2.12 C D). However, this hydrophobic “wall” is much smaller (350  ) than the one involved in 2 A  binding Rassfl ’. 2338 At this point it can only be speculated that this face might be involved in C binding proteins with a different motif to the one encountered in Rassfl C. The RassflC ’” sequence certainly provides clues on how the DHB domain identifies 2338 potential binding partners. It can be hypothesized that other interacting motifs contain a similar negatively-charged region juxtaposed with a short hydrophobic core. Some variability in these putative “DHB-interacting motifs” (DIMs) is expected, as the isolated DAXX’ ° (SIM-N) is also 2 able to bind to the DHB domain in the same region as Rassfl ’’ 2338 (section 4.5) despite having C a very different hydrophobic sequence. Indeed, based on the structure of the DHB/Rassfl ” 2338 C complex, it can be predicted that the intramolecular binding of these two DAXX regions occurs v 2338 in a reverse orientation with respect to RassflC. This is because, contrary to the RassflC peptide, the hydrophobic core in SIM-N is N-terminal to the negatively-charged cluster of residues (Table 3.3). Reverse binding of short motives is also observed with SUMO (Reverter and Lima, 2005; Song et a!., 2005), and Sin3B (Brubaker et a!., 2000; Swanson et al., 2004). 119  Chapter 3 Table 3.3 DAXX.  -  —  Structure of the DAXX/RASSF1C complex  Proteins previously reported in the literature to interact with the N-terminus of  3 (CAG33366) USP7 or HAUSP  1-160,  Full-length  (NP_003461)  347-570  (1102 aa)  MDM2 (NP_002383)  1-160, 1-260, 260-740,  CoIP  STAT3 (NP_644805)  1-188 1-240  NHEI  1-130,  (NP 003038)  13 1-400  ASK-i (NP_005914)  19  RassflC Hausp Hausp  23 828 856  RassflC 23 Full-length (497aa)  Co-IP  Mdm2 Mdin2  294-393 320-493 567-637  GST, Y2H.  GST, Co-IP Y2H,  Co-1P  RassflC 23 39 p53 312 p53 RassflC 23 458 Stat  (AAA45997) Axin  (AA113172)  No alignment  GST  No alignment  Co-IP  No alignment  293-411  SQEDSDSELEQYFThR SHTELDIKLGQHYVET  38 443  (Muromoto et al., 2006) (Jungetal.,  662  Co-IP  1-240  38 57 331  Askl Aski  RassflC 23 458 pp 71  DMAPI (CA123 197)  SQED---SDSELE-QYFTAR NDDLMLSPDDIE—QWFTEfl SSSPQPKKKPLDQEYFTLQ  38 678 1001 1320  Co-IP  2006)  EFSDTDSEGAQYV1VQ  FEEDPEISLPDYWKCT  SQEDSDSELEQY-FTR EEGDCESDLLEYDYEYO VSPDTELKVDPFSFKTR GTNTEOSELTDw-LRVN  985 1305  (Tang et al.,  38 141 313 283  23  Full length  -  SQEDSDSELEQYFTAR SSSPKEEQLEQVLDKQ  RassflC  43-197 439-501  1 188  YEG LRDLLQFFKPR  38 580  Co-IP  HIPK2 (NP 073577)  38 843 871  QRLNTDPMLLQFFKSQ  SQEDSDSELEQYFTR QERSKEPQLIAFYHKM  Full-length  1-197  1  SQEDSDSELEQYFTAR  23 565  70-2 16  1-686, 1598, 757832, 1-506, 576-832 1-520, 189-520  AADEDDDDLVI I SNATAM  RassflC Nhel  Aski pp7l  158 296 298  Mdm2  157-260 53 p (BAC16799)  SIM-N  SQEDSDSELEQY--FTR 38 TDSDEDGSAEEF’GAFAE 441  (Tangetal.,  2006) (Gostissa et  al., 2004)  2008) (Kitamura et al., 2009) (Hofmann et al., 2002)  (Li et al., 2007) (Hofiann et al., 2003) (Muromoto et al., 2004a)  Co-IP: Co-immunoprecipitation of exogenous protein, GST: GST-pulldown of exogenous protein, Y2H: Yeasttwo-hybrid. (b) Residues in red, blue and green are negatively-charged, positively-charged or hydrophobic, respectively. (c) Inverted sequence.  As mentioned at the beginning of this chapter, the reported binding partners of DAXX have generally been studied with coarsely-defined truncation fragments. Table 3.3 summarizes most proteins that have been reported to bind constructs of DAXX that included the DHB domain. A sequence alignment with Rassfl C 2338 shows that many of them do contain a potential DIM sequence. Moreover, Leu3 1, Tyr34 and Phe35 provide most of the intermolecular NOEs in the DAXX/Rassfl C complex, and most of the DIMs in these molecules have a high degree of conservation of these key residues (Table 3.3).  120  Chapter 3  —  Structure of the DAXX/RASSF1C complex  Constructs failing to give a good alignment covered regions of DAXX that might include parts of other domains (Chapter 2), or conversely, DAXX might contain recognition sequences in its unstructured regions for recruiting folded domains from other proteins. Further experiments are needed to investigate these possibilities. One DAXX binding partner of interest is the tumor suppressor protein p53. This so-called ‘guardian of the cell” is inactivated by mutation in about half of all known human cancers (Joerger and Fersht, 2007). p 53 has been shown to interact with DAXX in a number of studies (Gostissa et al., 2004; Kim et al., 2003; Zhao et a!., 2004). This interaction seems to be crucial to the ability of cells to respond to DNA damage (Gostissa et a!., 2004). , which contains the DHB domain, was shown to be involved in the interaction 80 DAXX’’ 2338 and with p53 (as well as other DAXX domains). A sequence alignment between RassflC p53 reveal two possible DIMs within p53, one between residues 39-57 and the other in the region 312-331 (Table 3.3). Consistent with this hypothesis, Gotissa et al. reported that region 294-3 93 of p53 is involved in binding DAXX. However, all of their deletion constructs lacked residues 1-74, and thus they might have missed the first DIM. The N-terminal DIM in p53 might have higher affinity for DAXX as its hydrophobic core is preceded by acidic residues similar to Rassf1C peptide, whereas the second DIM contains a number of Lys residues, which v the 2338 would decrease or abrogate the electrostatic interaction. Additionally, the first DIM resides in the unfolded N-terminal transactivation domain of p53, which is known to interact with Mdm2, with components of the transcription initiation complex, and with acetyltransferases p300/CBP (Joerger and Fersht, 2007). The Mdm2 interaction involves residues 15-29 of p53, which also form an amphipathic helix upon binding (Kussie et  121  Chapter 3  —  Structure of the DAXX/RASSF1C complex  al., 1996). Interestingly, Tang et al. reported a direct interaction between DAXX and Mdm2 by co-immunoprecipitation studies. This direct interaction was mediated by residues 157-260 of DAXX (Tang et al., 2006). It can be hypothesized that p53 could serve as a bridge between Mdm2 and DAXX, enhancing their direct interaction. It will be interesting to investigate this in the future. 3.6.4 Possible new DIM-containing interacting partners of DAXX 2338 as a query returned mostly proteins involved in A BLAST search using the RassflC transcription or signaling pathways. However, they have not (yet) been reported to interact directly with DAXX (Table 3.4). Of interest is the general transcription factor IIIB, which regulates RNA polymerase Ill-mediated transcription. This could provide a new mechanism by which DAXX regulates transcription independently of HDACs. It will be exciting to see whether these structural studies reveal new DAXX partners that could play an important role in the numerous functions attributed to DAXX to date.  122  Chapter 3 Table 3.4  -  —  Structure of the DAXX!RASSF1C complex  (a) BLAST search for sequences similar to 2338 RassflC .  Protein (Accession number)  Protein size Alignment with RassflC Location of possible DIMs (b)  Protein Function  Reference  38 395  Transcription initiation factor  (Teichmann etal..2000)  38 675  Scaffold protein with three PDZ domains, involved in transcriptional activation of p53.  (Tam etal., 2008)  Co-repressor of transcription factors involved in cell-cycle regulation and DNA repair.  (Papaioannou etal.. 2007)  Chromatin remodeling enzyme involved in transcription regulation.  (Lutz et al., 2006)  Transcription factor involved in the induction of genes encoding detoxifying enzymes.  (Wangetal., 2007)  419 aa. TFHLB NP 060780  23 381  —  —  SQEDSDSELEQYFTAR DENISDSEIEQYLRTP  —  —  for RNA polymerase Ill.  C-terminus.  2641 aa. PDZ domain containing protein AJPC  23 662  AAKO7 661  —  -  SQEDSDSELEQYFTAR DSLISESELSQYFAHD  —  -  Immediately after the second PDZ domain. 305 aa  Alien AAD30269  Chromodomain Helicase DNA (CHD) binding protein 6  24 18  —  —  QEDSDSE—-——LE—QYFTAR SEDSNSEPNVDLENQYYNS  -  —  N-terminus. 2715 aa. 24 487  —  QEDSDSELEQYFTAR 38 LDKKSDESLEQYFYSVA -1504 —  AA172397  Nuclear Factor (erythroid derived 2)-like 1 NPO 03195  38 36  772 aa. 24  67  —  —  QEDSDSELEQYFTAR IHPKSIDLDNYFTAR  -  —  38 81  N-terminus. (a)  Significant hits returned by BLAST (Altschul et a!., 1990) using the Rassfl C 2338 sequence as a query (top sequence) in the non-redundant-protein sequence database, and searching only human proteins.  (b)  Residues in red, blue and green are negatively-charged, positively-charged or hydrophobic, respectively.  123  Chapter 3  —  Structure of the DAXX/RASSF1C complex  3.7 Biological implications of the RassflC-DAXX interaction The molecular mechanism for the interaction between DAXX and Rassfl C presented here is /Rassfl C’ 60 ) and Lindsay et al. 220 consistent with previous studies by Kitagawa et al. (DAXX’’ ) (Kitagawa et a!., 2006; Lindsay et a!., 2009b). However, as discussed in 50 /RassflC’ 42 (DAXX’’ section 1.4.3 and summarized in Figure 1.6, the two groups proposed different functions for the DAXX/Rassfl C interaction. Kitagawa et al. suggested that the interaction resulted in sequestration of Rassfl C into PML NBs, and Lindsay et al. proposed that the interaction controlled a mitotic checkpoint in a cellcycle dependent manner. Lindsay et al. failed to reproduce Kitagawa’ s result, and DAXX and Rassf1 C did not interact during interphase, as the proteins were in the nucleus and cytoplasm, respectively. Other groups have also reported that Rassfl C is mostly located in the cytoplasm with RassflA (Estrabaud et al., 2007; Liu et a!., 2005). It is unclear at the moment what other mechanisms were involved in Kitagawa’ s study that resulted in Rassfl C nuclear localization, and DAXX-mediated sequestration into the PML-NBs during interphase. Additionally, Lindsay et al. found that the Rassfl C/DAXX interaction played an important role in the cell cycle progression. Particularly, this proved very important to understand how breast cancer cells can show resistance to taxol (a common agent used for chemotherapy). In short, upon taxol treatment, Rassfl C and DAXX interacted in taxol-sensitive cancer cells, which resulted in a quick exit from mitosis. This quick exit eventually leads to cell death due to the stabilization of microtubules and a net increase in tubulin polymerization caused by taxol. On the other hand, taxol-resistant cells were found to be DAXX-depleted. The lack of the Rassfl/DAXX complex caused cells to arrest in mitosis long enough for taxol to decay. After 124  Chapter 3  —  Structure of the DAXX/RASSF1C complex  depletion of taxol, these cancer cells exited mitosis and continued to proliferate. Interestingly, Lindsay et al. were able to convert taxol-sensitive cancer cells to taxol-resistant cells by overexpressing GFP-Rassfl C 1-50, which presumably competitively depletes DAXX and abrogates its interaction with endogenous Rassfl C. The role of DAXX in mitosis is just beginning to be investigated and a number of questions remain unanswered. The most obvious question is how the DAXX!RassflC interaction does to affect the progression of mitosis. Lindsay et al. found that RassflC dimerized with RassflA, which has been recently shown to physically interact with and become phosphorylated by the Aurora-A kinase (Rong et al., 2007; Song et al., 2009). The Aurora-A kinase has been highly conserved through evolution, and is carefully regulated (by ubiquitylation and phosphorylation) during mitosis (Fu et al., 2007). Interestingly, Lindsay et al. discovered that inhibitors for the Aurora kinase converted DAXX/RassflA-depleted taxol resistant cells to taxol-sensitive cells (Lindsay, 2009). It is unclear yet how DAXX/RassflA could inactivate the Aurora kinase. Since both proteins do not have any catalytic domain, it is fair to speculate that inactivation would be through recruitment of other complexes with enzymatic activity, or by competitive binding of the active domain of the kinase. The next question concerns the mechanisms that regulate these interactions. The observation that S TM-N and Rassfl C bind at the same site of DAXX 44 could provide a mechanism in ’ 55 which SUMO binding regulates association of the DHB domain with Rassfl C by relieving intramolecular binding of SIM-N (discussed further in Chapter 5). Indeed, the role of SUMO in mitotic checkpoints is just beginning to be uncovered (Gutierrez and Ronai, 2006). It will be  125  Chapter 3  —  Structure of the DAXX/RASSF1C complex  definitely exciting to see what further experimentation reveals in this new research area in the near future.  3.8  Materials and methods  3.8.1 Cloning Table 3.1 summarizes the constructs used for this chapter. Unless noted otherwise, all of the Rassfl C constructs were cloned via PCR amplification from a pBABE plasmid containing ° kindly provided by Dr. Alexander Ishov (University of Florida). Constructs that were 5 RassflC’ inserted into a PGEX-2T vector were cloned with BamHI and EcoRI recognition sequences. ’ and RassflC 2338 ’ were cloned using two complementary 82 The genes encoding Rassf1C DNA oligomers encoding the entire Rassfl C fragment sequence, with BamHI and EcoRI sticky ends (PAGE purified, Integrated DNA technologies, Iowa, USA). The DNA oligoniers were dissolved in deionized water to a final concentration of 200 jtM, and mixed in a ratio 1:1. 0.5 tL of this mixture were diluted in 99.5 iL of water and kept at 95 °C for 5 minutes. Then, the samples were cooled slowly to room temperature over 20 minutes. The annealed inserts were ligated into previously cleaved and dephosphorylated PGEX-2T. The Rassf1C ’’ and 2344 Rassfl ° 235 genes were cloned into PGEX-6P- 1 (which encodes a PreScission proteolysis-site C instead of thrombin site) via PCR (BamHI/EcoRI). RassflC’ ° was also inserted into a pET28a 5 vector with NdeI and Hindill restriction enzymes. 3.8.2 Sample purification 44 purification was carried out as described in Chapter 2. The purification of ’ 55 DAXX , RassflC 821 RassflC ”, and RassflC 2338 838 followed the same protocol as described in Chapter 4  126  Chapter 3  —  Structure of the DAXX/RASSF1C complex  , 50 with the GST-SIM peptides. The following sections describe the purification of RassflC’ 2350 C Rassfl ”, 234 and Rassfl . C  3.8.2.1 RassflC ° 15 -RassflC’ formed inclusion bodies. Thus, its purification followed a similar 6 His ° Expressed 5 44 in Chapter 2, although 6 M guanidinium hydrochloride ’ 55 protocol to that described for DAXX 2 binding and elution buffers. The eluted sample was purified by reversed was added to the Ni phase HPLC (0.1% TFA, water/acetonitrile gradient) to remove the salt. Preliminary tests -RassflC’ was insoluble at pH values below 8. However, after thrombin 6 His ° showed that 5 -tag, RassflC’ 6 ° became soluble at pH 6.5. Prolonged thrombin 5 treatment to remove the His treatment was found to be problematic, as MALDI-ToF-MS showed the presence of a significant . The latter results from cleaving at a pseudo-thrombin cleavage site 38 amount of RassflC’ between Arg38 and Thr39. For large scale production of ‘ N- and 13 5 N-labeled samples, HPLC-purified peptides were 5 C/’ re-suspended in Tris buffer (20 mM, 150 mM KC1, 2.5 mM CaC1 , pH 8.8) and treated with 1.5 2 units of thrombin (Novagen) for 1.5 hours. The sample pH was then adjusted to 6.5, and the lack of precipitation showed that the His -tag removal was complete. Excess DTT (10 mM final) and 6 benzamidine beads (200 iL, Sigma) was added to deactivate thrombin. The samples were then immediately re-purified by HPLC. MALDI-ToF-MS confirmed that the 5 N-RassflC’ was ‘ ° homogenous, with no RassflC’ 38 present. Unfortunately, the 5 C/’ sample contained a small 3 ‘ N amount of Rassfl C’ . 38  127  Chapter 3  —  Structure of the DAXX/RASSF1C complex  ° 235 ”’ and Rassf1C 234 3.8.2.2 Rassf1C ° were purified from a pGEX-6P-1 vector, which contains a 235 ’ and RassflC 2344 Rassf1C PreScission cleavage site between the GST-tag and the peptide. This was used to avoid the heterogeneous samples that resulted from cleavage at the pseudo-thrombin site at Arg3 8. The PreScission enzyme was kindly prepared by members of Dr. Strynadka laboratory. Samples were incubated with 200 tg of GST-tagged PreScission for 48 hours at room temperature. SDS-PAGE showed incomplete cleavage after 24 hours, and complete cleavage after 48 hours. MALDI-ToF 235 sample had a major peak at 3683 Da (expected mass 3682 Da), C MS showed that the Rassfl ° . RassflC 7 ’ 2 226 or RassflC and a smaller contaminant at 1843 Da, probably RassflC’  23-44w  showed a peak 3102 Da (expected 3100 Da) and another peak of same intensity at 2604 Da, 2342 Preliminary tests with these samples showed no significant C which corresponds to Rassfl . 55144 (Table 3.1), and thus further efforts to optimize their improvement in binding DAXX purification was unnecessary. 3.8.3 NMR-monitored titrations Table 3.1 outlines all the samples used for titrations performed in this chapter, with their respective concentrations and buffers. Concentration of the samples was determined by UV ° and 5 absorption at 280 nm using the following predicted molar absorptivities: RassflC’ ”’ (6990 M’ cm 2344 ’, and Rassf1C 2338 ); and 1 ); RassflC 1 , RassflC 821 838 (8480 M’ cm RassflC ° (1490 M’ cm’). 235 RassflC N-labeled species and increasing amounts 5 All titrations were performed with 500 tL of the ‘ of the unlabeled species until saturation was reached as determined by plotting the binding curve  128  Chapter 3  —  Structure of the DAXX!RASSF1C complex  Hon the flyH• All of the NMR-monitored titrations were in the fast-exchange regime, and the dissociation constants were calculated as described in Appendix 4. 3.8.4 Structure of the complex 3.8.4.1 Sample preparation After optimizing conditions, MOPS buffer (10 mM, pH 6.5, 0.1 mM EDTA) was employed /’ 5 C 3 ‘ -labeled ”. Specifically, N 2338 4 and RassflC DAXX 4 ’ for preparing the complex between 55 V (1.2 mM, 250 iL) was prepared and titrated with unlabeled DAXX 2338 RassflC 44 (3.4 ’ 55 4 ratio, -92% saturation was reached :RassflC 4 ’ 55 DAXX ” mM) to saturation. At a 1.5:1 2338 (Figure 3.1 2A) as determined by comparison with a previous titration (Figure 3 .4C-D). The estimated KD (25 .iM) was consistent with the result in HEPES buffer at pH 7.5 (Table 3.1). C/’ isotope-filtered NOESY-HSQC experiment of labeled RassflC 3 2D-’ N A control 5  23-38w  in  the absence of protein did not show any cross-peaks (not shown). In contrast, at 92% saturation 4 the spectra showed a considerable number of cross peaks (Figure 3.12 B-C). with 55 DAXX 4 ’  129  Chapter 3  —  Structure of the DAXX/RASSF1C complex  Preparation of DAXX ’’ complexes with complementary 2338 IRassf1C 55444 Figure 3.12 labeling for structural studies. The sections on the left (A-C) show results from the 5 C/’ 3 ‘ N ” / unlabeled DAXX 2338 RassflC 44 sample (Rd complex); whereas the sections on the right ’ 55 2338 sample (Dr complex). C illustrate results from the /unlabeled C 3 ‘ N 5 4 ’ 55 -DAXX 4 /’ Rassfl s (A,D) Titration curves show that samples at an unlabeled/labeled ratio of 1.5 were >90% saturated as estimated from the regression curves. The dissociation constant was approximately C/’ 3 2D-’ N 20 jiM. (B-C, E-F) Sections of the methyl region (C,F) or amide region (B,E) of a 5 isotope-filtered NOESY experiment. Cross peaks correspond to intermolecular NOEs. The lines illustrate the diagonal for reference. -  130  Chapter 3  —  Structure of the DAXX/RASSF1C complex  /’ C 5 C 3 ‘ N —Rassfi 2338 /’ DAXX’ C 2 ‘ 4 44  i -DAXx 44 c 3 ‘ N 5 lb. 4 liC/J Rassf1 C 233  A 0.4  Io.  0.3  <  0.2  0.  D •  I5  •  A37  • •  0.3  i1$ V125 YI2 1127  -—-  0.2  /‘  -  • __—.  0.1 0.1  M K-21 i 1 0.5  DAXX:RassflC ratio  1.5  RassflC/DAXX ratio  B  E  2.  2  E  0.  C,  O  4.  4 0  6  6 ;-  _-:-‘  .  -  7.5  7.5  C  7.0  F  --  2  2  S  0. 0.  rj,  4  6  6  .  —  1.5  ii  1.5  23 ‘H (ppm) RassfIC  1.0  DAXX ‘H (ppm)  -  131  Chapter 3  —  Structure of the DAXX/RASSF1C complex  ”’ (10.8 2338 C 3 ‘ N 5 4 ’ -DAXX 4 /’ (1.1 mM) and unlabeled Rassf1C The reversed titration with 55 mM) was carried out until reaching a similar level of saturation  (  96%) at roughly the same  iM) stoichiometric ratio as the previous titration (Figure 3.12D). Consistently, a similar KD (21 1 C/’ isotope-filtered NOESY-HSQC experiment showed a small 3 ‘ N was obtained. The 2D 5 number of cross-peaks, especially in the methyl region (Figure 3.12 E-F).  3.8.4.2 Resonance assignments C 3 ‘ N 5 ”/unlabeled -RassflC /’ DAXX 44 sample will be referred as ’ 55 For simplicity, the 2338 N-DAXX 5 C/’ 13 4 ’ 55 4 Rassf1C ’’ sample as the “Dr” 2338 the “Rd” sample, and the /unlabeled sample. 44 were assigned using the following experiments on the Dr ’ 55 Resonances from DAXX complex:  HCCH-TOCSY,  (H)CC(CO)NH-TOCSY,  HCC(CO)NH-TOCSY,  (HB)CB(CGCD)HD, (HB)CB(CGCDCE)HE, CT-HSQC, aromatic HCCH-TOCSY, ‘ C- and 3 N-NOESY-HSQC (see section 2.4.1). Assignments for the apo-protein were used as a starting 5 ‘ point since only resonances from residues near the binding site showed any significant changes. Rassf1C were assigned using the following experiments on the Rd v Resonances from 2338 complex:  (H)CC(CO)NH-TOCSY,  HCC(CO)NH-TOCSY,  (HB)CB(CGCD)HD,  (HB)CB(CGCD)HD-TOCSY, CT-HSQC, TOCSY-HSQC, ‘ C- and ‘ 3 N-NOESY-HSQC. Amide 5 resonances on the free peptide were obtained from HNCACB and (HB)CBCA(CO)NH experiments and were transferred to the complex following their shifts on the fast exchange titration. From these spectra, 96% of all non-labile nuclei in the complex were assigned (Appendices 8 and 9).  132  Chapter 3  —  Structure of the DAXX/RASSF1C complex  3.8.43 Structure calculation C/’ isotope-filtered 3 ‘ N N-NOESY-HSQC, and 5 5 C- and ‘ 3 Simultaneous three-dimensional ‘ NOESY-HSQC spectra for the Rd and the Dr samples (with 100 ms and 150 ms mixing times, N HSQC spectra before and after the 15 respectively) were collected over two weeks. Control ‘Hthree-dimensional experiments showed that the samples were stable. All spectra were processed with NMRPipe and analyzed with Sparky (Delaglio et a!., 1995; Goddard and Kneller, 2004). Peaks in the eight spectra were manually picked and partially assigned. For automatic NOE assignments and structural calculations, ARIA 2.2 was used (Habeck et al., 2004), specifying that filtered experiments contained intermolecular NOEs. Chemical shift tolerances are an important parameter in automatic NOE assignments. If they are too narrow, ARIA will likely miss the correct assignment for an NOE, and if they are too wide, too many possibilities allow the program discard the peak early in the calculation (Fossi et al., 2005). Thus, relatively wide tolerances (0.08-0.15 ppm) for the filtered experiments were used for the initial run. The initial structure was used to manually fix violations and further assign peaks. This process was repeated eight times, until no violations> 0.5  A were used for the calculation. The  majority of long range intramolecular NOEs were automatically assigned, whereas all of the intermolecular NOEs were manually assigned. The quality of the final ensemble was analyzed with Pro check_NMR and by its statistics (Table 3.2)  133  Chapter 4  SUMO-interacting motifs (SIM) in DAXX In this chapter, the identification of two SUMO-interacting motifs in DAXX, one at its Nterminus (SIM-N) and one at its C-terminus (SIM-C) is presented. Their interactions with SUMO- 1 and SUMO-2 were examined using NMR spectroscopy, and the following properties discovered. (1) Binding was mediated by both hydrophobic and electrostatic interactions. (2) SIM-N had a 4 fold higher affinity towards both SUMO paralogues than SIM-C. The difference in affinity is attributed to the difference in charged residues that flank their identical hydrophobic core sequences. (3) SIM-N bound SUMO-1 with one predominant orientation, whereas SIM-C adopted multiple binding modes. (4) Both SIMs were found to bind SUMO-1 and SUMO-2 with similar affinities under most conditions. In addition SIM-N was found to bind intramolecularly to the DHB domain and SIM-C to mediate the interaction of DAXX with the sumoylated Ets-1 transcription factor. Importantly, the latter did not involve any direct contacts between DAXX and Ets- 1, but rather derived from the non-covalent binding of the DAXX SIM-C to SUMO- 1, which in turn was covalently linked via an isopeptide bond in a Theads-on-a-string’ fashion to the unstructured N-terminal segment of Ets- 1. These results provide new insights into the binding mechanisms and hence biological roles of SUMO-interacting motifs.  134  Chapter 4— SUMO interacting motifs in DAXX 4.1  DAXX UMO-interacting motifs (SIM-N and SIM-C)  4.1.1 Identification of SIM-N and SIM-C As discussed in Chapter 1, DAXX has been reported to bind and repress a number of transcription factors, many of which are sumoylated (Lindsay et a!., 2008; Shih et al., 2007). For example, the transcription factor Smad4 is repressed by DAXX, and mutations that prevented Smad4 from being sumoylated completely relieve this effect (Chang et al., 2005). The hypothesis is that DAXX is able to bind sumoylated proteins via possible SIMs. In order to identify the SIMs within DAXX, the consensus sequence established by Song et al. (Val/Ile-X-Val/Ile Val/Ile) was searched (Song et a!., 2004). Close inspection of the DAXX sequence allowed the identification of two segments that contain such consensus motifs, one at the N-terminus (denoted herein as SIM-N) and one at the C-terminus (SIM-C) (Figure 2.2). Indeed, while this work was in progress, two groups independently identified these SIMs and confirmed their ability to mediate binding of DAXX to SUMO. Lin et a!. first reported the presence of a SIM-C within DAXX (Lin et a!., 2006). They used a yeast two-hybrid system to show that deletion of residues 73 2-740 or mutations within the hydrophobic core of the SIM 11VL 733 impaired the binding of DAXX to SUMO-l. ( ) 736 Moreover, the presence of SIM-C in DAXX was crucial for its ability to be targeted to PML bodies and to repress several sumoylated transcription factors, including Smad4 and AR. More recently, Santiago et al. reported the existence of SIM-N (Santiago et al., 2009). In their study, SIM-N was shown to bind both SUMO-l and SUMO-2/3 by a yeast two-hybrid system. Indeed, the authors observed that it was necessary to remove both SIM-N and SIM-C from DAXX to completely abrogate its SUMO binding capacity. This stands in contrast to the 135  Chapter 4— SUMO interacting motifs in DAXX work of Lin et al. in which drastic changes in the function of DAXX occurred upon disruption of only SIM-C (with SIM-N being unrecognized at the time). One interesting observation mentioned by Santiago et al. is that DAXX is able to bind SUMO-1, SUMO-2 and SUMO-3 through either of its SIMs, but not SUMO-4. They speculated that the presumed inability of SUMO-4 to be covalently attached to substrates accounts for their result, i.e. that DAXX cannot bind free SUMO, but it rather associates preferably with sumoylated proteins. 4.1.2 Role of DAXX SIMs in its localization As described in Chapter 1, DAXX localization is crucial to its function. In the studies described above, both groups showed that recruitment of DAXX into PML-NBs depends greatly on the ability of DAXX to bind sumoylated PML (Lin et a!., 2006; Santiago et al., 2009). A group in France initially identified Lys 160 as the sumoylation site in PML that controls DAXX recruitment (Zhu et al., 2005). More recently, the same group reported that PML is primarily isopeptide-linked to SUMO-2 at this lysine, with SUMO-1 being added at Lys65 or Lys490. (Lallemand-Breitenbach et al., 2008). Therefore, paralogue selectivity from each SIM might have biological importance in determining the localization of DAXX. 4.1.3 Research objectives The proposed model of Santiago et a!. in which DAXX SIMs cannot bind free SUMO is at odds with various reports in which SIMs of other proteins have bound free SUMO (Duda et al., 2007; Hecker et al., 2006; Sekiyama et al., 2008; Song et a!., 2005). The first objective was to determine the functionality of DAXX SIMs. Can they bind free SUMO? Is this binding independent of the protein to which SUMO is covalently linked?  136  Chapter 4— SUMO interacting motifs in DAXX It is peculiar that DAXX possesses a SIM at each of its termini. It is tempting to speculate that they might have different functions. Indeed, a recent report used a yeast-two hybrid assay to suggest that the SIM-N and SIM-C have preference towards SUMO-2 and SUMO-1, respectively (Chen et a!., 2006). Since it is established now that the specific SUMO paralogue on a protein can have distinct functions (discussed in section 1.3.4), the second objective was to determine the relative affinities of DAXX SIM-N and SIM-C towards SUMO-1 and SUMO-2. What are the structural and thermodynamic bases for differences in affinity/specificity of the SUMO paralogues? Section 4.2 explores these two questions. The available structures of SUMO-l with SIMs show two possible binding modes: one with a SIM adopting a 13—strand conformation to from a short parallel 13—sheet with strand 2 of SUMO-1 (Song et a!., 2005), and one at the same site but with an antiparallel orientation (Reverter and Lima, 2006) (Figure 4.5A). The third objective was to determine the binding mode of SUMO-1 and SUMO-2 with the two DAXX SIMs. Which binding mode do DAXX SIMs adopt to bind SUMO? In sections 4.3 and 4.4 an answer to this question is found. It is desirable to expand the knowledge of the biological function of SIM-N and SIM-C. SIMs can be involved in regulation of protein sumoylation, conformation, localization and interaction (discussed in section 1.3.4) (Kerscher, 2007). Until now all SIMs reported in the literature are specific for SUMO-binding. The fourth objective was to resolve whether DAXX SIMs can only bind SUMO or whether they are also involved in other mechanisms. Section 4.5 discusses how SIM-N is involved in intramolecular binding with the DHB domain, which opens up possibilities of a new regulatory function of SIMs involving competitive binding.  137  Chapter 4— SUMO interacting motifs in DAXX Finally, it has been reported that DAXX repression is mediated by DAXX SIMs (Lin et a!., 2006; Shih et a!., 2007). The last objective was to investigate the role of DAXX SIMs in mediating the interaction with transcription factors. Is there any cooperative binding involving other regions of DAXX with the model transcription factor Ets- 1? Section 4.6 investigates how surnoylation of the transcription factor Ets-l affects its ability to bind DAXX.  4.2  NMR-monitored titrations of DAXX-SIM-N and SIM-C with SUMO-1 and SUMO-2  4.2.1 Preamb1e SIM binding by SUMO-1 and SUMO-2 SUMO-l and SUMO-2 share 50% sequence identity and adopt highly similar ubiquitin-like structures (Figure 4.1). Not surprisingly, each utilizes the analogous cleft to bind SIMs (Hecker et al., 2006). This cleft, which is between the strand 2 and helix 1, consists mostly of hydrophobic residues surrounded by positive charges from a number of Lys and Arg residues (Figure 4.1). This “key lock” matches the complementary negative charges from Glu and Asp residues within SIMs. However, the exact position of the Lys/Arg residues does differ between SUMO-l and SUMO-2, suggesting that electrostatic interactions could dictate a paralogue preference towards various SIMs. Indeed, one of the first SIMs that showed higher affinity towards SUMO-2 could be converted to binding SUMO-1 preferably by changing the charges flanking the hydrophobic core of the SIM (TTRAP, Figure 4.1 C) (Hecker et a!., 2006). However, a recent study suggested that the SIM hydrophobic residues alone can determine paralogue specificity (USP25, Figure 4.1C) (Meulmeester et al., 2008). Interestingly, the SIM used for that particular case contained an unusual Val-Ile-Asp-Leu core, so presumably electrostatics still played a role in determining binding specificity. 138  Chapter 4— SUMO interacting motifs in DAXX  Figure 4.1 Differences between SUMO-1 and SUMO-2. (A) Sequence alignment of SUMO 1 and SUMO-2. Identical and similar residues are highlighted in black and gray, respectively. Secondary structure elements are shown as arrows (13-strands) and cylinders (ct-helices). (B) Comparison of the surfaces of SUMO-1 (1A5R.pdb) and SUMO-2 (1WM2.pdb), showing the side containing the SIM binding cleft (dashed circle). Positively-charged residues are shown in blue, negatively-charged in red, and non-polar residues in gray (electrostatic surface calculated with the “simplecharge” method of MOLMOL using default parameters). Cartoon representations of the molecules are shown for reference. (C) Sequence alignment of SIMs reported to have SUMO-paralogue specificity (top) and selected SIMs that showed no SUMO paralogue preference (middle). The sequence of the studied SIM-N and SIM-C peptides (bottom). The sequence of the human SIM-C, identical to the murine version in its SIM, is shown for reference, although it was not used for these studies (gray). The hydrophobic core is shown in green, the Asp/Glu residues in red. -  139  _____  ____  Chapter 4— SUMO interacting motifs in DAXX A  10  30  20  50  40  132  31  I  ccl  S UMO- 1 SUMO- 2 AIEKPIE  SQPI  131 1 60  70  133 I  30  ccl 40  50  90  135  •PN  134  f33  Øii  132  20 80  ci2  SUMO-1 NS SUMO-2 RQ  a2 70  60  B  Ø11  10  80  SUMO-1  90  SUMO-2  C Protein RANBP2 TTRAP wr TTRAP mutant USP25 U5P25 MCAF1  ELM PIAS1 SENAThXIN SP100 TOPORS ZNF237 hDAXX SIM-N mDAXX SIM-C hrAXX SIM-C  Sequence Preference [.]DNEKEC Cliv WEKKPTVEK?U<ADTLKLP [. SUMO-1 RCGGLPNN I 11W WEFLGKPKHCQYTWDTQ SUMO-2 RCCGLPNN 111W WEFEDEPKHCQYTWDT SUMO-1 GSQADTN VIDL TGDDRDDL SUMO-3 TN VIDL T SUMO-3 G VIOL TMDDEE SUMO-3 SSESE QIDL TEEQKDDSEWLSSD VC1 DDGPI SUMO-2  KNKK SRGQ QASD RSPV 0000000  VEVI VIII IIVI VITI  DLTIOSSSDEEEEEP SDSODDDDE SSEOSEGSTDVD OSDSDKDSEVKGD  vvFI ESI  MATS IIVL DDDDEDEAR 19 718 IYKTSVATQCDPEE IIVL SDSD 739 719 TCITS’JEE IIVL SDSD 740  140  SUMO-1/2 SUMO-1/2 SUMO-1/2 StJMO-1/2 SUMO—1/2 SUMO- 1 / 2 SUMO-1 or SUMO-1/2  Reference Tatham et al. 2005 Hecker et al. 2006 Hecker et al. 2006 Meulmeester et al. 2008 Meulmeester et al. 2008 Sekiyama et al. 2008 Zhu et al. 2008 Hecker Hecker Hecker Recker Recker  et et et et et  al. al. a1. al. al.  2006 2006 2006 2006 2006  Chapter 4— SUMO interacting motifs in DAXX In fact, all of the peptides reported to bind preferentially to SUMO-2 contain an Asp at the third position of the V/I-V/I-X-V/I SIM (MCAF 1, TTRAP, USP25 and BLM Figure 4.1 C) (Hecker et al., 2006; Meulmeester et a!., 2008; Sekiyama et al., 2008; Tatham et a!., 2005; Zhu et al., 2008). Only the section containing the so-called 1R2 (internal repeat 2) of RANBP2 and the mutant TTRAP have been reported to bind preferentially to SUMO-1 in vitro (Figure 4.1C) (Hecker et al., 2006; Tatham et al., 2005). Overall, SIMs reported to show specificity for a particular paralogue have the negativelycharged residues mixed with neutral amino acids. In contrast, most known SIMs have Asp/Glu stretches mixed only with Ser residues, and show no paralogue predilection (selected examples from (Reeker et a!., 2006) on Figure 4.1C). Thus, SIMs with apparent paralogue specificity have one of two characteristics: an asymmetric hydrophobic core containing a charged residue; and/or juxtaposed charged residues mixed with non-polar residues. In contrast, most SIMs with canonical hydrophobic cores and contiguous juxtaposed charged residues have no apparent paralogue specificity. It is interesting that both SIMs from DAXX contain the identical hydrophobic core Ile-Ile Val-Leu, which is rather symmetric and non-polar. Thus, any specificity would have to reside on the juxtaposing charged residues. SIM-N has a stretch of seven Asp/Glu residues following the hydrophobic core (Figure 4.1 C), which makes it an unlike candidate for exhibiting paralogue specificity. SIM-C, on the other hand, has much shorter Asp/Glu stretches on both sides of the hydrophobic core, mixed with only a Pro residue on one side and two Ser residues on the other (Figure 2.1). SIM-C might be a better candidate for having paralogue specificity. To address this question, NMR spectroscopy was used to characterize the structural and thermodynamic bases  141  Chapter 4  —  SUMO interacting motifs in DAXX  for the interaction of isolated peptides containing the DAXX SIM-N and SIM-C with purified SUMO-1 and SUMO-2. 4.2.2 NMR characterization of SIM-N, SIM-C, SUMO-1 and SUMO-2 An expression system to obtain 2O aa peptides encompassing SIM-N (residues 1-19 of hDAXX) and SIM-C (residues 718-739 of mDAXX, identical in its SIM to hDAXX) was N HSQC spectra of 5 designed (Figure 4.1C, see materials and methods for details). The ‘H-’ labeled SIM-N and SIM-C show little dispersion in the amide hydrogen dimension, indicating the lack of any predominant secondary structure (Appendix 3). Using HNCACB and C, and ‘ 3 CBCA(CO)NH experiments (Figure 2.5A), the signals from the backbone ‘H, ‘ N 5 nuclei of both peptides was assigned. Similarly, the assignments of SUMO- 1 and SUMO-2, reported previously in the literature, were mostly confirmed, although some errors in the literature assignments were detected and corrected (Appendix 3). 4.2.3 NMR-monitored interactions b The interactions of the DAXX SIM peptides and the SUMO paralogues were monitored by following changes in ‘H-’ N-labeled species upon titration with the 5 N HSQC spectra of each ‘ 5 unlabeled species. Importantly, both isolated SIMs bound both purified SUMOs in vitro as shown by spectral changes. Chemical shifts are exquisitely sensitive to structural perturbations accompanying ligand binding. Although interpreting these shift changes in terms of explicit direct or indirect structural changes is difficult, a qualitative, low resolution view of the binding  b To calculate the concentration of the samples via UV-spectroscopy, samples used for NMR-monitored titrations had a non-native Trp residue at either the N-terminus (SIM-C”, SUMO1” and SUMO2v) or at the C terminus (SUMO-N”). These constructs are marked with a superscript “w”. Control experiments with SUMO 1 !SIM-C (without Trp) showed identical results to SUMO- 1 w/sIM..cw experiments (not shown), indicating that the non-native Trp did not interfere with binding. See materials and methods for details.  142  Chapter 4— SUMO interacting motifs in DAXX interface can be obtained by mapping residues showing the largest spectral perturbations onto the sequence or structure of the protein. 4.2.3.1 Mapping of the binding interface Chemical shift perturbations confirm that the binding of SIM-N”’ and SIM-C”’ occurred at the cleft between strand 2 and helix 1 of both SUMOs (Figure 4.2). Conversely, the hydrophobic  core, Ile-Ile-Val-Leu, of SIM-N” and SIM-C” showed the greatest chemical shift perturbations when bound to SUMO-1”’ and SUMO-2’’. This is in agreement with reported structures of SUMO/SIM complexes (Duda et a!., 2007; Hecker et a!., 2006; Sekiyama et a!., 2008; Song et al., 2005). In one particular titration, SUMO-l”/SIM-C”, peaks broadened beyond detection both on SUMO-l and SIM-C (Figure 4.2C, 4.2F). As explained in section 4.3, this appears to be due to conformation exchange between multiple binding orientations.  4.2.3.2 SIM-N binding to SUMO-1 and SUMO-2 Upon titration with unlabeled SIM-N’’, the  N 5 lHNl  signals from many amides in ‘ N-labeled 5  SUMO-1’ and SUMO-2’’ showed small shift changes and progressive loss of intensity with the concomitant appearance of new peaks arising from the resulting complex (no added KCI, 17°C). This behavior is indicative of relatively high affinity binding at the edge of the slow exchange regime (kex < Av) on the chemical shift timescale (see Appendix 4 for an explanation on titration curves). For those amides showing fast exchange behavior (i.e. with small chemical shift changes M changes so kex  >  Av, Appendix 6), plots of A versus SIM concentration indicated essentially  stoichiometric binding under the experimental conditions and hence KD Appendix 5).  143  <  5tM (Table 4.1;  Chapter 4— SUMO interacting motifs in DAXX  Figure 4.2  -  Mapping of the binding interface of SUMO-1 and SUMO-2 with SIM-N and  SIM-C. (A,C) Amide chemical shift perturbations (calculated as  ,J(AoH)2  + (O.2A615N) ) 2  of  SUMO-l”’ and (B,D) SUMO-2” upon binding SIM-N” (A,B) and SIMCv (C,D). Also shown are SIM-N’ ” (E,G) and SIM-C” (F,H) amide chemical shift changes upon binding SUMO-1’ 1 (E,F) and SUMO2w (G,H). The signals of residues marked as * were broadened beyond detection in the complex. (I) Residues with chemical shift changes greater than 0.2 ppm upon binding SIM-N and SIM-C are shown in red on the cartoon structures of SUMO-l (1A5R.pdb) and SUMO-2 (1WM2.pdb). Arg39 of SUMO-1 broadened beyond detection in the complex with SIM-C (blue).  144  Chapter 4— SUMO interacting motifs in DAXX SUMO-1”  SUMO-2”  S  ÷  CM  0.5  C  D 0.6  0.4  &  + CM  0.3  0.4  0.2  0.1  0.2  JJIJ 20  40  60  80  SIM-N’  SIM-C” 0.6  0.8 S <  F +  0.6  0.4  9  0.4 0.2  0.2  -  _II:*II  ---  I  YKTSVATQCDPEEI IVLSDSD 0.6  0.8 S .  0.6  H + CM  0.4  C  • 0.4 0.2  0.2  719  I  724  729  Residue  Residue  SUMO-1  SUMO-2  +SIM-N  -i-SIM-C  ÷SIM-N 145  734  739  ÷SIM-C  Chapter 4— SUMO interacting motifs in DAXX As described above, it was hypothesized that the binding would be dependent on the overall sample ionic strength (salt concentration) due to the electrostatic contribution of the SIM-SUMO interactions. Indeed, as the KC1 concentration was increased from 0 mM to 100 mM to 200 mM, the binding progressively weakened in all four cases (Figure 4.3). This allowed the determination of KD values from the dependence of Aö on added ligand concentration (for details see Appendix 4). This result confirms the importance of the electrostatic contributions to SIM SUMO binding. Indeed, in a separate experiment, weak binding of a SIM-N mutant lacking completely the hydrophobic core was observed (section 4.4), showing that electrostatic interactions are sufficient for binding at very low ionic strengths. Inspection of Table 4.1 reveals that SIM-N” bound to SUMO-1”’ and SUMO-2”’ with similar affinity at all tested salt concentrations. Thus SIM-N” did not display paralogue specificity between SUMO-1 and SUMO-2. This is attributed to the symmetric composition of the hydrophobic core of SIM-N, together with the lack of non-polar residues on the acidic stretch of SIM-N (Figure 4.1C).  4.2.3.3 SIM-C binding to SUMO-1 and SUMO-2 The NMR-monitored titrations of 15 N-labeled SIM-C’ with unlabeled SUMO-1’’ and SUMO-2” showed ‘3-4 fold weaker binding than observed with SIMNv under similar conditions (100 and 200 mM KC1; Table 4.1). The signals of the hydrophobic core, IIVL, also exhibited significant broadening due to conformational exchange (section 4.3), which progressively worsened at higher salt concentrations (Figure 4.4). (For a detailed discussion on  146  Chapter 4— SUMO interacting motifs in DAXX N SUMO-1’ 5 ‘  SIM-N”  +  N SUMO-2” 5 ‘  A  B  +  SIM-N  — -  • ,v•  123 124  E13  L24  124  F87 F64  K37 126 R70  125  z  2  K1l  M59  128 E89  126  H43  127 8.9  8.8  N SIM-N’ 5 ‘  8.7  8.7  8.6  SUMO1v  +  8.5  8.3  8.4  N SIM-N” 5 ‘  +  8.2  SUMO-2’  1224 1116  17  Eli  1116  Ml  0  l24  A19  A 19 A1870  2  z  E17  Ml  A18’b  A2  126  018  2  -  128.  OJ 3 l  122E  ---  1116  E17  17  17  1)16 Elf  0  Ml  Ml  124J  A19  A19  0  A18,  B 0 A2  126  A40 -  -  A2  18  0  A4  0  C.)  2  V9i  V9  OLIO  128  130 8.6  8.4  8.2  8.0  ‘H (ppm)  ‘H (ppm)  Figure 4.3 NMR-monitored titrations demonstrated that SIMNw bound both SUMO1w and SUMO2w. (A-B) Superimposition of a section of the ‘H-’ N HSQC spectra of SUMO-1’ 5 and SUMO-2’ in the absence (red) and presence of increasing amounts (cyan to black) of unlabeled SIM-N” at 0 mM KC1. (C-F) Superimposition of a section of the ‘H-’ N HSQC 5 spectra of SIM-N” in the absence (red) and presence of increasing amounts (cyan to black) of unlabeled SUMO-l” and SUMO-2’” at 100 mM and 200 mM KC1. -  147  Chapter 4— SUMO interacting motifs in DAXX  SIMCv +  SUMO-1”  +  B 127  SUMO2sv  V734  ,1733  D739  D739 128 129i,  /  Z  C  V734  1733  1733 D739  D739  °A724  ‘  128  E ((4  .j  127  I  /  A724  _-‘  c-)  c-)  0  L735  %  129  @  E  V734  127  I  L735  128  F  1733  0  V734 1733  o  D739  A724  c.) D’7  129  E C  8.6  8.4  8.2  8.0  8.6  ‘H (ppm)  8.4  8.2  8.0  ‘H (ppm)  Figure 4.4 NMR-monitored titrations demonstrated that SIM-C’’ bound both SUMO-1’’ and SUMO-2’’. Superimposition of a section of the ‘H-’ N HSQC spectra of SIM-C” in the 5 absence (red) and presence of increasing amounts (cyan to black) of unlabeled SUMO- 1” and SUMO-2” at O mM, 100 mM and 200 mM KC1. -  148  Chapter 4— SUMO interacting motifs in DAXX lineshape changes due to ligand binding see Appendix 6). Furthermore, at 0 mM salt, SIM-C exhibits —3 fold higher affinity towards SUMO- 1’’ than SUMO-2’. This preference is reduced at 100 mM and 200 mM salt concentrations, enforcing the hypothesis that charges on the SIMs are crucial in determining paralogue specificity (Table 4.1). Dissociation constants between SIMNw and SIM-C’’ with SUMO1w and Table 4.1 suMo2i.a -  [KCI] (mM)  SIM-N’’ KD ( iM) 1 SUMO-2’’ SUMO-1’’  SIM-C’’ KD (jiM) SUMO1v SUMO2w  0  Stoichiometric (<5)  Stoichiometric (<5)  4.5±1.4  16±7  100 200  8.1± 1.7 36±3  8.7±0.9 38±6  30±7 110±14  24±6 170±24  a pH 6.5, and 25 °C. Reported KD values and errors correspond to the mean and standard  deviation of the individually fit titration curves of at least 3 residues. 4.3  Comparison with values from the literature Table 4.2 summarizes the dissociation constants between SUMO and SIMs in the literature.  At low ionic strength (20 mM phosphate buffer), the reported KD values are all between 1-10  jiM, which is similar to the KD values of DAXX SIMs (10 mM phosphate buffer,  <  5 jiM for  SIM-N’, 5-15 jiM for SIM-C”). Only Sekiyama et al. studied KD, values as a function of ionic  strength, and did not interpret their data as showing any significant changes in the KD values (Table 4.2) (Sekiyama et a!., 2008). This is surprising, since the SIM peptide in that study (G  TMDDEE) contained a stretch of charged residues, which when they mutated,  dramatically disrupted binding to both SUMO-1 and SUMO-3. Closer inspection of their ITC binding curves reveals that the data became progressively noisier at higher salt concentrations (due to smaller heat release), and thus their reported KD values carry a greater error. Indeed, at 300 mM NaCl, SUMO-3 did not show any significant binding to the MCAF1 peptide (Sekiyama  149  Chapter 4— SUMO interacting motifs in DAXX et al., 2008). Thus, despite their interpretation, ionic strength seemed to play a significant role in the interaction affinities. As summarized in the previous section, NMR-monitored titrations allowed a more accurate measurement of KD values as a function of ionic strength. In contrast to the previous study, the progressive weakening of the interaction between DAXX SIMs and SUMO- 1 and SUMO-2 at higher salt concentrations (Table 4.1) is consistent with electrostatic forces playing a major role in SUMO/SIM interactions.  Table 4.2  (a) (b)  (c) (d)  -  Summary of reported dissociation constants of SIMs/SUMO complexes.  KD  SUMO paralogue  SIM  SUMO-1  PIASX-Pa  7  20 mM Tris, pH 7.6, 30 C  SUMO-1  PIASX-N  9  20 mM Tris, pH 7.6, 30 °C  SUMO-3  PIASX-N  5  20 mM Tris, pH 7.6, 30 C  SUMO-1  PIASX-Pb  6  20 mM phosphate, pH 7.4, 30 °C  SUMO-1  MCAFI  14  20 mM phosphate, pH 6.8, 25 °C  SUMO-1  MCAF1  12(c)  20 mM phosphate, pH 6.8, 25 °C  Reference  Buffer  150 mMNaC1  SUMO-1  MCAF1  8(c)  20 mM phosphate, pH 6.8, 25 °C  SUMO-3  MCAF1  1  20 mM phosphate, pH 6.8, 25 °C  SUMO-3  MCAF1  1(c)  SUMO-3  MCAF1  N.D.  20 mM phosphate, pH 6.8, 25 °C 150 mMNaC1 20 mM phosphate, pH 6.8, 25 °C 300 mM NaC1  300 mM NaCI  d  (Song et a!., 2004) (Song et a!., 2004) ADDIN 2004) (Song et a!., 2005) (Sekiyarna et al., 2008) (Sekiyama et a!., 2008) (Sekiyama et al., 2008) (Sekiyama et a!., 2008) (Sekiyarna et al., 2008) (Sekiyama et al., 2008)  All reported dissociation constants were measured with ITC. PIASX-Pa: KVDVIDLTIESSSDEEEEDPPAKR; PIASX-N: VDVIDLTIE; PIASX-Pb: KVDVIDLTIE MCAF-1: GVIDLTMDDEE  Noisy binding curve. Not determined due to no significant heat release.  150  (a)  Chapter 4— SUMO interacting motifs in DAXX  4.4 Two orientation binding of DAXX SIM-C to SUMO-1 To date, five structures of SIMs with SUMO-l have been reported (Baba et al., 2005; Duda et a!., 2007; Reverter and Lima, 2005; Sekiyama et al., 2008; Song et a!., 2005). In all cases, the SIM adopts a n-strand conformation, pairing with strand 2 of SUMO-1. However, this pairing can either be in a parallel or anti-parallel orientation (Reverter and Lima, 2006; Song et al., 2005) (Figure 4.5A). It has been suggested that the juxtaposition of charged groups relative to the hydrophobic core is responsible for determining the binding mode of a given SIM (Kerscher, 2007). However, SIM-C contains acidic residues on both sides of a rather symmetrical hydrophobic core (Figure 4.1 C), thus precluding an obvious prediction of its binding mode with SUMO-1. One way of answering this question is to measure intermolecular NOEs between SIM-C complexed with SUMO-1, as these would be very different in each binding mode. However, N5 N-HSQC spectrum of ‘ 5 upon titration with unlabeled SIM-C”, several amide signals in the ‘ labeled SUMO-1’ surprisingly broadened beyond detection upon reaching saturation (Figures N-labeled SIM-C” 5 4.2C,4.2F). The same behavior occurred during the reciprocal titration of ‘ with unlabeled SUMO-1’’. This of course precluded the measurement of the desired intermolecular NOEs.  151  Chapter 4— SUMO interacting motifs in DAXX  Figure 4.5 SIM-C bound SUMO-1 in both parallel and anti-parallel orientations. (A) SUMO-l is known to bind SIMs either in a parallel (left, SUMO-1:PIASx, 2ASQ.pdb) or an anti-parallel orientation (right, SUMO-1 :RanBP2,1Z5S.pdb). It has been proposed that the charged residues juxtaposed with the hydrophobic core of SIMs are involved in dictating the orientation. (B) Three unlabeled peptides with a cysteine residue at a different position were TEMPO nitroxide spin labeled. (C) PREs are measured as the spectral differences for a sample in the presence of the paramagnetic nitroxide and the reduced, diamagnetic hydroxylamine. (B) OC (left). Upon reduction of 74 N HSQC spectrum of SUMO-1 saturated with TEMPOSIMC 5 ‘H-’ the spin label, peaks that had been broadened beyond detection re-appeared (right, red boxes). HN transverse relaxation rate, AR 2 (> 30 s (E) Residues that showed the greatest PRE change in 1 ‘,blue) or that re-appear (AR > 100 s 2 , red) upon reduction of the spin label from TEMPO-SIM 1 oc are located on both sides of the SUMO-1 74 sc and TEMPOSIMC 73 C, TEMPOSIMCs sc, from which triangulation of the PRE 73 binding surface. (F,G) AR 2 for TEMPOSIMCs derived distances between the spin label (balls) and individual amides (G1u33, G1n55, Met59, green; Val38, Leu44, Leu47, red) show that the peptide bound SUMO-l in two possible orientations. Similar results were obtained with the other two spin-labeled SIM-C peptides (see Appendix 10 for details on the calculations). -  152  Chapter 4— SUMO interacting motifs in DAXX A  +  PIASx  +  RANBP2  B WT (C727) 740C S738C  GSIYKTSVATQCDPEEIIVLSDSD GSIYKTSVATQADPEEIIVLSDSDC GSIYKTSVATQADPEEIIVLSDCD  C NH  NH  -r >  II&--CH.- S—-S--  H ‘  /  E )  D ((p)  121  -  3/  ir  122  ‘  123  124  8.7  8.6  8.58.4  78  ‘H (ppm)  -  5  ‘H (ppm)  +TEMPOSIM-C  F  +TEMPO- +TEMPO oc S1MC 74 SIMC c 738  G  80 60 40 20  I  I ii 20  •N().  i 40  1,1  I I 60 Residue  80  153  Chapter 4— SUMO interacting motifs in DAXX It was hypothesized that msec-Ilsec timescale conformational exchange between 2 or more binding modes within the SIM-C/SUMO-1 complex led to the observed spectral behavior. To confirm this hypothesis, paramagnetic relaxation enhancement (PRE) measurements using a nitroxide spin label placed on one end or the other of the SIM-C peptide were undertaken. PRE originates from a dipolar interactions between an unpaired electron of a paramagnetic center (spin label) and the nucleus of an observed ‘H (Su and Otting, 2009). Similar to the NOE, a PRE is proportional to the inverse sixth power of the distance between ). However, due to the large magnetic moment of the 6 the spin label and detected nucleus (r unpaired electron, the effect is measureable over distances much larger than possible for a NOE (up to 20  A vs. -5 A) (Clore et al., 2007). A wild-type cysteine residue (Cys727) was used to  place a TEMPO nitroxide spin label N-terminal to SIM-C. This residue was also mutated to an Ala and added a new single cysteine at the C-terminus of SIM-C at either position 738 or 740 to place the spin label on the other side of SIM-C (Figure 4.5B). N-HSQC spectra of ‘ 5 N-labeled 5 PREs were measured qualitatively and quantitatively via ‘ SUMO- 1 titrated separately with the three SIM-C derivatives in their paramagnetic nitroxide forms and then subsequently reduced to their diamagnetic hydroxylamine forms (Figure 4.5 C). As expected, upon reaching saturation, a number of peaks in the spectra of SUMO-1 were broadened (often beyond detection) due to the bound nitroxide, yet reappeared upon addition of reducing agent (Figure 4.5D). Qualitatively, SUMO- 1 amides experiencing the largest intensity changes were closest to the SIM-C-binding site. However, recall that several amides are undetectable in the unmodified SIM-C/SUMO-1 complex, thus complicating the analysis of these spectra.  154  Chapter 4— SUMO interacting motifs in DAXX To extract distance restraints necessary for positioning the spin labeled SIM-C relative to , of corresponding amide 2 SUMO- 1, the differences in transverse relaxation rates, z\R  11 1 N  nuclei  in the presence of the label in its paramagnetic and diamagnetic states were also measured 2 values (and calculated distances), it is clear that SIM-C (Figure 4.5 F). Based on these PRE R must be bound in both parallel and anti-parallel modes in order to account for strong PRE effects on both ends of the SIM-binding site of SUMO-1 (Figure 4.5E,G). This result strongly suggests that the broadening observed in the titration is due to conformational exchange between two detectable binding modes that inter-converted on the chemical shift time scale (Appendix 6). This agrees with the model in which the juxtaposing acidic residues determine the binding mode of the peptide. When they are asymmetrically placed with respect to the rather symmetric hydrophobic core of the DAXX SIMs, the peptide binds in one mode (see for example SIM-N, next section). When they are symmetrically placed, the peptide can adopt two bound orientations.  4.5 Determination of the binding mode of DAXX SIM-N with SUMO-1 In contrast to SIM-C’’, SIM-N” bound SUMO-1’’ and SUMO-2” with higher affinity and without exchange broadening effects under saturating conditions. It was thus hypothesized that, consistent with its asymmetric charge distribution, SIM-N” binds SUMO in only one preferential orientation. To verify this hypothesis, 5 C/’ filtered-edited NOESY-HSQC experiments were 3 ‘ N used to measure intermolecular NOEs between unlabeled SIM-N’’ and labeled SUMO-1 and unlabeled SIM-N’’ (Table 4.3).  155  Chapter 4— SUMO interacting motifs in DAXX  N labeled SUMO-1. 5 C/’ Table 4.3 Intermolecular NOEs between unlabeled SIM-N and 13 -  unlabeled SIM-N  N SUMO-1 15 C/ 13 134  H35 1135 F36 F36 (or 35)  HN 2 Ho’, H HH , 1 2 aromatic  18  Ha, H, H; V9 H  18 18 LlO LlO LlO  Ha,H 2 2 H, fl7 62 H H, H ; V9 W” 2 2 8H H, H 62  K37 K37 K37  RN  ,H 1 H 2  V9 LiO  W” LlO 1162 ; 29 62 H  V38  116(10r2)  LiO  1161, 1162  V39 V39 T42 H43 L47 L47  H H 2  Dii LiO  2 H  L47 S50  H 6 1 HN  R54 R54 R54  LiO Dii LlO LiO LlO  H” HN H  1131,  32 H ;DllH 8 H 2 a 11 2  61 H H , 82 61  82 H  1162  LiO  82 He’, H  1 H  18  fl61  2 H  17 or 18  2 I-F  61 H  18  61 H  The intermolecular NOEs on the structure of SUMO-1 and the sequence of SIM-N were then mapped (Figure 4.6A). 11e8 gave NOE interactions with Arg54 and 11e34, His35 and Phe36, whereas Aspi 1 gave NOEs to Thr42, His43 and Lys39. This clearly demonstrates that the SIM N” peptide binds in a parallel conformation, i.e. similar to that observed with PIASx (Figure  4.5A, left). This situates the negative charge of the acidic residues on the positive side of SUMO 1 (Figure 4.6B).  156  Chapter 4— SUMO interacting motifs in DAXX  Figure 4.6 SIM-N bound SUMO-1 in a parallel orientation. (A) Six representative ‘H-’H strips from a 3D 5 C/’ filtered-edited NOESY-HSQC spectrum showing intermolecular NOEs 3 ‘ N C/’ 3 ‘ N between unlabeled SIM-N and three residues from strand 2 and three from helix 1 in 5 labeled SUMO- 1. (B) Mapping of intermolecular NOEs to the structure of SUMO- 1. Strand 2 and the helix are shown in a cartoon of SUMO- 1 in purple (left) and as isolated elements, with cyan highlighting the residues that receive intermolecular NOEs from SIM-N. Each line represents the presence of at least one intermolecular NOE. Positively-charged and negatively charged residues are shown in blue (left) and red (right), respectively. (C) NMR-monitored -  titration of SUMO-l with SIM-N. Arrows show peaks shifting as the concentration of SIM NGS  2 ) +(O.2AöN) increases. (D) Comparison of chemical shift perturbation (calculated SUMO-l residues upon addition of SIM-N (top) and SIMNGS (bottom). (E) Mapping of residues of SUMO- 1 showing the greatest chemical shift perturbation (<0.1 2ppm) upon binding SIM-N. as,J(M,i)2  from  157  Chapter 4— SUMO interacting motifs in DAXX A  B 0  iS H611 LIOHO2 (.10 H01 1  LIOH62  I8Hy2  L10 1102  0  LII) 1162 LII) Hol  ,-  1M 2A 3T 4A 5N  IS H311 IS fly 2  6S 71 2  2  2  3  3  3  2  2  2 DII HI’  4 .  iSilco  4  DII HO 3  5,  4  L10H c  .  0.6  -J 9,1,  134 HyZ  1(37 uN  2.  Dl1H -  .7  8.9  K39 H2  7.5  H43 HN  3.1  S50 uN  R54 110  0.6  SuM-N”  C  0.5  Q94 124  I  \  K37  0.4  124  •(S,  .  I2  V26  S  1,0  M59 5  ((.2  1143  27  ‘H (ppm)  0.!  ((II  +  E  SIM-N  (15  0.4  J0.3  0.2  0.1  0,0  . t L,J  .1 .fl  20  4!)  Residue  158  611  i 1 iih., ((0  Chapter 4— SUMO interacting motifs in DAXX If the charge of the juxtaposing acidic residues in SIM-N is solely responsible for establishing the binding mode of SIM-N on SUMO-l, it would be expected that increasing the ionic strength of the solution would weaken the electrostatic contribution of those residues and N 5 perhaps lead to multiple binding modes as with SIM-C. Indeed, it was observed that the lHNl  signals from several amides in the hydrophobic core of SIMNv were broadened beyond detection in the ‘H-’ N HSQC spectra of the SUMO-1 complex recorded in the presence of 200 5 mM KC1 (11e8, Va19 and LeulO, Figure 4.3E), similar to SIM-C at 100 mM KC1 (section 4.3). Curiously, under these conditions SIM-N’ bound SUMO-1 with a KD similar to that between SUMO-1’’ and SIM-C” at 100 mM KC1 (Table 4.1). Broadening of peaks could also result from being in intermediate exchange between the free and bound states under non-saturating conditions (Appendix 6). However, the titration curves of peaks that moved in fast exchange indicated that saturation was reached. Thus, the broadening is most likely the result of conformational exchange in the complex. Interestingly, SUMO2v in 200 mM KC1 produced broadening on the peaks of SIM-N” as well, albeit not beyond detection (Figure 4.3F), despite having similar kex and Axo (Appendix 6, compare Figure 4.3C with F4.3D). This suggests that the binding mode has not reversed at that ionic strength yet, despite having a very similar KD to SUMO-1” (Table 4.1). This phenomenon was also observed with SIM-C’’, where SUMO-2’’ showed broadened but detectable peaks when SUMO1w did not (Figure 4.4). It appears that the different hydrophobic and charge distribution between the paralogues is enough to favor one detectable orientation on SUMO-2’’ whereas SUMO-1’ accommodates multiple orientations.  159  Chapter 4— SUMO interacting motifs in DAXX Although flanking charged residues dictate the orientation of binding, the closest SIM-N’’ SUMO- I contacts, as detected by intermolecular NOE interactions, involved the hydrophobic residues of the peptide. Accordingly, it was hypothesized that mutation of these residues would completely disrupt binding. To test this, a construct with the hydrophobic core, Ile-Ile-Val-Leu, mutated to Gly-Ser-Gly-Ser (SIM-N) was produced. Surprisingly, the mutant was still able to bind SUMO-l at low ionic strength, although understandably, weakly and in the fast exchange limit (Figure 4.6C). The dissociation constant of the mutant was determined to be 440 ± 30 tiM, compared to the  <  5iM for the wild type (Table  4.1). The chemical shift perturbation with SIMNGS was much less than with the wild type 5 produced a smaller perturbation to the structure of (Figure 4.6D), suggesting that SIM-N° SUMO- 1. Interestingly, residues showing the greatest changes when binding SIMNGS are clustered on the binding cleft (Figure 4.6E), particularly on the part rich in basic residues (Figure 4.6B). This result highlights the contribution of the charged residues of SIM-N in binding SUMO-1.  4.6 Intramolecular binding of SIM-N to the DHB domain With the goal of investigating the behavior of SIM-N in a more “native context”, i.e. in the presence of the DHB domain, NMR spectroscopy was used to characterize , 44 which ’ 1 DAXX contains both SIM-N and the DHB domain. Surprisingly, the ‘H-’ N HSQC spectrum of this 5 species in 100 mM KCI indicated extensive aggregation (Figure 4.7A). This behavior was not alleviated with addition of excess DTT to ensure complete reduction of all four cysteine residues.  160  Chapter 4— SUMO interacting motifs in DAXX  A OmMKC1  300 mM KC1 4  110  a  .  .  2  z  .#  120  Co  130  100 mM KC1  400 mM KCI  110  :.  .  I  z  0.0  o  .  120  •  O0*  •0  .  : ;  130  200 mM KC1  500 mM KCI  110  0  I  0  .  0 oe  0.0 .  00  0  0  0  9  0  0  120  0  0  .  00 ,  :  0  0 9  0  . 0  130 10  9  8  7  10  9  ‘H (ppm)  8  7  ‘H (ppm)  B [KCIJ  (j SIM-NO  SIM-N  t  ° 0 0  --—-‘O [-.1  K  -®  Figure 4.7 DAXX’ 144 self-association was ionic strength dependent. (A) The 5 H- HSQC 1 N spectrum of DAXX 44 shows broad and missing peaks at low salt concentrations. As the KC1 ’ 1 concentration was increased, lHNI 5 N peaks became sharper and reappeared. (B) At low salt concentration, and the high concentration of DAXX 144 (100 1 iM) intermolecular binding of multimers was presumably favored, while at high salt concentration, the monomer was favored. -  161  Chapter 4— SUMO interacting motifs in DAXX Progressively increasing the sample ionic strength to 500 mM KC1 yielded progressively 44 (Figure 4.7A). A simple sharper ‘H-’ N HSQC signals, indicative of monomeric DAXX’’ 5 144 due to explanation for this behavior is an ionic strength-dependent self-association of DAXX’ intermolecular cross-linking interactions of the added N-terminal residues with the DHB domain N-DAXX’ was purified and changes in its 5 ‘ (Figure 4.7B). In order to map such an interaction, 56 4 were monitored. Binding did DAXX 4 ’ H- HSQC spectrum upon addition of unlabeled 55 1 N 15 N-DAXX’ most affected corresponded to those of SIM-N (Figure 5 ‘ occur and the residues of 56 4 net positive, this is DAXX 4 ’ 4.8A). Since at pH 6.5 SIM-N is negatively-charged, and 55 consistent with an electrostatically-driven interaction. 4 with 55 N 5 ‘ 4 -DAXX This interaction was confirmed through the reverse titration of ’ 4 with SIMN? (not shown). 55 N 5 ‘ 4 -DAXX 56 (Figure 4.8B), and with a titration of ’ DAXX’ Following the peaks in the fast exchange regime (100 mM KC1), the dissociation constant was determined to be 170  ±  60 j.iM (Figure 4.8C). However, in the context of the full-length protein  this value will likely be lower as the linker between SIM-N and the DHB domain might improve binding. 56 binding Mapping residues showing the largest chemical shift perturbations due to DAXX’ onto the structure of DAXX 44 showed that, apart from the C-terminus (His 137, Ala 139, ’ 55 Lys 140, and Lysl4l), the loop between helix 1 and helix 2 (Thr78, Ala79, Asp8O), and helices 2 and 5 (Phe87, Leu88, Gln92, Tyr124, Asn128, Glu129, Thr132, A1a136) were most affected by the interaction (Figure 4.8D-E). Clearly this region is very positively charged (Figure 4.8F),  162  Chapter 4— SUMO interacting motifs in DAXX  55144 through SIM-N. (A) Superimposition of 56 interacted with DAXX Figure 4.8 DAXX’ 56 in the absence (150 !IM in 500 iL, red) and presence of the 15 H- HSQC spectra of DAXX’ 1 N 44 (added 250 iL of 1.4 mM stock, black). Residues showing the most ’ 55 unlabeled DAXX 4 N-DAXX 5 ‘ 4 ’ significant changes correspond to those of SIM-N (labeled). (B) Titration of 55 56 confirmed weak binding (added 210 iL of 2.2 (140 .iM in 500 iL) with unlabeled DAXX’ . (C) A 56 mM stock). Arrows indicate peaks undergoing changes upon addition of DAXX’ representative peak, K 141, used for the determination of the dissociation constant for the resulting complex. The dissociation constant (170 ± 60 tiM) was calculated using chemical shift 44 calculated as ’ 55 DAXX changes in five amides. (D) Chemical shift perturbations of , /5)2 . (E) Mapping of residues involved in 56 +(O.2&55N)2, upon addition of excess DAXX’ -  binding DAXX’ 56 on the structure of the DHB domain. Residues showing the greatest changes (>0.045 ppm) are shown in red. (F) Proposed intramolecular binding orientation. The 44 (green) and the ’ 55 hydrophobic residues of SIM-N face the hydrophobic residues of DAXX lA 55 negatively-charged residues of SIM-N (red) face the positively charged residues of DAXX (blue).  163  Chapter 4— SUMO interacting motifs in DAXX 0  A  110  B  0  0  0  110  I)  e  115  8  0 Z  115  •  120  G f2  1  0  t  0  8  —  —  125  0  V  -  0  z 120  -D13  9.0  1)14 1)16  D11/E13  -  --  F  7.0  3.20 122.8  Al9  M 5 K,,=i7060  Oc  •  7.5  8.0  ‘H (ppm)  C  17 -  E17  8.5  0  1)12  123.0  0.12  1 AI8’  125  123.2  o  —  18  123.4 123.6  L10  3  004  8.6  123.8  8.2  8.4  8.0  7.8  8.11)  8.15  8.05  50  II (ppm)  ‘H (ppm)  100  150  204)  Volume ( L) 1  E  A79  D  0.14  0.12 T132 11.1  _.a—  AIi6..15KI4()  55  0.08  0.06  144  F  0.04  Iii  002  56  61  66  71  76  79  85  90  93  %  11  106  109  114  119  125  128  id. .  133  13.)  1)3  Residue 19  164  Chapter 4— SUMO interacting motifs in DAXX ” 2338 confinning the previous result that the interaction is mainly electrostatic. As in the RassflC titration (section 3.3.3), Lys6O was affected but displayed a dissociation constant two orders of magnitude larger than the other residues (KD = 13 mM), suggesting that its chemical shift change was independent from the binding to SIM-N. One interesting observation is that helices 2 and 5 were also involved in binding RassflC ”, the residues affected by 2338 (Chapter 3). However, despite being of a similar size as RassflC ” (compare Figure 4.8 with Figure 3.6). 2338 SIM-N binding are less clustered than with RassflC This could be explained if SIM-N did not form a helix, but instead it bound in an extended conformation. It is hypothesized that the negatively-charged residues at the C-terminus of SIM-N 2338 C (residues 11-17) faced the positively-charged face of the DHB domain similar to Rassfl ’, but the hydrophobic residues at its N-terminus (residues 2-10) had contacts with the hydrophobic 2338 as well as with the hydrophobic cluster between C cavity of Phe87/Ty 124 (filled by Rassfl ”), helices 1 and 2 (Thr78/Ala79; Figure 4.8F). Lastly, SIM-N binding to the DHB domain produced significant broadening in the signals, 2338 binding did not produce such broadening effect (compare Figure 4.8B C whereas Rassfl ’’ with Figure 3.4B). This suggests that the dynamics of the two complexes were significantly ” upon binding the 2338 different. It is possible that the helix formation, undergone by RassflC DHB domain to fill the well defined hydrophobic cavity of Va184, Phe87, Tyr124 and 11e127, prevented big conformational changes, whereas an extended conformation of SIM-N was free to undergo conformational exchange. A full relaxation study of the two complexes could provide some clues on the viability of these proposed mechanisms.  165  Chapter 4— SUMO interacting motifs in DAXX The use of the same binding interface of the DHB domain with SIM-N and Rassfl ’ 2338 C leads to a tantalizing model where a competing intramolecular SIM-N and DHB interaction mediates the intermolecular interaction of DAXX with RassflC and SUMO-l/2 (Chapter 5). This model will be tested through further NMR-monitored titration experiments and mutational studies.  4.7 Characterization of the interaction between sumoylated Ets-1 and DAXX SIM-C A prevalent function of DAXX is to repress transcription factors. One such factor is Ets-1, which contains a DNA-binding domain at its C-terminus (residues 331-415), and a helical bundle PNT domain (residues 42-135) that mediates MAPK docking and binding to the general transcriptional co-activator CBP/p300. At its N-terminus, Ets-1 contains an unstructured region (residues 1-42) where it can be regulated via sumoylation and/or phosphorylation (Lee et a!., 2005; Lee et a!., 2008; Macauley et a!., 2006; Mackereth et a!., 2004). Ets- 1 was shown through a yeast two-hybrid screen and a luciferase reporter assay to interact and be repressed by DAXX, respectively (Li et a!., 2000b). Li et a!. identified residues 567-740 of DAXX to be responsible for the interaction with residues 1-139 of Ets-1, which they named the “DAXX interaction domain (DID)”. However, attempts to reproduce that interaction in vitro were unsuccessful, as the N 5 H 1 -’ HSQC spectrum of ‘ 138 N 5 -Ets’ did not show any perturbations upon addition of excess unlabeled mDAXX 566739 (Figure 4.9D). Recently, our group and others showed that Ets- 1 is sumoylated at Lys 15 and Lys227 (Ji et al., 2007; Macauley et al., 2006; Nishida et al., 2006). Furthermore, our group also demonstrated that Lysl5-sumoylated Ets-1 can be described as “beads-on-a-string” with the Ets-l and SUMO 166  Chapter 4— SUMO interacting motifs in DAXX 1 components behaving independently and simply linked via a flexible isopeptide bond (Macauley et al., 2006). Since the DAXX-Ets interaction was identified in a two-hybrid screen in yeast, where sumoylation is known to occur (Panse et al., 2004), it was hypothesized that the interaction reported by Li et al. was actually due to DAXX binding via SIM-C to a SUMO covalently linked to Ets- 1. In the simplest model, the interaction between SUMO-1 and mDAXX 566739 would be . As discussed in Chapter 2, mDAXX 38 , also known 566739 independent of the presence of Ets’’ as the SPT-domain, was intrinsically unstructured, and binding to Ets-1 would likely occur without any cooperative effects involving all three species (SPT-domain, SIM-C and Etsi 1-139, as in Figure 1 .4A). To corroborate that this is indeed the case, it was first checked whether SUMO 1 binds mDAXX 567739 only through its SIM-C. Indeed, the ‘HN HSQC spectrum of SUMO-1 showed identical changes when titrated with 15 566739 or SIM-C (Figure 4.9A-B), yet no changes when titrated with mDAXX mDAXX 566727 (which lacks SIM-C) (Figure 4.9C). Then, the effect of mDAXX 566739 on sumoylated Ets’’ 38 was checked. The latter was prepared from unlabeled SUMO-1 and 38 N-labeled-Ets using an 5 ‘ in vitro sumoylation reaction (Macauley et al., 2004). Addition of mDAXX 566739 did not cause  any spectral changes, showing that the binding of its SIM-C to SUMO-1 does not perturb Ets’’ 38 (Figure 4.9D). These results therefore demonstrate that there is no cooperative binding between the PNT domain and the SPT region of DAXX (Figure 4.9E).  167  Chapter 4— SUMO interacting motifs in DAXX  567739 via the covalently attached Figure 4.9 Sumoylated Ets-1 bound SIM-C of mDAXX N-labeled SUMO-1 in the absence 5 N HSQC spectra of ‘ 5 SUMO. Superimposition of the ‘H-’ , (B) DAXX 729-740, or (C) DAXX 567-727 567739 (red) and presence of unlabeled (A) mDAXX (black). Chemical shift perturbations demonstrate that SUMO-1 bound only to DAXX truncation N HSQC 5 fragments containing SIM-C (residues 728-740). (D) Superimposition of the ‘H-’ 1-138 567 N-Ets-1 in the absence (red) and presence of unlabeled mDAXX spectra of sumoylated 15 (black). The lack of any detectable chemical shift perturbations in the amide signals of Ets- 1’ 138 demonstrates that the Ets-1 and DAXX fragments did not bind with any appreciable affinity. Note that the SUMO moiety of this covalently-linked species was unlabeled. (E) Model for the 38 showing that there is no interaction between the binding of mDAXX 567739 to sumoylated Ets PNT domain containing fragment of Ets-1 and the SPT region of DAXX. (F) Current model of Ets-l repression by DAXX. Sumoylation mediates the interaction of Ets-1 with DAXX. This leads to repressed transcription, possibly through the recruitment of histone deacetylases (HDACs), which cause chromatin remodeling. -  168  ___  Chapter 4— SUMO interacting motifs in DAXX  120  120  122  122  I29  .  ..I4  124  37  124  z  1144  z 126  126 ‘7’  128  128  0  9.6  9.4  9.2  ‘H (ppm) 120  V3  C  L1/3  0  D  091 1•5I  1  110  1 o  K37  E  G.0  •  a  *  115  0  0 0  z  z  a  o  0*  120  E89  126  0  0  0  0  V26  L44  8.8  R63  122  124  9.0  073/029  Ci:  I36  ,  o  *  a  l43 a I/IS  125 128  0  a,  127  9.0 9.6  9.4  9.2  9.0  0  0.  8.0  7.0  ‘H (ppm)  8.8  ‘H (ppm)  F  E  SPT HDAC S 740  Repression  169  Chapter 4— SUMO interacting motifs in DAXX  4.8 Summary and significance In this chapter, two SIMs in DAXX, which are able to bind SUMO- 1 and SUMO-2 in vitro, have been identified. It was found that there is no significant SUMO paralogue specificity for either SIMs. However, SIM-N binds both SUMO-1 and SUMO-2 with higher affinity than SIM C. Also, SIM-N binds SUMO-l in one detectable orientation whereas SIM-C binds SUMO-1 in multiple modes. Additionally, SIM-N is the first SIM demonstrated to bind a domain other than SUMO. It was discovered that SIM-N also bound the DHB domain of DAXX, although with ‘-20 fold lower affinity than SUMO under similar conditions. Even more interestingly is the observation that the interface used by the DHB domain to bind SIM-N was the same as the one utilized to bind Rassfl C (Chapter 3). This opens a new model to explain how DAXX is regulated. It is possible that SUMO-1 or -2 relieve the intramolecular binding between SIM-N and the DHB domain so that it can interact with other partners such as Rassfl C. Such a model is discussed in Chapter 5. The data suggest that SUMO- 1 functions as an independent tag on Lys 15 of Ets- 1, and by inference other transcription factors, recruiting DAXX through SIM-C (and likely SIM-N). Such recruitment of DAXX can then bring HDACs (Hollenbach et al., 2002), which have been shown to associate with DAXX, and which in turn modify the tails of histones producing chromatin remodeling and gene repression (Kuo et al., 2005) (Figure 4.9F). It is clear that the data presented in this chapter bring up many new questions. In Chapter 5 some of them and future experiments are discussed.  170  Chapter 4— SUMO interacting motifs in DAXX  4.9 Materials and methods 4.9.1 Constructs Genes encoding residues 1—97 of human SUMO-1 (NP_003343), residues 1-93 of human SUMO-2 (P61956), residues 1-138 of murine Ets- 1 (AAN3 8317), full-length human UBC-9 (P63279), residues 567-740 and 567-727 of murine DAXX (NP 03 1855) were cloned via PCR into the pET28a expression vector (Novagen) using NdeI and XhoI restriction enzyme sites. The -tag with a thrombin cleavage site such that a 6 resulting constructs contained an N-terminal His Gly-Ser-His extension remained after proteolytic processing. New constructs were created with a Trp residue between the Gly-Ser-His and Met-i of SUMO-1 (SUMO-1’’) and SUMO-2 (SUMO 2j to allow quantification via UV absorbance. Genes encoding residues 1-19 (SIM-N) of human DAXX (CAG33366), residues 1-19 with IIVL’° mutated to 7 7 GSGS’° (SIM-N GSGS), residues 718-739 of murine DAXX (SIM-C or SIM-C WT), residues 718-739 of murine DAXX with C728A and S738C mutations (SIM 5738 residues 718-739 of murine DAXX with C728A mutation and an additional cysteine C c), (SIMC were cloned via PCR into pGEX-2T expression vector OC) residue at the C-terminus 74 (GE Healthcare) using BamHI and EcoRI restriction enzyme sites. A Trp residue was added at the C-terminus of DAXX’’ 9 and at the N-terminus of DAXX . The resulting constructs 718739 contained an N-terminal Glutathione-S-Transferase (GST) at the N-terminus with a thrombin cleavage site such that a Gly-Ser extension remained after proteolytic processing. All clones were verified by DNA sequencing. 44 was cloned and purified as described in Chapter 2. The clone of the yeast El ’ 55 DAXX activating enzyme (AOS1/UBA1) was generated as described in (Bencsath et al., 2002). A 171  Chapter 4— SUMO interacting motifs in DAXX peptide containing residues 729-740 of human DAXX was synthesized and HPLC purified by EZBiolab (Westfield, USA) and its purity was verified by mass spectrometry and NMR spectroscopy.  4.9.2 Protein expression  Freshly transformed E. coil BL21 (2DE3) cells were grown overnight in 25 mL of Luria Broth, collected by centrifugation, and transferred to 1 L of minimal M9 medium enriched with 1 g of N labeling, or 5 1 (Spectral Stable Isotopes) as the sole nitrogen source for uniform ‘ (99%)C 4 NH 15 600 to 1 L of Luria Broth (for unlabeled proteins). When cells reached an 0D  0.6, protein  expression was induced with 1 mM IPTG. Cells were then grown overnight (for labeled proteins) or for 5 hours (for unlabeled proteins) at 30 °C, harvested by centrifugation, and frozen at -70 °C prior to further purification. 4.9.3 Protein purification  Thawed E. coil pellets were resuspended in binding buffer (5 mM imidazole, 50 mM HEPES, 500 mM NaCl, 5% (v/v) glycerol, pH 7.5) with a protease inhibitor cocktail tablet (Roche), and lysed by passage through a French press (1000 psi) followed by sonication (duty cycle 50%, 10 mm). After centrifugation at 15,000 rpm in a Sorval SS32 rotor for 30 mm, the supernatant was passed through a 0.8 pm filter (Millex). For pET28a-encoded proteins, the lysate was applied to a 5 mL His-Trap metal affinity column (Amersham Biosciences) connected to an AKTA chromatography system (GE Healthcare). The column was washed with 200 mL of washing buffer (binding buffer plus 60 -tagged proteins eluted with a gradient to 500 mM imidazole. 6 mM imidazole), and the His Fractions containing the protein, as detected by absorption at 280 nm, were pooled and buffer 172  Chapter 4— SUMO interacting motifs in DAXX exchanged with a 3K MWCO Amicon concentrator into a thrombin cleavage buffer (20 mM -tag was removed by incubation with 6 , pH 8.4). The His 2 Tris-HC1, 150 mM NaC1, 2.5 mM CaC1 thrombin  (  1.4 units, Novagen) at room temperature for -5 hours. Proteolysis was monitored by  iL p-aminobenzamidine MALDI-ToF-MS. The reaction was terminated by incubation with 200 1 -tag, the supernatant 6 beads (Sigma) for 1 hour. To further purify the sample and remove the His was diluted 10 fold in buffer without salt, applied to an ion exchange column (HiPrep 16/10), and eluted with a salt gradient (20 mM Tris, pH 8.0, 2 mM DTT, 0  —  Q  FF  1 M NaC1).  Fractions absorbing at 280 nm were evaluated by SDS-PAGE and those containing the sample were concentrated using a 3K MWCO Amicon centricon (Millipore). In each case, the protein purity was judged to be >95% by SDS-PAGE, and the protein identity confirmed by MALDI ToF-MS. For GST-tagged peptides, the lysate was applied to four 5 mL GSTrap HP affinity columns (GE Healthcare) connected to an AKTA chromatography system (GE Healthcare). The lysate was kept on ice-cold water to prevent proteolytic cleavage and slowly loaded to the room temperature column at 0.7 ml/min (GST binding to the column is slow and temperature dependent). Once loaded, the column was washed with 100 mL of PBS/EDTA buffer (137 mM PO 5 mM EDTA, pH 7.3), and the GST 2 KH , HPO 1.4 mM 4 2 Na , NaC1, 2.7 mM KC1, 4.3 mM 4 tagged peptides were eluted with glutathione buffer (10 mM Tris, 20 mM glutathiorie, 5 mM DTT, pH 8.0), prepared fresh. Fractions containing the GST-tagged peptides, detected by absorption at 280nm, were pooled and buffer exchanged with a 10K MWCO Amicon , 2 concentrator into a thrombin cleavage buffer (20 mM Tris-HC1, 150 mM NaC1, 2.5 mM CaC1 pH 8.4). The GST was removed by incubation with thrombin  (-  1.4 units, Novagen) at room  temperature for —5 hours. Proteolysis was monitored by MALDI-ToF-MS. The reaction was 173  Chapter 4— SUMO interacting motifs in DAXX terminated by incubation with 200 tL p-aminobenzamidine beads (Sigma) for 1 hour. The supernatant was passed through the 10K MWCO Amicon filter, which retained the GST and uncleaved GST-peptide in the concentrate. The cleaved peptides were collected in the flowthrough. Concentrates were washed with 40 mL of thrombin cleavage buffer. The flow-through was injected into a C18 HPLC column, washed thoroughly with 0.1% TFA water, and eluted with an H 0:acetonitrile gradient (0.1% TFA). Fractions absorbing at 280nm were evaluated by 2 MALDI-MS, and those containing the desired peptide were lyophilized. Protein concentrations were determined by UV light absorption spectroscopy using predicted 278  cm’ for SUMO-2 and 1 values (Gasteiger et al., 2005) of 9970 M’cm’ for SUMO-1, 6990 M  , 5500 M’cm’ for DAXX’’ 718739 DAXX , 23000 M’cm’ for Etsl-138, 29700 M’cm’ for 9 UBC9, and 67500 M’cm’ for Aosl/Uba2.  4.9.4 NMR spectral assignments  NMR data were collected using a 600 MHz Varian Inova NMR spectrometer and processed using NMRpipe (Delaglio et al., 1995) and analyzed by SPARKY (Goddard and Kneller, 2004). ’ and ‘ H 1 N resonance assignments for the amide groups of SIM-N and SIM-C were determined 5 from HNCACB and CBCA(CO)NH experiments of 5 C 3 ‘ N -labeled /’ samples. 4.9.5 NMR titrations All NMR-monitored titrations were carried out in NMR sample buffer (5% D 0, 10 mM 2 HPO buffer, 0.1 mM EDTA, 10 mM DTT, pH 6.5 buffer with 100 mM KC1 unless otherwise 2 K 4 stated). SUMO-1 and SUMO-2 were exchanged into this buffer by concentrating/diluting 1:10 with a 3K MWCO Amicon Centricon at least 3 times. Lyophilized peptides were dissolved in NMR buffer, with small amounts of NaOH added to re-adjust pH back to 6.5. 174  Chapter 4— SUMO interacting motifs in DAXX H-’ HSQC spectra were recorded at 25 °C using an Inova 600 MHz 1 N Sensitively-enhanced 5 N-labeled species were titrated with increasing amounts of 5 spectrometer. 500pL samples of the ‘ the unlabeled species. Table 4.4 summarizes the sample conditions used for all the titrations presented in this chapter. Table 4.4  -  Sample conditions used for titrations presented in Chapter 3.  Labeledspecies SUMO1lv OmMKCI SUMO2v OmMKC1 SIM-N 100mMKC1 SIM-N 100mMKC1  SIM-N 200 mMKC1 SIM-N 200 mM KCI SIM-C OmMKCI SIM-C OmMKCI SIM-C 100 mMKCI SIM-C 100 mMKCI SIM-C 200 mMKCI SIM-C 200 mMKC1  Concentration  (jiM) 130 100 120 120 90 140 110 110 130  130 90 130  Unlabeled species  SIM-N OrnMKC1 SIM-N OmMKCI SUMO-l’’ IOOmMKCI SUMO-2’’ 100mMKC1  SUMO1#  200 mMKC1 SUMO-2” 200 mM KC1 SUMO-1’’ OmMKC1 SUMO-2’’ OmMKCI SUMO1# 100 mMKCI SUMO2v  100 mMKC1 SUMO-1’’ 200 mMKC1 SUMO-2” 200 mMKC1  175  Concentration  (m1  Final point volume (j.L); ratio ‘ N 5 species :unlabeled species  3  32;l:l.5  3  27;l:l.6  2.5  130;l:5.5  2.5  130;1:5.5  2.3  112;l:5.8  8.7  120;1:15.5  1.7  142;l:4.4  1.7  l42;l:4.4  2.2  220;l:7.8  1.7  250;l:6.8  2.3  240;l:12  8.7  180;1:24  Chapter 4— SUMO interacting motifs in DAXX 4.9.6 Dissociation constants 1 N and ‘ N signals in the fast exchange 5 Dissociation constants (KD) for the complexes with H regime were obtained from least-squares fitting of chemical shift changes as a function of total ligand concentration (Johnson et al., 1996) using Sigma Plot (see Appendix 4 for details).  4.9.7 Spin labeling To ensure full reduction of all Cys residues, 20 mM DTT was maintained through the SIMC until HPLC purification. Peptides were OC SIMC and 74 C purification of the SIM-C, 738 HPLC purified, lyophilized, re-suspended in 50 mM phosphate buffer, pH 7.4; and subsequently reacted for 24 hours in the dark at room temperature with MTSL (Toronto Research Chemicals), previously dissolved in acetone, at a peptide:MTSL ratio of at least 1:5. MALDI-ToF-MS showed the signal for the reaction to be -90% complete. Peptides were re-purified by HPLC to remove any unreacted peptide and side products. Spin-labeled peptides are denoted as TEMPO TEMPOSIMC and 74 c TEMPOSIMC respectively. oc, SIM-C, 738  4.9.8 Paramagnetic relaxation enhancement measurements N-labeled SUMO-l was dialyzed into non-reducing NMR buffer with 100 mM KC1, 5 C/’ 13 and the TEMPO-modified peptide was titrated in to saturation (determined from previous titrations, as the spin label absorbs at 280nm, thus precluding accurate concentration measurement by UV-absorbance). A series of relaxation experiments were carried out to determine the R 2 of the amide IHN (Donaldson et al., 2001). Then, a mixture ascorbic acid (in SIMC were added to reduce the spin oc, NMR sample buffer, 2 mM final), and 10 mM DTT for 74 label and the same measurements repeated.  176  Chapter 4— SUMO interacting motifs in DAXX The reported average global correlation time of 8.6 ns for SUMO-1) (Macauley et al., 2004) was used to calculate the effective distance (reff) from the amide proton to the paramagnetic center according to the following equation (Johnson et al., 1999):  6  K( ‘  3r. i 1 1+a)  K is 1.23 x 1 032 cm 2 for a nitroxide radical, r is the distance between the electron and the s 6 proton, and  H 0  is the Larmor frequency of the proton (3.8 x 1 0 rad s’). The calculated distance  was used to triangulate the location of the spin labels using three residues on each side of SUMO- 1 by solving three equations of a sphere (with the amide proton at the center and the radius equal to the effective distance) with the program Maple. H” of Va138, Leu44, and Leu47 (Figure 4.5G, red) in the For example, the position of the 1 PDB file (model 1, 1 A5R.pdb), and their effective distances to the nitroxide radical were determined as:  LEU LEU VAL  44 47 38  X 81.420 83.481 79.324  z —5.838 —2.193 4.386  ref f  —0.168 —4.234 —5.251  13.3 13.1 15.4  A A A  Appendix 1 OA shows a sample sheet from the program Maple solving the three resulting sphere equations, which provided two possible solutions, one of which could be discarded as it was located on top of other SUMO atoms (Appendix 1 OB).  177  Chapter 5  Concluding remarks and future experiments In this thesis, the first structural characterization of DAXX has been presented. The modular structure of DAXX, with both folded and intrinsically disordered regions, is consistent with its role as a scaffold protein that mediates transcription repression, apoptosis regulation and mitosis control via specific protein-protein interactions. 2338 complex (Chapter C The structure of the DHB domain (Chapter 2) and the DHB/Rassfl ” 3) defined how DAXX interacts with flexible DAXX interaction motifs (DIMs). The study of the SIMs in DAXX (Chapter 4) provided important clues to how DAXX binds SUMO and sumoylated transcription factors. Together, these provide a structural foundation for understanding protein partnerships involving DAXX and prompt new ideas on how DAXX could be regulated. This final chapter summarizes the proposed structural configuration of DAXX. Furthermore, a model for the regulation of DAXX is presented based on the observations from previous chapters. Finally, important remaining questions that require further experimentation are discussed.  178  Chapter 5  —  Concluding remarks and future experiments  5.1 Structure of DAXX The overall goal of this thesis was to present a structural characterization of the domains of DAXX and their role in mediating protein-protein interactions. Secondary structure prediction algorithms, presented in Chapter 2, suggested the presence of at least two well-defined and mostly c-helica1 structural domains at the N-terminus of DAXX (residues 60-145 and 175-400), joined by an unstructured linker. A long intrinsically disordered C-terminal segment of the protein was also predicted (Figure 2.1). Importantly, these predictions agreed with the sequence conservation of DAXX orthologues found in different vertebrates (Figure 2.2). Figure 5.1 A summarizes the truncation constructs presented in this thesis, and Figure 5.1 B shows the proposed modular organization of DAXX. In this model, the first half of DAXX contains at least two helical domains. The first is the DHB domain (residues 55-144), whose structure and function in mediating protein-protein interactions were presented in Chapters 2 and 3. The second, a putative DAXX helical domain (DHD, residues 175-400), could not be expressed in soluble form in E. coli. Smaller fragments within this region, studied in Chapter 2 61342 and DAXX (DAXX’ ), partially confirmed the existence of folded elements, but further 347420 studies are needed to determine their exact structure and function. These experiments could include the production of full-length DAXX using an insect cell expression system (Zhao et al., 2004)), followed by partial proteolytic analysis and mass spectrometry to identify structural domain boundaries. Alternatively, different eukaryotic expression systems, where protein modification and processing patterns are more similar to those in vertebrates, could render this domain soluble (Hunt, 2005; Schmidt, 2004).  179  Chapter 5  —  Concluding remarks and future experiments  Figure 5.1 Domain structure of DAXX. (A) Summary of the principal constructs studied in ° 34242 61342 (sand), DAXX this thesis: SIM-N and SIM-C (red), the DHB domain (green), DAXX’ (orange), and mDAXX 566739 (magenta). A putative DAXX helical domain (DHD) between residues 175-400 is shown in cyan. (B) Model of the structural organization of DAXX. DAXX contains the characterized DHB domain, along with a second putative cs-helical DHD. A model for the second DHD, created by Robetta is shown (with same color scheme as diagram on the top) (Kim et al., 2004). The intrinsically disordered regions, including two SIMs (red with sidechains), are to scale with the folded domains, and shown in an arbitrary extended conformation. In reality, they would sample a much smaller average radius of gyration. (C) List of reported DAXX binding partners, along with coarsely mapped regions of DAXX mediating the interaction. References: (Boellmann et al., 2004; Emelyanov et al., 2002; Gostissa et al., 2004; Hofmann et al., 2002; Hofmann et al., 2003; Hollenbach et al., 1999; Jung et al., 2008; Lalioti et al., 2002; Li et al., 2000a; Li et al., 2007; Li et al., 2000b; Lin et al., 2003; Lin and Shih, 2002; Murakami et al., 2006; Muromoto et a!., 2004a; Muromoto et al., 2004b; Ohiro et al., 2003; Penman et al., 2001; Pluta et al., 1998; Song et a!., 2008; Tang et al., 2006; Tang et al., 2004; Wethkamp and Klempnauer, 2009; Yang et al., 1997; Zhao et al., 2004). -  180  QO  B  17  55  DHB  MSP5S HIPK2 71 pp DMAP 1 TSGIOI ATRX 53 p 53 p LANA NI-TEl Axin RassflA C/EBPJT HAUSP MDM2  Interactor  C  243  DIII)’  Reference and Shih, 2002) (Lin 1-250, 250-501 {Hofrnann et al., 2003) 1-188 (Hofrnann et al., 2002) 43-197, 439-501 (Murornoto et aL 2004a) 1-240, 241-492 (Muromoto et al., 2004b) 1-240, 241-492 (Tangetal..2004) 1-160 (Z1’iao et a!.. 2004) 1-50 1 1-188, 189-400. 600-740 (Gostissa et a!., 2004) (Murakami et aL. 2006) 1-500 (Jung et aL, 2008) 1-130, 131-400 (Li et al., 2007> (Song et a!., 2008) 1-240 (Wethkarnp and Klernpnauer, 2009) 190-400 (Tang et al., 2006) 1-160, 347-570 (Tang et al., 2006) 157-260  DAXX binding region  DHB  144 161  SIM-N  1  [  A  Interactor Ets-1 Fas PML Tf3RI 1 Pax3 CENP-C GLUT4 PaxS p53 GR HSF1  420  DAXX binding region 567-740 626-740 67 1-740 489-740 635-740 636-740 66 1-740 626-740 433-493, 625-740 501-740 560-740  Reference (Li et al., 2000b) (Yang et al., 1997) (Li et al.. 2000a) (Perlrnan et aL, 2001) (Hollenbach et al., 1999) (PlutactaL, 1998) (Lalioti et aL. 2002) (Emelyanov et a!., 2002) (Ohiro et al., 2003) (Lin et aL. 2003) (Boellmann et al,, 2004)  SJM-C  724 740  I.  cL  I  -t  0  I  Th  Chapter 5  —  Concluding remarks and future experiments  In addition to the linker between the observed and proposed DHB and DHD domains, intrinsically disordered regions of 50 and 340 residues are also predicted at the N- and C44 and mDAXX ’ 1 566739 confirmed the intrinsic terminal segments of DAXX. Analysis of DAXX disorder of these regions. Each of these disordered regions contains a SIM, characterized in Chapter 4. Despite the lack of structural elements, numerous proteins have been reported to interact with the Ser-Pro-Thr rich C-terminal DAXX (Figure 5.1 C). Indeed, intrinsically disordered regions have been shown to serve as “linear peptide recognition motifs” in a number of proteins, and are often the site of post-translational modifications that regulate the function of a protein (Uversky et al., 2008). For example, DAXX contains a nuclear localization signal in this region (Yeung et al., 2008), at least two phosphorylation sites (Ecsedy et al., 2003; Song and Lee, 2003), and multiple sumoylation sites (Jang et al., 2002; Lin et al., 2006), so the flexibility of the C-terminus could play a role in facilitating access to modifying enzymes and interacting partners. At the same time, we also speculate that, as with the case of Ets 1, many of these reported interactions may not be direct but instead may be mediated by an unrecognized intervening SUMO tag covalently attached to the partner protein (see below).  5.2 Significance for future truncation constructs of DAXX Figure 5.1 C summarizes a number of studies that used truncation constructs of DAXX to partially define regions mediating interactions with each partner. In most cases, these constructs are rather coarse and mostly likely arbitrarily defined, e.g. based on convenient restriction sites for cloning, poor knowledge of the domain structure of DAXX, etc. Most investigators have assumed that DAXX contains two PAH domains, as originally proposed by Hollenbach et al. a decade ago based on sequence comparisons with Sin3 (Hollenbach et al. predicted a PAH1 182  Chapter 5  —  Concluding remarks and future experiments  domain within residues 64-108 and a PAH2 domain within residues 192-240 (Hollenbach et al., 1999)). This original proposal was put forward before any structural characterization of Sin3 PAH domains and the DHB domain was done, and it is now clear that a new way of thinking for the structural domain boundaries of DAXX is needed. For example, the structure of the DHB domain ) 44 presented in this thesis significantly differs to Sin3 PAH domains both in ’ 55 (DAXX overall fold and function, as demonstrated in Chapters 2 and 3. Furthermore, the originally proposed DAXX PAH2 domain resides on a putative coiled-coil region, where secondary and tertiary structure prediction algorithms do not support the existence of a four-helix bundle similar to the Sin3 PAH domains or the DHB domain (see Figure 2.2 and 5.1B). Instead, a much longer DHD covering residues 175-400 is proposed here. Although further experiments are needed to characterize this structural region, it is clear that the original hypothesis of the existence of two PAH domains within DAXX was not correct. Additionally, constructs of the C-terminus of DAXX studied in the literature included SIM C, and it is unclear if the reported interaction was direct or indirect via SUMO, as it was demonstrated for Ets-1 (Chapter 4). Indeed, GLUT4, PML, p53, GR, HSF1, CENP-C are reported to be sumoylated (Chung et al., 2004; Giorgino et a!., 2000; Gostissa et a!., 1999; Hong et al., 2001; Sternsdorf et al., 1997; Tian et a!., 2002), so a SUMO-dependent interaction with DAXX is certainly possible. Further experiments with similar DAXX truncation constructs lacking SIM-C are needed to clarify whether their interaction is indeed SUMO-mediated. Furthermore, Figure 5.1C shows that a number of studies reported simultaneous interactions of various proteins with multiple truncation constructs of DAXX. It is likely that the DHD and  183  Chapter 5  —  Concluding remarks and future experiments  DHB domains interact with different parts of some of these proteins. Indeed, multiple domain interactions could increase the overall affinity and specificity of the binding. Therefore, the structure of the DHB domain might be only part of the puzzle for many interactions, and more studies are needed to determine its role in vivo. These studies include the functional characterization of truncation constructs lacking specifically the putative DHD and/or the DHB domain. Alternatively, now that the one interaction surface of the DHB domain is known, point mutants could be studied. For example, the hydrophobic core (Val84, Phe87, Tyr124, 1le127), or the positively-charged surface (Arg9l, Arg94, Lys 135, Lysl4O) could be mutated. Such mutations could disrupt specific DHB controlled interactions and provide important clues of the scaffolding function of DAXX. Overall, the work presented in this thesis will be the foundation for future structural experiments and at the same time, it will facilitate the design of better constructs in future protein-protein interaction studies of DAXX.  5.3 Proposed mechanism for activation of the DHB domain 5544 bound Rassfl ” DAXX 2338 via electrostatic and hydrophobic interactions along helices C 2 and 5 (Chapter 3, Figure 3.10). Surprisingly, this region was also involved in intramolecular binding to SIM-N (Chapter 4, Figure 4.8). This raises the question of whether the DHB domain and S TM-N also interact within the context of the full-length protein. It is very tempting to hypothesize that an auto-regulatory mechanism for DAXX results from the intramolecular association of the DHB domain and the SIM-N. This could competitively sequester either or both regions of DAXX from their binding partners (i.e. RassflC and SUMO, respectively). 184  Chapter 5  —  Concluding remarks and future experiments  Figure 5.2 summarizes a possible mechanism for such auto-inhibitory regulation of DAXX. In this “competitive/co-operative coupled equilibria” (CCCE) model, DAXX exists in conformation equilibrium between SIM-N/DHB associated or separated (Figure 5.2A). The former is inactive due to competitive binding of SIM-N to the binding interface of DIMs (like RassflC during mitosis). The latter is free to bind either SUMO (Figure 5.2B), which shifts the equilibrium to DHB-free DAXX and enhances RassflC-binding (Figure 5.2D); or RassflC (Figure 5.2C), which shifts the equilibrium to SIM-N-free DAXX and enhances SUMO-binding (Figure 5.2E). In this model, interaction of DAXX with SUMO enhances RassflC binding and vice versa, making their overall interaction co-operative. Cells could control this mechanism by regulating the available concentration of each protein (by gene expression, localization, turnover, etc.) or by post-translational modifications of either DAXX or RassflC. For example, phosphorylation of Ser23, Ser27 or Ser29 of RassflC would make the DIM more negative and presumably would increase the affinity of Rassfl C towards the positively-charged interaction surface of the DHB domain. Interestingly, a phosphorylation prediction server returned very high scores (>0.97, 1 being a perfect phosphorylatiori site) for Ser23 as a putative phosphorylation site for casein kinase II, DNA-Protein Kinase and ATM kinase, and Ser27 and Ser29 as putative phosphorylation site for casein kinase II (Blom et al., 1999; Blom et al., 2004). Alternatively, post-translational modifications on DAXX could alter its binding affinity as well. For example, very recently Lys122 has been reported to be an ubiquitylation site in DAXX (Fukuyo et al., 2009). Certainly, ubiquitin at Lys 122 would produce steric hindrance for RassflC binding and SUMO binding.  185  Chapter 5  —  Concluding remarks and future experiments  Figure 5.2 Proposed auto-regulatory mechanism of the DHB domain. The competitive/co operative coupled equilibria (CCCE) model. In the context of the full-length protein, the DHB domain and SIM-N are in equilibrium between free and bound states (A). Such equilibrium can be shifted by competitively binding either SUMO (B) or a DIM-containing protein, such as RassflC during mitosis (C). SUMO binding would increase the amount of DHB-free DAXX, thereby enhancing RassflC binding (D). Similarly, RassflC binding would increase the amount SIM-N free DAXX, thereby increasing the SUMO binding (E). The folded domains and unstructured regions are to scale. -  (*) The dissociations constants are those measured with minimum isolated constructs (DAXX 55 44 DAXX’’ [SIM-N] and RassflC , 100 mM KC1). However, in the context of larger 838 constructs they might change due to the linker between the DHB domain and SIM-N.  186  Chapter 5  ®1L  KD  =  —  Concluding remarks and future experiments  170 tM*  *  V DHB  160-740  187  Chapter 5  —  Concluding remarks and future experiments  Future experiments are needed to study this CCCE model. For example, the KD values for each step need to be measured in the context of larger fragments, since the values on Figure 5.2 are from isolated minimum constructs. In larger constructs, the SIM-N would be covalently attached to the DHB domain, as in the native protein, and could thereby bind it with higher 44 to bind SUMO and/or affinity. Future NMR experiments studying the ability of DAXX’’ ’ will be carried out to test this model. 2338 Rassf1C At the biological level, point mutations could provide clues to the veracity of the CCCE model. Ideally, a mutant disrupting one interaction and not the other would be ideal. For example, the C-terminus of the DHB domain (Lys 140 and Lysl4l) is more disrupted by the intramolecular binding of SIM-N than by Rassfl C binding. Thus, point mutations at these flexible residues are good candidates for disrupting intramolecular binding without disrupting the structure of the DHB domain, and without significantly affecting Rassfl C-binding. With those mutants, the CCCE model would be shifted towards free DHB domain, enhance RassflC binding, and a phenotype change of this mutant during mitosis could be observed. Alternatively, it was demonstrated that mutations at the hydrophobic core of SIM-N decreased drastically its binding to SUMO. However, such mutations might disrupt the electrostatically-driven intramolecular binding of SIM-N differently. If this is indeed the case, such mutant would shift the CCCE model towards SIM-N-bound DHB domain, reducing Rassfl C binding, and producing a different phenotype. To date, no SIM has been reported to display such multiple functions, and it will be exciting to see if the experiments suggested above lead to the discovery of new SUMO-mediated cell cycle mechanisms.  188  Chapter 5  —  Concluding remarks and future experiments  5.4 Future experiments in defining DHB domain interacting motifs (DIMs) The work presented in Chapter 3 is the first in defining the physicochemical properties of a DIM. Sequence alignment of Rassfl ’ 2338 with proteins reported to interact with the N-terminal C region of DAXX suggests the presence of a common DIM (Table 3.3). Expression and purification of these putative DIMs is needed to corroborate this hypothesis and answer important questions. For example, do they bind the DHB domain in vitro? What are the dissociation constants? What are the structural bases for binding? Is reverse binding indeed possible? Good candidates for such experiments are Mdm2 , Hausp 296313 , Hausp 828843 , and 856871 28 C 3 . 3 , which show a high degree of conservation with Rassfl N. 44348 Stat Fascinatingly, a number of proteins involved in transcription, but not yet reported to interact with DAXX, have also been identified through BLAST using RassflC DIM as bait (Table 3.4). Until now, the transcription repression mediated by DAXX has been suggested to occur through the recruitment of HDACs (Figures l.4B and 3.9F). However, the first candidate in Table 3.4 is the transcription initiation factor IIIB (TFIIIB). TFIIIB activates RNA polymerase III, which catalyzes transcription of a variety of short RNA molecules with essential functions in cellular metabolism. TFIIIB activity is carefully regulated during the cell-cycle, and is believed to be activated by phosphorylation by ERK and other kinases, although the detailed mechanisms for its regulation are still unknown (Mauger and Scott, 2004). Protein-protein interaction between DAXX and TFIIIB could act as a repression mechanism as proposed in Figure 1 .2B. Further in vivo and in vitro experiments are needed to investigate whether DAXX and TFIIIB proposed interaction is real and functional.  189  Chapter 5  —  Concluding remarks and future experiments  Finally, further experiments are needed to investigate if other DIMs exist. A yeast-two hybrid 4 as bait could provide clues on whether Rassfl C-defined DIM is unique screen using 55 DAXX 4 ’ or one among many.  5.5 DAXX SIMs It is interesting that DAXX contains two SIMs, one at its N-terminus and the other at the Cterminus (Chapter 4). The distance between them suggests that they function independently, and not in synchrony as it is the case for RNF4, which binds polySUMO-2/3 chains with adjacent SIMs (Tatham et al., 2008). Indeed, there are significant differences between them. There is overwhelming evidence that SIM-C is active in vivo, and mediates DAXX-dependent repression to sumoylated transcription factors (Lin et al., 2006). Excitingly, a new paradigm for regulating SIM interactions has emerged very recently. Stehmeier et al. have discovered that the casein kinase 2 (CK2), an essential kinase in numerous signaling pathways, can regulate a number of SIMs by phosphorylating serine residues adjacent to the hydrophobic core (Stehmeier and Muller, 2009). SIM-C (and Rassfl C) contains a perfect 737 which has been conserved in all the organisms that ( ), 740 recognition sequence for CK2 SDSD contain DAXX (Figure 2.2). Chapter 4 has shown the importance of electrostatic interactions in SUMO/SIM interactions, and it is expected that phosphorylation at either serine would change the ability of DAXX to recognize sumoylated proteins significantly. First of all, SIM-C can bind SUMO in multiple orientations due to the symmetric distribution of negatively-charged residues in SIM-C. Phosphorylation of the serines would likely affect this, inducing a single binding mode like in the SIM-N/SUMO interaction. Second, SIM-C phosphorylation will definitely affect the dissociation 190  Chapter 5  —  Concluding remarks and future experiments  constant between SUMO and SIM-C, most likely decreasing it. Future NMR-monitored titration experiments of SUMO with phosphorylated versions of SIM-C will help to clarify these hypotheses. On the other hand, the role of SIM-N in mediating transcription repression is unclear, since only DAXX’ , was unable to repress Smad4-, AR-, or CBP-mediated 732 , and not DAXX’ 740 transcription (Lin et a!., 2006). This is despite the fact that its dissociation constants for SUMO-l and SUMO-2 are 3-4 times smaller than those of SIM-C (Table 4.1). The CCCE model provides a mechanism to explain this result, that is, SIM-N is unable to substitute SIM-C since it can only be active under special circumstances due to its intramolecular binding to the DHB domain. Interestingly, its sequence does not contain a CK2 recognition site, but RassflC does. It is plausible that CK2 could regulate activation of SIM-N via activation of Rassfl C (as discussed above). Future experiments investigating the effect of point-mutations on Rassfl C CK2 sites (Ser23, Ser27 and Ser 29) are needed to corroborate this hypothesis.  5.6 Remaining questions about the DAXX/RassflC interaction As mentioned in the introduction, members of the Ras-association domain family are also scaffold proteins. The most characterized member of the Ras-association domain family is Rassfl A, due to its important role as tumor suppressor. Rassfl C shares most of its sequence with RassflA, differing only in its 50 N-terminal amino acids (Figure 1.5). The function of these unique residues is just emerging, and no structural characterization had been done on this region. In light of the results presented in Chapter 3, it is clear that these residues are unstructured, and mediate protein-protein interactions through small recognition sequences such as the DIM. The structure of the DHB/Rassfl ’’ 2338 complex suggests that electrostatic and hydrophobic C 191  Chapter 5  —  Concluding remarks and future experiments  interactions are essential in DIMs. Although it is still unclear how these interactions are regulated, conspicuous CK2 phosphorylation sites within the RassflC DIM provide possible avenues for regulation (discussed in section 5.2). Chapter 3 has provided a structural mechanism for the direct binding of Rassfl C 2338 with the DiTB domain of DAXX. Based on the biological significance of this interaction (discussed in section 3.7), there are several hypotheses to be examined. For example, will expression of GFP Rassfl C 2338 return similar results to those found with GFP-Rassfl C’ ? Furthermore, will point 50 mutations within the binding interface of the DHB domain disrupt Rassfl C association and hence render cells taxol-resistant? Or, are there interactions from other domains of Rassfl C and DAXX that increase affinity/specificity? It will also be interesting to investigate whether gene therapy in taxol-resistant cells is viable. If DAXX expression were reinstated, it could make the cells taxol-susceptible. If indeed so, then a minimum construct of DAXX including the DHB domain could be sufficient to render the cells sensitive to taxol. Overall, the DHBfRassf1C ’’ complex has provided the molecular basis for the 2338 function of specific domains in DAXX and RassflC.  5.7  Concluding remarks Taken as a whole, DAXX is a very interesting protein. It is a scaffold that brings together  diverse complexes that fulfill specific functions in specific places and times. Despite being involved in important processes such as transcription regulation, apoptosis control, mitosis management, and other cellular mechanisms, the molecular basis for its scaffolding properties and regulation are still unknown. The structure of the DHB domain, its interaction with Rassfl C, and the properties of its SIMs are the first stones in the structural knowledge needed to decipher 192  Chapter 5  —  Concluding remarks and future experiments  this enigmatic protein. There are still a number of important unanswered questions that will require further structural and functional studies in this exciting field.  193  References  References Agathanggelou, A., Cooper, W.N., and Latif, F. (2005). Role of the Ras-association domain family 1 tumor suppressor gene in human cancers. Cancer Res 65, 3497-3508. 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J Biol Chem 283, 29405-29415. 213  Appendices  Appendices Appendix 1  ° 34742 Circular dichroism (CD) spectra of DAXX  Appendix 2  Expression tests of DAXX constructs  Appendix 3  N HSQC spectra 5 Annotated ‘H-’  Appendix 4  Calculation of dissociation constants in NMR-monitored titrations  Appendix 5  Dissociation constant plots from SIM/SUMO NMR-monitored titrations  Appendix 6  Exchange regimes  Appendix 7  Assignment of the DHB-domain  Appendix 8  ”’ in the DHB/RassflC 2338 ” complex. 2338 Assignment of RassflC  Appendix 9  ” complex 2338 Assignment of the DHB domain in the DHB/RassflC  Appendix 10 Triangulation of the spin label  214  Appendix 1  —  CD of DAXX ° 34742  42 347 DAXX Appendix 1 Circular dichroism (CD) spectra of °  A  I  -2  S -7 C.?  -12 —  C.?  47  I.  -22 190  200  B S  230  220  210  250  240  260  Wavelength (nm) -8 -8.5  -9 -9.5 I.  -10 -10.5  1  -11  10  20  30  40  50  60  70  80  90  Temperature (°C) (A) CD spectrum of DAXX ° (10 tiM, 10 mM phosphate, pH 6.5, 100 mM KC1, 0.1 mM 34742 EDTA, 25 °C, path-length 0.1 cm). The spectrum demonstrates that this construct was predominantly disordered, yet may adopt a small fraction of secondary structural elements. In particular, the CD signal at —222 nm is diagnostic of x-helical conformations. (B) Thermal denaturation of DAXX ° monitored by CD spectropolarimetry. The loss of CD signal at 222 34742 nm with increasing temperature also confirmed the presence of a small fraction of helical secondary structure. 215  Appendix 2— Expression test of DAXX constructs  Appendix 2 Expression tests of DAXX constructs  Summary of all DAXX constructs used for expression tests. The solubility results are summarized in Figure 2.3. The red star highlights the resulting DAXX fragment. In cases where multiple colonies of the same construct were expressed, only the colony used for DNA sequencing is labeled. The protein markers (M) had the following masses: 14 kDa, 18 kDa, 25 kDa, 35 kDa, 45 kDa, 66 kDa, 116 kDa. The lanes are labeled with the following code: un = uninduced 3h  =  three hours after induction with 1 mM IPTG.  5h  =  five hours after induction with 1 mM IPTG.  9h  =  nine hours after induction with 1 mM IPTG.  on  =  overnight (approximately sixteen hours after induction).  t  =  total (soluble  s  =  i  +  insoluble).  soluble. insoluble.  EtOH  =  5% glycerol, 2.5% ethanol added to the media.  The methodology for preparing samples of each lane is described in section 2.6.2.  216  Appendix 2— Expression test of DAXX constructs  Identifier 740  A  1-160  B  46-160  C  46-420  I)  55-243  E  161-243  F  161-342  C  161-420  H  181-335  I  181-365  J  181-385  K  181-407 *151.365  NI  *151.407  N  206-335  0  206-365  P  206-385  Q  206-407  R  246-342  S  261-420  T  261-342  I-I  279-342  1  347-420  w  rn566-739  x  47-560  4-—, ( 4—, +— 4.— <> ÷—* < .  4-> 4->  217  Appendix 2— Expression test of DAXX constructs  —  —  ‘—I  -  a  —  -  — —  -  -  ---  —  -  un  -  —  M 6ht 6hs 6hi ont un 6ht 6hs A 1-16030°C  B 46-16030°C  ———“—,  M  —=—=-----  3ht  3hs 3hi  un Sht M 5hs ont ons oni Shi 5hs ont ons oni ont ons oni  C 46-420  ——  —  •  s.  —  a  un  6hi ont  C 46-42022 C  C 46-420 15 °C  C 46-420 15°C EtOH  I  .---  -r 4I_*:  l’L  .  -Iti un M Sht Shs 5h1 E 16 1-243 30°C  un Sht Shs Shi un Sht 5hs Shi D 55-24330°C  M on  F 161-34230°C  ont ons  oni  r  ont ons oni  F 161-342 18°C F 161-34222°C  — :_. —  S  ——I——  —= -‘i  fA  un 3ht  3hs  3hi M  G 161-42030°C  un Sht Shs  M ont  ons oni Sht Shs  G 161-42022 °C  218  ont ons oni ont ons  G 161-420 15 °C  oni  G 161-420 15°C EtOH  Appendix 2  —  Expression test of DAXX constructs  —  un  M 9ht 9hs 9ht 9hs 9ht 9h 9ht 9hs 9ht 9hs 9ht 9hs J 181-385 30 °C  —  ——  K 181-407 30°C  I  —  •1  —  uii 9ht M 9hs 9ht 9hs 91i1 9h 91i1 9hs ‘Jul ‘JIts 9ht 9hs  N 206-335 30°C  0 206-36530 °C  P 206-385 30 C  91i( 9Iis I. 151-365 311 (  1 ‘liii gins I  SI \I—’  ,nsIrIIi  151-407  9ht Ohs I) 2116-407  30 (  311  219  9h1 9ht 9I I’ 2116-355 311  Appendix 2— Expression test of DAXX constructs  —  -  *  *  un M Sht Shs Shi un Sht Shs Shi un Sht Shs  R 246-342 30°C  -  W  —  T 261-342 30 “C  5h1  un  U 279-34230 “C  Sht  M Shs Shi  un  V 347-42030 “C  ::: iz Iz  S 261-42030 “C  —  4—---  — —  -*  ‘:‘  ‘T  * un  M ont  ons  oni  T 261-342 18°C  ont  OflS  Ofli  tin  T 261-342 22°C  ont  Ons  Ofli  D 55-243 18 “C  ont ons  —  e  : ont un ons M oni ont  ons  S 261-420 18 “C S 261-420 22 “C  220  Ofli  D 55-243 22 “C  —  M tin 3ht 3hs  3hi  X 47-56030°C  Sht 5Iis Shi  Appendix 3  —  Annotated spectra of SUMO/SIMs  1 15N HSQC spectra Appendix 3 Annotated H  (A) SUMO-1 (155 j.iM, 17°C)  (B) SUMO-2 (1.1 mM, 25 °C) (C) SIMNw (120 jiM, 25 °C)  (D) SIM-C’’ (125 jiM, 25 °C). All samples were in NMR sample buffer (10 mM phosphate, 100 mM KC1, 0.1 mM EDTA, NH groups are connected by horizontal 5 ‘ pH 6.5). The signals from Asn and Gln side-chain 2 lines. Aliased peaks are shown in red.  221  _______  Appendix 3  —  Annotated spectra of SUMO/SIMs  ‘G68  105  A -  I66 R63  Q29 K25  ,  Q92  110W  F64  Y51  /  M40  .1, -  K39—  A72 1.24 /  C-T76  DI K16t4.E$ /. 21(17  0G14 0G19  G56N74 S31  MV D< E11 134 L13 1--Y9i’  QG96  V57—  0  •S61  N60  G28  -0  ••\Q55  1 115  K78\...G97 ‘sso OT’o V87  T98 tj izz  z  OS9os2  S32  C52 0  cJL8O  M82 K48  E5 k54 V9O 69  120.  01175 -OT42 L62  E79  L.L47  0 0 MR*  ;..L6S  0  125  L44  E89 -  ci ,.  (1435  -....;  0A6  E93  :1143  T4l  l27 0 E67  (.27  B  T7’ -  j.G24  110 @D63 .A23  2 G52  .G9  -2-.H37  C92  0Q51  1’8  01)80  z  -  ..R36 1(45 L$3  vio  ‘  1F60  120  —162 K61  C 42  25 F31Q  L40.. -N68  K21  y47  -  -. N14  1)16  .  :Rs9  0  158  L76  V86  .-..  -Q(.0  MI  OQ9*_j3” cGU .F87  p  .  K42  R  N15  .  Q)i8  -  -- — _.__  — QG93 S41J 1 0Q57 (.._fI2 2 ,) —iM•78 74 . 8 fl)7_S C48.  01311  I  % R56  ..S54  115  -.  446  - L4  (1)7J  Q89 —A2 Q31 0 KU  125  V30  0 184  9.5  9.0  8.5  8.0  ‘H  (ppm)  222  7.5  7.0  6.5  Appendix 3  C  —  Annotated spectra of SUMO/SIMs  ON5N-H  115  NS  I 120 D13+—D12 D14 9 1)1I.E15  Z  A19  125 44 :A  0W20  18  .  ‘vQ LIO  8.5  8.0  7.5  7.4) Q724N-IF  D  0  .  115  S738 T721  0  S736 S722  E  120  0  E730  QC727 ‘7Z3 E731 0  Q726 -1732 V719  125 V734 1733  .JA72.l L735  c  1)739  7.5  8.0  ‘H (ppm)  223  7.0  Q726N.Ff  Appendix 4  —  Dissociation constants  Appendix 4 Calculation of dissociation constants in NMR-monitored titrations  For a protein binding a ligand, the equilibrium can be written as: P+LPL  (1)  where the dissociation (KD) and association (Ka) constants are defined as:  —  [P][L]  —  [PL]  Ka  KDli  (2)  [P], [L], and [PL] indicate the molar concentrations of the unbound protein, unbound ligand, and bound protein-ligand complex.  In titrations that occur in the fast exchange regime (Appendix 6), the chemical shift (ö ) of a 1 given nucleus at each titration point (I) is the population-weighted average of its chemical shifts in the free () and fully bound  (sb)  species. Thus, its chemical shift can be expressed in terms of  the fraction of the bound species (fbI, where fbI + ffi  fbl  or  1) as:  (3) =  =  fbi (b  —  f) +  (3a)  This fraction can also be expressed in terms of concentrations of the free ([Pf]) or bound ([PL]) species:  —  fbi —  [PL] [Pf]+[PL]  224  (4)  Appendix 4  —  Dissociation constants  Experimentally, we know the total, rather than free or bound, concentrations of protein and ligand. The total amount of protein ([Pt]) and ligand ([Li]) can be expressed as the sum of the free and bound species: [Pt] = [Pr] + [PL]  (5)  ] + [PL] 1 [Lb] = [L  (6)  The above equations can be combined to derive a quadratic equation for the fraction of bound species at each titration point: from equation 2:  [PLI  substituting into equation 4  f  from equation 6  [Lf]  substituting into equation 7  fbi  =  Ka[Lfl  Ka[Pf][Lf] =  =  [Pf]+Ka[Pf][Lf]  =  [Lb]  —  1+Ka[Lf]  [Lf] =  (7)  1  —+[Lf]  [PL]  (8)  = ±[Lb[PL1  substituting equation 5 into equation 4  substituting into equation 8 fbi  [P1[LIKa  or [PL]  fbi =  = +[Lt]fbi [Pt]  =  = fbi  [Pt]  [Ltlfbi[Pt]  =  1  (1+Ka[LtbKafbj[Pt])  Ka([Ltbfbi[Ptl) 1+Ka[Lt]Kafbj[Pt]  which can be rearranged into the quadratic equation:  fbi  + fbiKa[Ltl  —fbi  —  —  fbi’<a[’t]  2 [Pt] Kafbi  =  Ka([Lt]  —  fbi[Pt])  + Kafbi 2 [Pt] + Ka[LtbfbiKa[Ptl = 0 225  Appendix 4 2 + (—KD ([Pt])fb  [Lv]  —  [Pt])fb, + [Lb]  —  —  Dissociation constants (9)  0  =  Quadratic equations have the solutions: 0  2 + bx + c ax  x=  2 —4ac —b±gb  (10)  2a  Using the solution that will give a number between 0 and 1:  —  —(—KD—[Lt]—[Pt])—,J(—KD—[Lt] —[Pt]) —4([Lt] [Pt]) 2  fbi  2[Pt]  —  Substituting into equation 3a:  —  2 —4([Ltl [Pt]) KD+ [Lt]+[Pt]—lJ(KD+[Lt]+ [Pt])  —  2[P]  ,.-  ’f)+( b f 5  (12)  Finally, to account for dilution during the titrations, [Pt] is substituted by PV/(x+V), [Li] by Lx/(x+V), where V is the initial volume (in j.tL) in the NMR tube, P is the initial concentration of the analyte solution in the NMR tube (in M), L is the initial concentration of the titrant (in M), and x is the volume of titrant added (in jiL) to give the following equation:  =  (öb  —  +  (13)  x+V  For the KD calculations reported in Chapters 3 and 4, a plot of ö versus volume (x, in iL) was used. In all plots, ö was calculated using J(L8IH was used solve equation 13 for KD and  i  )2  +5 AöI 2 (O. N  )2  so  was zero. Sigma Plot  non-linear least squared fit using initial concentrations  226  Appendix 4  —  Dissociation constants  of protein (P), peptide (L) and initial volume (V). Initial estimates for the variables KD and  b  2 of the fit was usually greater than 0.95. The reported errors were provided by inspection. The R are standard deviations of independent fitting of signals.  227  Appendix 5  Appendix 5 titrations  —  Dissociation constant plots for SIM/SUMO  Dissociation constant plots from SIM/SUMO NMR-monitored  The dissociation constants reported in Table 4.1 were derived using data from the titrations shown in Figures 4.3 and 4.4. Residues used for the calculation of each KD are summarized in the following plots. Equation 13 from Appendix 3 was used for each regression, using protein and peptide concentrations summarized in Table 4.3. The plots for ‘ N SUMO-1” and SUMO-2’’ with unlabelled SIM-N’’ showed stoichiometric 5 binding, so only an upper estimate for the KD was obtained (Table 4.1).  228  Appendix 5  Dissociation constant plots for SIM/SUMO  —  SIMCw + SUMO-V  + SUMO-2’  S  2  1  1  4  3  2  3  4  0.3  S ‘&  c.)  0.2  E 0.1  2  4  8  6  • V  0739 D737  G  E730  4  2  •  I  6  0739 0737  V  •  S736  A  L735 E730 0728  —  E V  V V  (•l  V  0.1 Ov  A  A  A  -  A--  -  A  --  3  6  9  12  4  SUMO-1”:SIM-C ratio  8  12  16  20  SUMO-2” :SIM-C” ratio  229  24  Appendix 5  Dissociation constant plots for SIM/SUMO  —  SUMO-F’ +SIM-N’  SUMO-2’ +SIM-N’  •  0.15  v  I  <>  I  A  I  V  V  I  I I I I  •  I  c.)  N68  V  E  I]  V30 F87 C48 E81 184  A  A  •  0.10  0 A  •  v  0.05  O O V  A  27 H35 C52 H75 K48  0  E  I V  V U V  0 V  SIM-N”:SUMO-1’ ratio  +  SUMO-1”  SIM-N” :SUMO-2” ratio  SIM-N’ +  SUMO-2”  0.3  c-) E  00-  0.2  E 0.1  2  4  2  4  0.3 C-) 0.2  E 0.1  2  4  4  SUMO-i”:SIM-N” ratio  8  12  SUMO-2’:SIM-N” ratio  230  Appendix 6— Exchange regimes  Appendix 6 Exchange regimes Equation 1 from Appendix 4 can also be written in terms of rate constants: PL  (14)  where the dissociation (KD) and association (Ka) constants can be re-written in terms of rate constants as:  L’  ff 0 k  ,  01 k  on  off  The apparent exchange rate is defined as: 011 + k ff 0 kex = [L] k  (16)  In a typical NMR-monitored titration experiment, only the protein or the ligand is labeled. As an example, assume that the labeled species is the protein, and that only one signal is being observed. In this case there will be an observed resonance frequency for the nucleus in the free protein (v ) and a different chemical shift for the same nucleus in the bound species 1  (Vb).  The  difference in frequencies between the two protein states is:  VVb—Vf  The exchange regimes of binding events observed by NMR are defined as: slow exchange if kex< öv intermediate exchange if kex fast exchange if kex> öv  231  (17)  Appendix 6— Exchange regimes The lineshape of the peak resonance is different for each exchange regime. The equation describing the lineshape of a resonance undergoing a 2-site exchange process is described in (Sandström, 1982):  QR + T-+--+ \Tzf b1 2 T  1(v)  0 C  =  (18)  +R P 2  where  1 b 2 \TzfT  J  2 T  Tzb  Q =r(2v—v(ff—fb))  R  =  IXv =--—v 2  =  = kon(KrJ+Lf)  f and T2b are the 2 Co is a normalization constant proportional to the total protein concentration, T apparent transverse relaxation times for the nucleus in the free and bound protein states, respectively. ff and fb are the fraction of free and bound protein, respectively. Thus,  =  fb  (equation 11)  2[Pt]  ff=l—fb  (19)  Lf=Lt—fbPt  (20)  232  Appendix 6— Exchange regimes Using equation 18 with Co  =  -1 e6, and assuming k 0 to be diffusion controlled (1  x  108 M’s’),  the exchange regime is determined by the lifetime of the bound state (as reflected by k ff) and v. 0 The interplay of these two terms is shown in the following simulations (using KD ff/1 0 k  X  =  ff/k k 0  108 M’s’).  Different KD values:  K=1nM k = 1e8 M’s’ P,= 100 tM =0.01 S 1 T, T,,= 0.01 S Vr = 100 Flz 0 = 160 Flz v  K,, = 1 .M k,, = 1e8 M’s’ P,= 100 tM = 0.01 S 0 T, T,0.01 s = 100 Hz =l6OHz 0 v 280  Slow  Intermediate  300  ThO0  , = 100 jiM 1 K k, = 1e8 Ms P=lOOjiM T00ls = 0.01 S 0 r v=lOOHz =l6OHz 0 v  0000 2000  233  Fast  =  Appendix 6— Exchange regimes Different v:  K{)= 1pM  k,= le8M’s’ P= 100pM f= 0.01 s 2 T T,= 0.01 S 1 = 100 Hz V = 180 Hz 280  300  10000  7500  = 1pM 0 K ,k, = 1e8 M’s’ 0 P= 100pM = 0.01 S 1 T, T7, 0.01 S =lOOHz 1 v = 160 Hz 1 v  5000 I) 2500  260  280  300  = 1pM 0 K = =  1e8 Ms’ 100 pM  =0.01 s 1 T, ,= 0.01 2 T Vb  280  234  300  =  S  100 Hz 120 Hz  Appendix 6— Exchange regimes In addition, T 2 values typically decrease in complexes due to slower correlation times for molecular motions. The effect of changing T 2 values is shown below for fast exchange:  = 100 tM 0 K k = 1e8 M’s’ 0 1 = 100 tM P = 0.01 S 1 T, b= 0.01 S 2 T 1 = 100 Hz v 160 Hz  l0000 7500  0 = 100 jiM K 1(00 = 1e8 M’s P, = 100 jiM 1,1=0.01 s , = 0.005 s 1 T, v. = 100 Hz 5 = 160 Hz V  5000 ---  0 0  200  300  10000 7500  K 100 jiM = 0 k, = 1e8 M’s’ = 100 jiM 1 P = 0.01 s 1 T, T, = 0.0025 S 1 = 100 Hz v =l6OHz 5 ‘i  s000 2500  0  2043  1-’ 200  300  235  Appendix 6— Exchange regimes The above simulations are for a simple two-state conformation equilibrium. In the case of conformational exchange involving both ligand binding and two possible bound orientations (PL and PU), a general three-state equilibrium can be written as:  P+L  (jk)  PU  (21)  If the ligand is in sufficient excess to ensure complete saturation, only the resonances from the nucleus in the PL and PL’ states will be observed. The lineshape of these signals is described by equation 18 as well (i.e. 2-state exchange between bound conformations). However, the apparent exchange rate is now independent of ligand concentration (kexki+ki). Assuming a  SvpLpL’  =  100 Hz, T2pL=T2pL’=0.05s, PpL=PpL’=O.5 (then k ), the following graph shows the effect of 1 different values for k . 1  50000 >40000 •k=lOs  30000  ik=lOOs” 20000 10000 0 0  280  236  • k,  =  200  • k  =  400 s  • k  =  1000 s  • k,  =  10000 s  ‘  Appendix 6— Exchange regimes Therefore, if the interconversion between bound conformations occurs in the intermediate exchange regime with kex  övpLpL’,  the signals from the given nucleus in the saturated complex  can be extremely broadened and thus difficult to detect.  237  oc  o  (J  (JI  U  (Il C  J1 Vi (J Vi  (JI Ui Ui  (JI ii  (Il .  fJI . Ui  Ui  Ui .  Vi  C’  Vi  C’  t-a ‘-C  Vi  a  .  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In  •0  © 0’  . . <  r— 00  C—  C-i  i—  0’  r  N 00  \O 0 0’  t 00  CD  00  0’  N  ‘.0  C  —  C  00  0’  N  C  00  0’  N  If)  00  C  -  N  N  C’)  .C  0’  N  ‘.0  N  c  0’  r  0’  0’  N  N  1)  N GO  *  0’  N GO  N i;  —  N 00  N  C—  00  CD C)  Cl  Cl  C’) C’)  00  .  CD C)  00 00  C)  00  00  00  C C)  00 c C-  N GO  C  r)  r  *  C00  .  \C \C  < C)  00  r’)  0’ N  0’ N  GO  C) 00  © 00  0’  —  0’  —  .  0’  C00  0’  —  0’  N  —  8 0’  —  0’  N  C)  —  0’  —  —  0’  —  0’  —  0’  —  C)  0’  00  —  —  0’  0  —  N  0’  C’)  CD  r— 00  If)  N  00 0. C  *  00 C  c  0’  ‘r  0’  r-—  C  r 0’  N  ‘.0 00  e  .  0’  00 C’)  N •  N  0’  8 0’  N 00  C’) CC)  C)  *  C)  a 00  *  C)  0’ 00  C)  z  0’ 00  In  n  0’  0’  N N  c  C C)  0’ 00  00 00  ci 0’  00 00 ‘.0  ci 0’  00 00  r 0’  C)  ci 0’  ‘.  C  ci 0’  If)  N  ci 0’  N  00  ci 0’  If)  0’  ci 0’  N  0’ 00  *  0’ 00  N  0’ 00  *  0’ 00  N  N  0’  .  ‘.  00  z  0’ GO  N 00  C)  N GO  .C)  N  0’  0’  0’  C’)  N  0’  N  0’  8  N  N  CN  -  C’)  C C)  ‘.6  C’)  C)  = 0’  00 00  If)  C)  0’  00 0  C)  0’  CD  © 0’  N  <  C)  C’)  00  C  C—i  00 N 0 N  If)  ‘.0 00  C’)  0’  C 00 N  ‘.0  C’)  ‘.0  ‘.0  C’)  0’  N  Cl  N C)  ‘.0 00  .  CD  N  C C)  ‘.0 00  ‘.  C CD x x x  If)  *  N 00  C’)  *  C)  N 00  00 0.  C)  N 00  00  C’)  N GO  N  N  CC)  00  N  .<  ‘.0 00  ‘0  0’ 0  Cl  N  Cl  )-I  Cl C  -<  ‘.0 00  NC-I  ‘.c  CC)  00  C’)  00  00  C’)  C  C4  00  C’)  r’)N  Cl  00  00  00  N  00  00  0’ 00  CC)  C C)  ‘.0 00  Cl  00  0’ 00  C Cl  CD C)  0’ 00  If)  ,.  0’ 00  N Il-i  C’)  C)  c  C)  ‘.0 00  ‘.0 0’ 0 N  < C)  ‘.0 00  Cl  00  —  \ 00  Cl  00  o 00  N  00  00  ci  00  00  0):; N  0’ 00  00  .  00 00  In  0’  If)  CD  00  ci  00  —  ci 00  0’  C  N  CD  00  —  If)  N 0 0’  c 00  N .0  If)  00  00 GO  00 Go  In 00  Cl  z z 00  00  In  If)  C’  Cl  *  If)  00 GO  *  N 00  N  x  00  00 00  *  x  N  C’) C  I,)  00  00 00  N 00  CD C)  00  00  If)  C’)  00 00  C  <  .C  C C)  C 0 00 N  CD C)  00 00  00 00  Ifl  00  N  C—)  00 00  .  Cl  C  If)  0 N  CD C)  00  < C)  I,)  N  < C)  00  C  If)  00  ‘)r’)  Cl  00  —  00  If) 00  0’ \C  C’) C  00 00  00 N  Cl  0’  C)  00  00 00  C’)  N  )  00  z 00  —  00  00  —  00  C’)  00 N  00 00  N  \O N  .  00  C) 00  Q N  N  .  C)  n 00  < C)  0.c  z 00 00  ‘.0 C’? N  N  00 00  N  CD  *  Cl  N  N 00  C’)  *  x  CD  00  .  00  .  If)  ‘.0  © 00  C) 90  ‘.0  N  CGO  C’)  c 0.  .<  e 00  —  C) 00  O<Z  0OCCl0O  C  .  C  00  I 00  C— 0  z t, 00  0’ N  O<Z  0’ N  Appendix 7— Assignments of the DHB-domain Residue  Atom Shift  (ppm)  Residue  Atom Shift (ppm)  Residue Atom  Shift (ppm)  Residue  Atom Shift (ppm)  93  HB*  2.06  97  HB*  3.94  100  HD1*  0.83  105  HA  4.31  93  HE2 1  7.42  97  RN  8.90  100  RD2*  0.54  105  RB  2.50  93  HE22  6.78  97  N  124.00  100  HG  1.58  105  RB’  1.42  93  HG*  2.22  98  CA  58.12  100  RN  8.03  105  HD*  7.07  93  RN  8.08  98  CB  41.40  100  N  118.54  105  RD2  7.06  51.33  105  HE*  7.23  93  N  115.46  98  CD!  24.84  101  93  NE2  114.03  98  CD2  23.78  101  CB  20.21  105  HE2  7.23  94  CA  55.49  98  CG  27.18  101  CO  176.47  105  RN  7.91  94  CB  31.26  98  CO  180.40  101  4.36  105  HZ  6.89  94  CD  43.47  98  HA  4.21  101  HA RB*  1.68  105  N  119.32  94  CG  27.70  98  RB  1.79  101  RN  7.28  106  CA  63.46  94  CO  175.41  98  RB’  1.62  101  N  121.39  106  CB  27.75  94  HA  4.31  98  HD1*  0.98  102  CA  59.45  106  CO  177.98  2.11  98  HD2*  0.94  102  CB  66.93  106  HA  3.73  94  HB  CA  94  HB’  1.63  98  HG  1.76  102  CO  175.57  106  RB  3.35  94  HD*  3.12  98  HN  9.40  102  HA  4.61  106  RB’  2.56  94  HG’  1.76  98  N  123.67  102  HB*  4.38  106  RN  7.21  94  HN  7.26  99  CA  61.40  102  RN  7.50  106  N  120.97  94  N  115.56  99  CB  39.07  102  N  115.85  107  CA  56.47  95  CA  51.85  99  CD*  130.75  103  CA  54.04  107  CB  38.33  95  CB  19.93  99  CE*  130.42  103  CB  18.16  107  CG  175.84  95  CO  176.10  99  CO  179.68  103  CO  179.74  107  CO  177.96  2.82  107  HA  4.33  95  HA  4.50  99  CZ  128.57  103  HA  95  HB*  1.39  99  HA  4.29  103  RB*  1.14  107  RB  2.87  95  HN  7.17  99  RB  3.31  103  RN  8.92  107  HB’  2.80  95  N  123.09  99  HB’  2.90  103  N  126.17  107  RD21  7.55  96  CA  57.80  99  HD*  6.55  104  CA  59.86  107  HD22  6.89  96  CB  32.29  99  HD2  6.55  104  CB  29.20  107  RN  8.10  96  CD2  118.46  99  HE*  6.40  104  CG  36.37  107  N  118.46  96  CEI  139.17  99  RE2  6.38  104  CO  179.07  107  ND2  112.62  96  CO  177.66  99  RN  7.36  104  HA  3.95  108  CA  65.19  96  HA  4.34  99  HZ  6.63  104  HB  2.15  108  CB  38.91  96  RB  3.15  99  N  119.57  104  1.92  108  CD!  14.94  96  RB’  3.07  100  CA  56.99  104  RB’ HG*  2.36  108  CG1  29.42  96  HD2  7.21  100  CB  41.42  104  RN  8.41  108  CG2  16.90  96  HEI  7.84  100  CDI  25.72  104  N  118.25  108  CO  177.80  96  HN  8.68  100  CD2  22.74  105  CA  57.54  108  HA  3.72  96  N  119.26  100  CG  26.42  105  CB  37.31  108  RB  1.97  97  CA  62.47  100  CO  177.90  105  CD*  131.57  108  RD1*  1.12  97  CB  62.47  100  HA  3.81  105  CE*  130.89  108  RGI*  1.23 0.99 7.94  97  CO  177.24  100  RB  1.87  105  CO  175.65  108  HG2*  97  HA  3.95  100  RB’  1.51  105  CZ  128.23  108  RN  241  Appendix 7— Assignments of the DHB-domain Residue  Atom Shift (ppm)  Residue  Atom Shift (ppm)  Residue Atom  Shift (ppm)  Residue  Atom Shift (ppm)  108  N  121.32  112  RG2*  0.78  116  N  121.51  120  HG  2.11  109  CA  57.55  112  RN  8.51  117  CA  58.99  120  HG’  2.06  109  CB  42.87  112  N  118.55  117  CB  30.62  121  CA  54.40  109  CDI  25.92  113  CA  58.63  117  CD  43.63  121  CB  18.77  109  CD2  24.07  113  CB  42.49  117  CG  29.33  121  CO  179.76  109  CG  27.09  113  CDI  24.92  117  CO  178.43  121  HA  4.36 1.51  CO  178.35  113  CD2  25.43  117  FIA  4.04  121  RB*  109  HA  3.62  113  CG  28.08  117  HB*  1.84  121  RN  8.58  109  RB  1.54  113  CO  178.02  117  RD*  3.11  121  N  119.99  109  HB  0.97  113  HA  4.01  117  RG*  1.62  122  CA  54.93  109  HD1*  0.24  113  HB*  1.67  117  RN  7.80  122  CB  32.21  109  HD2*  0.57  113  HD1*  0.97  117  N  113.44  122  CD  29.06  109  HG  0.77  113  HD2*  0.96  118  CA  60.29  122  CE  42.28  109  RN  8.57  113  HG  1.53  118  CB  64.14  122  CG  25.66  109  N  120.59  113  RN  8.29  118  CO  175.14  122  CO  175.78  110  CA  62.61  113  N  120.65  118  HA  4.44  122  HA  4.53  110  CB  62.66  114  CA  61.43  118  HB  4.00  122  RB  1.81  110  CO  176.94  114  CB  62.80  118  RB’  3.95  122  HB’  1.77 1.66  109  110  HA  4.02  114  CO  178.49  118  FIN  7.60  122  RD*  110  HB*  3.86  114  HA  4.11  118  N  112.65  122  HE*  2.95  110  RN  8.46  114  HB*  3.97  119  CA  53.78  122  HG  1.51  110  N  113.19  114  RN  7.53  119  CB  30.68  122  HG’  1.26  111  CA  59.34  114  N  111.65  119  CD  43.93  122  RN  7.98  111  CB  30.06  115  CA  59.03  119  CG  27.32  122  N  116.67  Ill  CD  43.36  115  CB  28.84  119  HA  4.95  123  CA  59.45  111  CG  27.56  115  CD  43.70  119  RB*  2.01  123  CB  42.25  111  CO  178.45  115  CG  27.69  119  RD  3.32  123  CD1  23.83  111  HA  4.10  115  CO  177.85  119  RD  3.26  123  CD2  26.78  111  HB  1.90  115  HA  3.90  119  HG  1.71  123  CG  26.85  111  HD*  3.19  115  RB  1.33  119  HG’  1.65  123  CO  177.83  111  HG  1.65  115  RB’  1.03  119  RN  8.22  123  HA  4.05  111  HG’  1.43  115  RD  3.01  119  N  121.29  123  RB  1.88  111  RN  7.69  115  HD’  2.95  120  CA  65.30  123  1.57  1.64  0.90  111  N  122.40  115  RG*  120  CB  31.66  123  RB’ RDI*  112  CA  66.64  115  RN  8.09  120  CD  50.31  123  HD2*  0.67  112  CB  31.20  115  N  121.53  120  CG  27.36  123  HG  1.31  112  CGI  21.75  116  CA  55.02  120  CO  179.17  123  RN  7.52  112  CG2  22.20  116  CB  19.37  120  RA  4.54  123  N  120.16  112  CO  177.18  116  CO  179.10  120  RB  2.51  124  CA  62.77  112  HA  3.40  116  HA  3.99  120  RB’  2.05  124  CB  36.79  1.54  120  RD  3.73  124  CD*  132.94  8.43  120  HD’  3.41  124  CE*  118.10  112  RB  1.89  116  HB*  112  HGI*  0.58  116  RN  242  C  C C))  I  CC  C))  E  rM  0?  .0  C  C C  C)) C  C 0. -C C)) C  0? = 01  .  —  Il) —  i) —  I))  00 0’ 00 N  C  f  —  —  Il) —  If) —  If) —  If) —  If)  —  ‘0 N  z 0)  Cl  < L) Cl 0)  L)  00  ‘0  N 0. 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N  00 0) —  I1  C 00  C  C)  ,  0) —  If)  0’ N  C)  C C) C)  ‘0 0’  = 0)  —  0)  N N  .  —  C) C)  N N  ‘0  N 0) —  — — 00 C C C C0’0ONC  N 0) —  N C) c. N  z 0) 0) —  00  0) N  0) Cl  ‘0  N  Cl  N  a’  0’ Cl  00  —  ‘0  c z z x x x x C) x  C  <  -  N N  C)  0) 0)  If) od N  C r  N  0) 0)  c) N 0’ N  —  C)  c: c::’ 0) 0)  6  C)  C L)  a’ Cl  0’ Cl  a —  Cl —  If)  NN—N  If)  Cl —  ‘0 —  Cl  .odod’00)C0’0’0000—  N  Cl  c  C  f)  —  (-)  N  C.  L)  —  0) 0) —  C’ N  0) 0)  < (-)  ‘0  If  00  -  —  XZc)L)c  —  —  N ‘0  If)  —  If) Cl —  Nc’)—CC  If) Cl *  ‘)  0’ Cl  00  N r C ‘0  < L)  N  Cl  C z z 00  Cl  0’  r) 00  00  —  Cl  00 Cl  00 00 00  Cl 0)  00  N n  —  N  *  00  If)  .  00  Cl  C  Cl 0)  c x x Cl 0) —  C ‘C  -  N N  N N  If) Cl —  0’ — 00 N N C’ C N .C’00’0000  00 Cl  N  —  0  4  ‘0  C  C L)  Cl 0)  .  C  N  C  —  Cl  0) —  N a. N  *  00  Cl  .  If) Cl —  ‘0  If) Cl —  —  00  Cl  x x x  <  1-  C N ‘0 N  N  N  c L) Cl 0) —  0)  Cl  .  —  —  0)  0)  N N  c-)  00  L)  m  L)  C  L)  00 Cl  c)  z 00  .  N  0’ N  0)  —  —  —  0)  —  NNsC’000—  —  Cl  00  Cl  00  Cl  —  N Cl  C  N  —  sc’  0 00  N Cl  00  —  0)  00  *  N Cl  If  —  N N  N Cl  Cl —  0)N  Cl  —  r 00  —  0)  .  C  N Cl —  ‘0 N  —  < z c-)  © 0) —  C N  *  N ..4  N c: ct = x Q x x N r’I  N  00 — N 0) 0) 0) N C C C odCC0’NN0000If  —  v Cl  00 0) —  0’  00 0) —  ‘0  00  00 0) —  00 00  x  —  0)  —  0)  00  N C  *  N  00 0) —  N  Z  If)  C  00  C  00 0)  00 0) —  00  00 0) —  If‘0  .  00  *  x  —  C)  *  N  .  C  —  00  0)  .  0) r  0)  —  —  ‘0  0)  z x x  C)  0)  —  ‘0 N  C  0)  .  N  0)  —  00  N  00  C  C  0)  c.  00  0)  00  0)  0)  00  *  N  x x x  0)  *  0)  N  <  —  C  N Cl —  0)  —  N  0)  C  Cl —  .  0)  Cl —  N  N  Cl —  N  Cl —  Appendix 7— Assignments of the DHB-domain Residue  Atom Shift (ppm)  Residue  Atom Shift (ppm)  Residue  Atom Shift (ppm)  Residue  139  CA  53.08  140  HG*  1.44  142  CD  29.29  143  139  CB  19.25  140  HN  8.36  142  CE  42.21  139  CO  Atom Shift (ppm) HA  4.36  143  HB*  1.61 0.90  177.65  140  N  120.19  142  CG  24.81  143  HD1*  CA  56.31  142  CO  176.19  143  HD2*  0.84  139  HA  4.27  141  139  HB*  1.35  141  CB  33.32  142  HA  4.32  143  HG  1.63  139  RN  8.86  141  CD  29.29  142  HB  1.83  143  RN  8.39  139  N  127.01  141  CE  42.21  142  HB  1.76  143  N  124.78  142  HD*  1.70  144  CA  54.72  140  CA  56.19  141  CG  24.80  140  CB  33.08  141  CO  176.17  142  HE*  3.01  144  CB  40.37  140  CD  29.25  141  HA  4.28  142  HG*  1.43  144  CO  178.33  CE  42.24  141  HB*  1.78  142  RN  8.35  144  HA  4.41  140  CG  24.87  141  HD*  1.69  142  N  123.49  144  HB  2.75  140  CO  176.42  141  HE*  2.98  143  CA  55.25  144  HB’  2.70  140  HA  4.31  141  HG*  1.40  143  CB  42.56  144  HD21  7.52  140  HB  1.84  141  RN  8.22  143  CD1  25.06  144  HD22  6.80  140  HB  1.74  141  N  122.94  143  CD2  23.44  144  RN  7.93  140  HD*  1.70  142  CA  56.31  143  CG  27.03  144  N  124.00  140  HE*  3.00  142  CB  33.22  143  CO  176.15  144  ND2  112.79  140  244  Appendix 8  —  2338 complex C Assignments of Rassfl ” 2338 in the DHB/Rassfl ” C  38 complex. 23 DHB/RassflC $w in the w 3 23 Appendix 8 Assignment of RassflC Sample concentration of the Rd complex was 1 mM, NMR sample buffer (10 mM MOPS 0.1 mM EDTA, pH 6.5, 25 °C). Hydrogen atoms with degenerate shifts are renamed as Q. ,  Residue Atom  Shift (ppm)  Residue Atom  Shift (ppm)  Residue Atom Shift (ppm)  Residue Atom Shift (ppm) HN  7.90  HZ  7.07  35  N  118.80  35  QD  7.10  QE  7.16  CA  62.20  CB  69.79 21.77  22  CA  43.63  27  HB3  3.85  32  CA  58.00  22  HA  3.87  27  HN  8.20  32  CB  29.97  35  23  CA  58.75  27  N  115.98  32  CG  36.53  23  CB  63.96  28  CA  55.00  32  HA  3.98  23  HA  4.50  28  CD  41.23  HN  8.10  QB  3.93  28  HA  4.65  32  N  119.61 1.95  36  24  CA  56.60  28  HB2  2.75  32  QB  24  CB  29.22  28  HB3  2.67  32  QG  2.22  36  CG2  24  CG  34.00  HN  8.46  33  CA  56.77  36  HA  24  HA  4.30  N  122.60  33  CB  29.04  36  HB  7.80  28  -  -  4.06 4.06  24  HB2  2.09  29  CA  59.55  33  CG  33.71  36  HN  24  HB3  2.01  29  CB  63.78  33  HA  4.07  36  N  113.86  24  HE2I  7.60  29  HA  4.34  33  HE21  7.37  36  QG2  1.07  24  HE22  6.84  29  HB2  3.90  33  HE22  6.78  CA  52.59  29  HB3  3.85  33  HN  8.00  37  CB  19.40  HN  8.23  33  N  118.77  37  HA  4.08  116.31  33  NE2  112.12  1-IN  33  QB  1.84  N  HN  8.68  24  N  122.17  24  NE2  112.69  29  N  24  QG  2.35  30  CA  57.37  -  7.90 125.39  25  CA  56.93  30  CB  30.12  33  QG  2.07  37  QB  1.19  25  CB  30.05  30  CG  36.61  34  CA  58.68  38  CA  55.83  25  CG  36.49  30  HA  4.23  34  CB  38.50  38  CR  30.97  25  HA  4.29  I-IN  8.39  34  CD1  132.60  38  CD  43.25  25  H132  2.05  30  N  122.39  34  CEI  117.92  CG  27.03  25  HB3  1.99  30  QB  2.04  34  HA  4.27  38  HA  4.16  HN  8.45  QG  2.28  34  HB2  2.69  38  HB2  1.61  25  N  121.09  31  CA  56.04  34  HB3  2.77  38  HB3  25  QG  2.29  31  CB  42.10  34  HN  7.84  26  CA  54.71  31  CD1  25.21  34  N  119.62  26  CR  41.22  31  CD2  24.16  34  QD  6.76  26  HA  4.62  31  CG  27.20  34  QE  6.61  QG  1.35  26  HB2  2.72  31  HA  4.11  35  CA  58.18  39  CA  58.39  26  HB3  2.65  31  HB2  1.55  35  CB  38.33  39  CR  30.23  26  HN  8.28  31  HB3  1.51  35  CD1  131.38  39  CD1  126.44  26  N  120.97  31  HG  1.51  35  CE1  131.15  39  CE3  121.08  27  CA  58.92  HN  7.97  35  CZ  129.12  39  CH2  123.88  27  CB  63.87  31  N  120.78  35  HA  4.45  39  CZ2  114.17  27  HA  4.41  31  QD1  0.83  35  HB2  3.04  39  CZ3  121.27  27  HB2  3.89  31  QD2  0.77  35  HB3  2.90  39  HA  4.44  25  -  245  -  1.44  FIN  7.83  38  N  119.54  38  QD  -  2.94  Appendix 8 Residue Atom  -  ’’ complex 2338 ” in the DHB/RassflC 2338 Assignments of RassflC  Shift (ppm)  39  HB2  3.23  39  HB3  3.04  HD1  7.05  HE1  9.94  39  HE3  7.50  39  HH2  7.04  39  HN  7.51  39  HZ2  7.29  39  HZ3  6.97  39  N  126.54  39  NEI  128.44  246  2338 complex C Appendix 9— Assignments of the DHB-domain in the DHB/Rassfl ”  w complex 2338 Appendix 9 Assignment of the DHB domain in the DHB/RassflC Sample concentration of the Dr complex was 1 mM, NMR sample buffer (10 mM MOPS 0.1 mM EDTA, pH 6.5, 25 °C). Hydrogen atoms with degenerate shifts are renamed as Q. ,  Residue 53  Atom CA  Shift  (ppm)  56.38  Residue Atom 57  CD  Shift (ppm) Residue  Atom  Shift (ppm)  Residue  Atom  29.08  60  I-IN  9.13  64  CA  64  CB  Shift (ppm) 60.27  53  CB  30.53  57  CE  42.21  60  N  123.91  53  CD2  119.93  57  CG  24.93  60  QG  1.25  64  CG  53  CEI  138.14  57  HA  4.23  61  CA  58.03  64  HA  53  HA  4.66  57  HE2  2.71  61  CB  41.31  64  HB2  24.91  64  HB3  2.11  -  29.34 37.03  -  -  4.11 2.28  53  HD2  7.04  57  HE3  2.67  61  CD1  53  HEI  7.95  57  HG2  1.17  61  CD2  23.69  64  HG2  2.66  54  CA  55.58  57  HG3  1.01  61  CG  27.43  64  HG3  2.28  54  CB  32.65  57  RN  8.34  61  HA  4.15  64  RN  54  CE  16.87  57  N  123.08  61  HB2  1.70  64  N  54  CG  31.92  57  QB  1.63  61  HB3  1.55  65  CA  59.36  54  HA  4.45  57  QD  1.49  61  HG  1.66  65  CB  32.17  54  HB2  2.11  58  CA  59.54  61  RN  8.40  65  CE  42.16  118.16  65  CG  24.89  8.28 -  123.77  54  HB3  1.98  58  CB  29.39  61  N  54  HG2  2.51  58  HA  4.55  61  QD1  0.94  65  HA  54  HG3  2.45  58  I-IN  8.20  61  QD2  0.87  65  HG2  54  HN  8.40  58  N  124.09  62  CA  60.20  65  HG3  1.48  54  N  122.01  58  QB  2.87  62  CB  29.26  65  UN  7.82  54  QE  2.05  59  CA  59.50  62  CG  36.48  65  N  120.51  55  CA  45.29  59  CB  37.83  62  HA  3.94  65  QB  2.00  55  HN  8.42  59  CD1  133.09  62  HB2  2.12  66  CA  57.86  55  N  110.06  59  CEI  117.96  62  HB3  1.90  66  CB  43.77  55  QA  3.94  59  HA  4.58  62  HG2  2.41  66  CD1  56  CA  56.20  59  HB2  3.15  62  HG3  2.24  66  CD2  56  CR  33.30  59  HB3  2.34  62  RN  8.63  66  CG  27.38  56  CD  29.30  59  RN  8.39  62  N  122.19  66  HA  4.09  56  CE  42.20  59  N  126.79  63  CA  55.22  66  HB2  1.92  56  CG  24.92  59  QD  7.11  63  CB  37.44  66  HB3  1.16  -  4.15 1.59  -  -  27.38 23.09  56  HA  4.33  59  QE  6.80  63  HA  4.36  66  HG  56  HB2  1.79  60  CA  61.94  63  HB2  3.07  66  RN  56  HB3  1.66  60  CB  33.89  63  HB3  2.42  66  N  120.06  56  RN  8.13  60  CE  42.35  63  HD21  6.87  66  QD1  0.88  56  N  121.05  60  CG  24.94  63  HD22  6.34  66  QD2  0.77  56  QG  1.38  60  HA  3.96  63  RN  8.95  67  CA  62.57  57  CA  56.39  60  HB2  1.87  63  N  118.69  67  CB  57  CR  33.52  60  HB3  1.83  63  ND2  103.30  67  CD1  247  1.86 -  -  -  8.51  39.23 131.66  C/D  C  (I,  rJ)  C  U,  C. C.  S C  Oe  C’,  C C.  (ID  C  = C’,  ‘4-b  ‘.‘D  00 N  XX  00 N  Ij N  00 N  CO  If) N  C  N  C N N  N  .  N  u()XX  00 N  If) N N  If)  CO  N  If)  N  ‘0  00  0  00  00  If)  N  N  C’  N  If)  r  N  If)  N  If)  N  CC  00  L)L)XX  N  C’  N  r  If)  If)  CO  If)  N  m  —  —  N  C  rNN-CON-  II N  N  —  N  ‘C  IN  ‘C  ‘C  00  N  ‘C  ‘C  N  00  — 00  N —  — 00  — 00  CC N  00  .  CC N  N  CC N  — 00  N  00  c  ‘C  00  N  0  00  —  0  —  ‘C N  N  ‘C N  r)  ‘C  —  C’)  N  ‘C  N  C’)  N  CC  ‘C N  00  ‘C N  —  i,-,  ‘C N  N  r)  N  CC  00  ‘C  N N  N  N  ‘C  N  N  C’)  ‘C  C’)  CO  00  N N  C’)  ‘C  N  00  N N  N  ‘C  CO  C’)  ,-c<CZ C.) XXX  N  NNNN  ‘C N  N  N  ‘C N  C’  ‘C  N N  C.)  ‘C  ‘  N N  CL.  ‘tD  ‘C  N  -  N N  _,.  N N  ‘C  N  0  ‘C  CO ‘IC CO N C’ N .‘oáN  —  XX  N  C.)  N  —  ‘C  L)  —  N  c  N  —  N  N  —  ‘C  L)  N  —  L)  —  N  L.)  —  ‘C  N N  N  ‘C  N N  N  —  ‘C  N N  N  —  ‘C  N  N  —  ‘C  C’  —  r  ‘C  ‘C  N  N  ‘C  I — — rD CC N Nrr 0 ç  ‘C  —  N  N N  ‘-4 00  N N  r,  00  C’  ‘  N  CO  N  C’)  C’  N  C’)  N  N  00  N N  c  00  N N  N  r—  r’)  —  N  Co  N 00  CC  N  N 00  m  N 00  .  N N  —  N 00  X  N  N  N  c  N N  C.)  N  N N  N  N  N  i  C’ r’  N  N  N  0  N N  N 00  N N  N  N  —  N  C’)  N  —  C’)  N  CO 00  N  ID  N N  00  CO  N  00  00 N  ‘  ‘C  N  00 N  N  If) N  N  If) N  < C.) C.)  00  !) 00  00 N  00 N  ‘0  N  CO  N  ‘0  N  N  N  00  2  CD  2 C  S C.  CiD  S  C  0  © o  C  ‘I,  —  F 00  00  0  — o  —  00  00  r  8 —  00  00  C—  t 00  —  \C  S C. C.  2 C  S C. C.  C,)  S C  00  00  r  —  00  00  .  —  in  —  in  —  —  in  —  t 00  r 00  •  00  00  m  •  SC  00  SC  z  00 — — çrr  00  .  00  .  z ) 00  in  z —  o  00 00  in  — o  00 00  00  \C  a  ‘  00 00  .  in  —  —  00 00  —  00  00  r-  00 00  ‘r  00 00  •1-  00 00  00  .  r-  z 00  00  00  C  —  00 00  in 00  tm000’  00  t  00  ir  00 00  ‘.c  00 00  00  t-  ‘‘  .  e o,  c  co  0’  00 00  in  00  in  00  r—  z ri  C  r-  ri  0’ 00  —  0’ 00  r—  .  0’ 00  0  t  0’ 00  in  in  00  SC  00  SC  00  oc  SC 00  8  0’ 00  oo  r-  o  oe  o  00  i-  0’ 00  00  SC  00  SC  0’ 00  SC  —  00  0’ 00  00 0’ r.) 00 C — C C’ 00 .t — 0  0’ 00  z in  00  00  0’ 00  00 00  in  00  00 00  in 00  SC r— — c o 0 o°h  in 00  l 00 r—r1  in 00  00  z o  .  0’  0’  SC  00  C  00 0  C  00  00  SC 00  0’  o  00  00  0’  o  0’  r  00  SC  SC  00  SC  00  00  00  .  00  —  0’  .  0’  SC  00  00 0  00  0’  00  0’  00  8  —  0’  00  z  00  DHB/RassflC complex w Appendix 9—Assignments of the DHB-domain in the 2338 Shift (ppm) Residue  Atom Shift (ppm)  Residue Atom  Shift (ppm)  Residue Atom  Shift (ppm)  Residue  Atom  99  CA  61.41  103  RN  8.93  107  ND2  112.54  111  RN  7.66  99  CB  39.15  103  N  126.10  108  CA  65.23  111  N  122.44  99  CD1  130.50  103  QB  1.12  108  CB  38.99  111  QD  3.14  15.06  66.67  99  CE1  130.19  104  CA  60.10  108  CDI  112  CA  99  CZ  128.38  104  CB  29.20  108  CG1  29.46  112  CB  31.26  99  HA  4.24  104  CG  36.54  108  CG2  17.04  112  CGI  21.95  99  HB2  3.26  104  HA  3.94  108  HA  3.67  112  CG2  22.29  2.12  108  HB  1.92  112  HA  3.37  99  HB3  2.88  104  HB2  99  HN  7.34  104  HB3  1.90  108  RN  7.90  112  HB  1.88  99  HZ  6.60  104  RN  8.45  108  N  121.11  112  RN  8.51  99  N  119.44  104  N  118.24  108  QD1  1.09  112  N  118.41  108  QG1  1.17  112  QG1  0.57  99  QD  6.49  104  QG  2.34  99  QE  6.36  105  CA  57.54  108  QG2  0.95  112  QG2  0.76  100  CA  57.10  105  CB  37.28  109  CA  57.54  113  CA  58.63  100  CB  41.57  105  CD1  131.23  109  CB  42.96  113  CB  42.53  100  CDI  25.70  105  CE1  130.30  109  CD1  26.04  113  CD1  24.95  100  CD2  22.83  105  CZ  128.09  109  CD2  24.18  113  CD2  25.48  100  CG  26.56  105  HA  4.27  109  CG  27.20  113  CG  28.12  100  HA  3.79  105  HB2  2.47  109  HA  3.59  113  HA  100  HB2  1.84  105  HB3  1.40  109  HB2  1.50  113  HG  100  HB3  1.49  105  RN  7.88  109  HB3  0.94  113  RN  100  HG  1.54  105  HZ  6.86  109  HG  0.73  113  N  100  1-IN  8.01  105  N  119.21  109  RN  8.54  113  QB  1.65  100  N  118.56  105  QD  7.04  109  N  120.40  113  QD1  0.93  100  QDI  0.80  105  QE  7.20  109  QD1  0.21  113  QD2  0.94  100  QD2  0.51  106  CA  63.48  109  QD2  0.54  114  CA  101  CA  51.34  106  CB  27.82  110  CA  62.82  114  CB  62.77  101  CB  20.31  106  HA  3.70  110  CB  62.78  114  HA  4.09  101  HA  4.34  106  HB2  3.31  110  HA  4.01  114  RN  101  I-IN  7.24  106  HB3  2.52  110  RN  8.42  114  N  101  N  121.24  106  RN  7.18  110  N  113.11  114  QB  3.95  101  QB  1.66  106  N  120.92  110  QB  3.83  115  CA  59.16  102  CA  59.57  107  CA  56.56  111  CA  59.40  115  CB  28.76  102  CB  66.89  107  CB  38.44  111  CB  30.11  115  CD  43.62  102  HA  4.58  107  HA  4.29  111  CD  43.32  115  CG  27.85  102  HN  7.47  107  HB2  2.82  111  CG  27.56  115  HA  3.86  102  N  115.80  107  HB3  2.75  111  HA  4.06  115  RB2  1.30  102  QB  4.39  107  HD2I  7.52  111  HB2  1.89  115  HB3  1.02  103  CA  54.06  107  HD22  6.88  111  HB3  1.83  115  HD2  3.00  103  CB  18.31  107  RN  8.08  111  HG2  1.60  115  RD3  2.91  103  HA  2.81  107  N  118.47  111  HG3  1.41  115  RN  8.04  250  3.99 —  1.51 8.30  -  -  -  —  120.51  61.48  7.51 111.63  ci  —  .  c)  — t-  .  “C LI.  a  a !)  .C ci  z  ci  a  w  p c  w  —  c  c  0  I\)  I  C  -  “C 00  —  a  a )  a  a  IN.)  X C)  X C)  00  a IN) ci  00 00  9  — IN) ci  0 00  9  —  a  00 00 “C  a IN) ci  ) c, 00  c  — —  -  a  a  ci  cc  X  a IN) —J  o  9  -J  z  — I  C)  — •3  -  —  C)  — .a  -  C) ‘C ‘C > L’)  C)  C)  C)  —  -  — te  -  00  a  a  .  ci  X  a IN ci  ci L1  —  — —  —a C  a  00  00  IN-.)  C) C)  — IN —1  -)  X C)  —  —  —  -  — —  — — c  00 IN.)  .)  IN.) C “C  C) C)  — IN ci  LI.  00  X  — I.i  —  W  z  a  : 00 C  C)  — IN ci  00 00  X  a  IN.) ON  00 00 00  .)  C)  a IN ci  X >  — I.i  XXX  — —  — —  a IN) N  .  00  p  t-J  C)  J  —  aN  a IN)  cc  I  t’3  C)  a k—i  0’  C)  — —  ci LI.  a  ON  ,  ON ci  ON LI.  a  9N ci LI.  LC LJ  N LC LI.  C) ‘C ‘C X  — IN) ci  p c  C) C)  — I-.i  XX  — —  a  a  -  —  a  ON  IN.)  — IN) ON  t-J LI  -  C) X  -  2  a  —a  N C  a IN) ON  -  C  LI  — — 00  — — 00  — — 00  —  — — 00  -  — — 00  -  a  C  )  a  IN.)  .)  I  a IN) ON  .  aN  9  a  ci  J  3  IN-.)  X  — IN) ON  LI  9N  z  ,J  a  a  : ON IN.)  .  z  a IN) ON  I  ci \C  — t’J  cc —  a  90 LI. IN.)  tj  C)  a IN) ON  -)  X C) ‘  iJ  —  LI  z  a  èo —I  w  C)  a IN) ON  ON  X C) I’)  a  L  a IN)  IN.) C  —a  .)  C)  a IN) ON  —a ON  (-h)  X  —  —  X C) Zz)>  — — C  -  C) e >  00  LI  -.i  C)  — —  C)  — —  UI  a IN)  LI.  X  -.i  —  — 00  —  a IN) UI  I’) LI. LI LI  C)  C)  a  ci  -  — —  -  — —  a IN)  ci  LI. :‘C  “C  a IN)  ci 00  9  — ci  a IN)  ci LI.  9  C) ‘C ‘C C) >- C) IN-.)  a IN) ON  00 I’)  L’J  X X  — ,J -.e  — — 00  -  .)  ‘  a IN)  .  )  IN.)  z  a IN) UI  (  IN.) IN.)  .  e  C)  a  — — ci  -  LI.  C  a IN)  00  :-..l  a IN) UI  “C  I-) C  C)  I-  a  -  — —  -  2  a IN)  00 C  X  a IN) UI  IN.)  .)  C) X  a  -  -  — —  -  — — ci  -  -  — —  -  — — ci  -  — — ci  -  — — ci  -  a  a IN)  LI. ci  .)  a IN) UI  ci ci  LI.  a IN) LO  .)  IN.) -a  IN.)  C) C)  a IN) UI  00  00  a IN) 00  00  IN.)  C) C)  a IN UI  C “C ON  z  a  a  ci  a  C) ‘C >  a IIsi  —  C  z  a IN) 00  —a  C)  a IN) UI  .  00 (I.  —  a  _)  a IN) .  00 00 00  C) X  a -J a  tJ  a IN) .  .  LI.  -  C)  — -J —  —  —  C  a IN) 00  .  C  9N  ON  -  “C  a IN) 00  ci L)  9N  LI.  00  a  —a C ON  C) ‘C ‘C >- nj  a IN) UI  LI.  .)  X >-  a  a  t)  Z>C)>  — — ci  -  —  “C  00  a  .  .)  2:  a IN) ..  IN.)  IN.)  X C) )  =  a IN)  -‘  — —  “C ci  00  a  00  -  00  a IN)  00  IN.)  L’J  X C)  a IN  — e  ‘  -  00  a  IN.) 00  L  X  a IN)  .)  X  a IN)  — —  .  00  .)  IN-.) 00  IN-.)  a IN)  “C  aN  .)  IN.)  X  =  a IN)  00  — —  — — (Il  ON  00  a  C LJ  X  a IN) .  J  I  X X  — IN)  C  C  ci  a  .  a “C  nj  C)  a IN) .  00  IN..)  IN.)  X  a IN)  -C)  — —  ON  ci  a  w IN.) LI. LI.  C)  a IN)  LI. IN.)  X >  =  a IN)  z  — —  =  B  C  >  =  C  CD  C  CD  —  CM  —  CM  CM  —  —  CM  CM  —  CM  — —  CM  ,-  L’3  00  d  .  —  . .  a  —a  00  C  2  . =  —  00  00  a  C-k)  00  .  —  C  V  a  .  —  00  :  00  .  3  ‘c  t’J  —  C  c)  ,-  (-J  —  C  C.) C  ZZ  ‘.  —  00  ‘j  1j  n  c-  (-  —  =  (Th  —  . ©  —  )00C00  C  00  .  —  ‘  —  —  .  —  p C c  c  >  C)  . ©  —  —  —  .  —  d  —  —  C  :1 C  z  —  Cd \  —  XX--  —  zzc.)rI>  cj  —  a  c,  00 00  cd  —  —  d  a  .  k)  —  C  —  .J  —  -c w C  (-  —  •  —  C  tJ  c  c-:  ci  —  L)  —  —  —J  -  C  z  00  d  C  —  .  L1  —a  d 00  —  C_  —  J  .  —  C  00  t  ,  —  —3  .  —  cC  00  x  Cdd  t  a  —  00  —  —  00 —  d  .  —  CJ  -  x  00  a  —  a  C  L,  Q  00  C.e  a  —  a  w  00  90  c)  >  Th  00  J  a  —  a  t-a  00  z  —  —  c  J  —  —  .  C  a (J —  —  -  a  C-,  00  tn  -  —  —  —  -  — lc  —-a  00  -  Cid  —  C  —  a  —a  t)  .-  a  -  —  —  C  a —  —a  C  ‘-a  -J  x  -J  —  00  —  —a  a —  >  —  Cid  —  —  a a  —a  00  (Th tn  Cd  —  —  —  — a  —a C  t-)  t’J  (-  —a  —  —  —a  a  3 —a  00  L3  c  —.  (.d  —  —  Ci  —  —  —  g  t  .  3 00  a  -a —a  c  —  IJ  —  00  —  Q 0  >  r  —  c.  —  t-J  —  —  —a —a  —  a  c  00  a a  —a C  —  C_.  b-.)  —  -  a a  —a  CJ  i—a  2  —  Cdd  —  .  ‘.c  —  C  a a  .  C  x  d  —  —  C 00  a  -  a . a  —a ,.c -a  Th  (.ii  a  C  —  (..J  —3 -d  a  .  .  a  --a  eCo  c,  C—)  a  a  •—  —  Q  a  .  ©  —  o  C)  —  i CM  (  —  a  —  a  D  CM  d  —  —  —  I  Cd)  C  C  =  €  C/)  B  B  c—i)  C  C  C,,  L,J  CD  0  r;I) Cd)  -  C  C  (I)  Appendix 10  —  Triangulation of the spin label  Appendix 10 Triangulation of the spin label  A > >  restart; eql:=  > )2  eqi (x— 79.3241 + (y4.386)2 + (+ 5.251 237.16 (x_83.481)A2+(y+2.193)2+(z+4.234)A2=13.1A2; eq2:= =  eq3:=  eq2:=(.v—83.481)++2.193)+(2+4.2341=171.61 (x_81.420)A2+(y+5.838)A2+(z+O.168)A213.3A2;  eq3 := (.v— 81.420)2 + (v+ 5.838)2+ (z + .168)2 solve({eql,eq2,eq3},{x,y,z}); =  74.80499681. c =  =  -11.24742530. v  6.543513866. v  =  =  -9.059500877  2.712728785 .x  =  =  176.89  1.  89.08359047  B  Ii ‘1.’  14.4  L47 IHN  V38 IHN  L44HN/O  ,.•‘.  NO• (A) Sample of a Maple worksheet used for solving three equations of a sphere with the co HN as centers, and calculated reff as radius. (B) Position of ordinates of Va138, Leu44 and Leu47 1 the two solutions on the structure of SUMO-1. One solution is impossible as it overlaps with other atoms in the molecule. The only acceptable solution gives an approximate location of the spin label.  253  

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