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Dissecting the mechanism of ETV6 polymerization Huang-Hobbs, Helen 2013

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 Dissecting the Mechanism of ETV6 Polymerization by Helen Huang-Hobbs B.Sc., McGill University 2009  A Thesis Submitted in partial fulfillment of the requirements for the degree of Master of Science in The Faculty of Graduate and Postdoctoral Studies (Chemistry) The University of British Columbia (Vancouver) December 2013 ? Helen Huang-Hobbs, 2013 i Abstract ETV6 (or TEL), a member of the ETS family of eukaryotic transcription factors, normally functions as a transcriptional repressor and putative tumor suppressor. ETV6 is modular, containing a SAM (or PNT) domain and a DNA-binding ETS domain joined by a flexible linker sequence. The ETV6 SAM domain self-associates in a head-to-tail fashion, forming helical polymers proposed to generate extended repressive complexes at target DNA sites. ETV6 is also frequently involved in chromosomal translocations yielding unregulated chimeric oncoproteins with the SAM domain fused to the catalytic domain of a tyrosine receptor kinase such as NTRK3. Cellular transformation likely results from SAM domain-mediated polymerization and constitutive activation of the kinase domain. In the case of the ETV6-NTRK3 fusion (EN), this transformation is linked to congenital fibrosarcomas. Our goal is to investigate via mutations within its SAM domain, the thermodynamic and dynamic mechanisms underlying the altered transformation properties of ETV6-NTRK3. These studies have been carried out using monomeric variants of the isolated SAM domains with "head" or "tail" point mutations that prevent self-association, yet allow for formation of a mixed dimer with a native binding interface. Specifically, we used a combination of NMR spectroscopy and isothermal titration calorimetry to study the effects of additional mutations on their dimerization. Consistent with its involvement in a crystallographically-observed interdomain salt bridge, mutation of Lys99 was found to weaken the association of ETV-SAM monomers in solution, and to disrupt cellular transformation by EN. This supports the role of the SAM domain self-association in the activation of ETV6-NTRK3, and helps define the mechanisms underlying cellular transformation by similar chimeric oncoproteins. ii Preface Chapter 1 and 4. Versions of these data (Table 2, Figure 6, Figure 22, Figure 23, Figure 24) have been published within the manuscript: Naniye Cetinbas, Helen Huang-Hobbs, Cristina Tognon, Gabriel Leprivier, Jianghong An!, Steven McKinney, Mary Bowden, Connie Chow, Martin Gleave, Lawrence P. McIntosh and Poul H. Sorensen. Mutation of the Salt Bridge-forming Residues in the ETV6-SAM Domain Interface Blocks ETV6-NTRK3-induced Cellular Transformation. The Journal of Biological Chemistry. 2013; 288:27940-27950. ? the American Society for Biochemistry and Molecular Biology. Mass Spectroscopy (Figure 13, Figure 26) was run by Jason Rogalski of the CHiBi Proteomics Core Facility. For the NMR studies, I prepared the samples myself and the experiments were set up with the assistance of Mark Okon. Preliminary results from Eric Escobar were consulted in the design of this project.  Figure 6 was produced by our collaborators (Naniye Cetinbas, Cristina Togon, and Poul Sorenson)  iii Table of Contents Abstract ............................................................................................................................................ i!Preface............................................................................................................................................. ii!Table of Contents........................................................................................................................... iii!List of tables.................................................................................................................................... v!List of figures................................................................................................................................. vi!Abbreviations............................................................................................................................... viii!Acknowledgements......................................................................................................................... x!Chapter 1! Introduction ............................................................................................................. 1!1.1! ETS PROTEINS AND THEIR SIGNIFICANCE ............................................................................. 1!1.2! THE SAM DOMAIN .............................................................................................................. 2!1.3! THE ETV6 GENE AND PROTEIN............................................................................................ 6!1.4! RECEPTOR TYROSINE KINASES AND THEIR ROLE IN CELLULAR REGULATION ....................... 9!1.5! ETV6-NTRK3 FUSION GENE ............................................................................................ 11!1.6! SUMOYLATION OF ETV6................................................................................................... 13!1.7! THE ROLE OF K99 AT THE BINDING INTERFACE ................................................................. 14!1.8! GOALS AND HYPOTHESIS ................................................................................................... 17!Chapter 2! Materials and methods........................................................................................... 18!2.1! DESIGNING CONSTRUCTS OF ETV6 PNT DOMAIN ............................................................. 18!2.1.1! Monomeric N90SA93D-ETV61-125 ............................................................................. 18!2.1.2! ETV643-125 constructs ................................................................................................. 21!2.2! PROTEIN EXPRESSION AND PURIFICATION.......................................................................... 22!2.3! NMR SPECTROSCOPY........................................................................................................ 24!2.4! ISOTHERMAL TITRATION CALORIMETRY............................................................................ 25!Chapter 3! NMR studies of the ETV6 PNT domain ............................................................... 26! iv 3.1! PROTEIN NMR: A GENERAL INTRODUCTION...................................................................... 26!3.2! AN NMR ASSIGNMENT OF ETV6 PNT DOMAIN ................................................................ 26!3.2.1! An assignment of the backbone residue signals of a full-length construct................ 28!3.2.2! A shortened construct and doubled peaks ................................................................. 31!3.2.3! Secondary structure analysis..................................................................................... 41!3.3! COMPARING THE VARIOUS MONOMERIC POINT MUTANTS BY NMR................................... 45!3.4! AN NMR TITRATION OF ETV6 PNT DOMAIN MONOMERS................................................. 49!Chapter 4! Binding studies of the SAM-ETV6 domain using ITC......................................... 52!4.1! INTRODUCTION TO ITC ..................................................................................................... 52!4.2! K99 AFFECTS BINDING OF MONOMERS............................................................................... 53!Chapter 5! Concluding remarks and future directions ............................................................ 59!5.1! GOALS OF THIS THESIS....................................................................................................... 59!5.2! FUTURE WORK................................................................................................................... 62!Bibliography ................................................................................................................................. 63!Appendices.................................................................................................................................... 68!Appendix A: Chemical shift assignments..................................................................................... 68!Appendix B: SSP Scores from NMR assignments ....................................................................... 74!Appendix C: ITC titration results ................................................................................................. 78! v List of tables Table 1. The primers used for PCR .............................................................................................. 19!Table 2. Binding thermodynamic parameters determination from ITC measurements................ 57!Table 3. A summary of the chemical shift residue assignments for N90SA93D-ETV61-125 ....... 68!Table 4. A summary of assignment of chemical shift residues for A93D-ETV643-125. ................ 71!Table 5. SSP score values for A93D-ETV61-125 ........................................................................... 74!Table 6. SSP score values for A93D-ETV643-125 .......................................................................... 76!Table 7. ITC results for individual experiments ........................................................................... 78! vi List of figures Figure 1. Structures of several SAM domains ................................................................................ 4!Figure 2. The structure of the ETV6 SAM domain with a native dimer interface ........................ 5!Figure 3. Head-to-tail SAM domain polymerization...................................................................... 8!Figure 4. Structure of the ligand binding domain of TrkC and the kinase domain of TrkB ........ 10!Figure 5. Modular structures of wild type ETV6, ETV6-NTRK3 chimera, and wild type NTRK3 ............................................................................................................................................... 12!Figure 6. A summary of results from our collaborators in the Sorenson lab................................ 16!Figure 7. Schematics of the initial ETV6 1-125 and subsequent ETV6 43-125 constructs ................ 20!Figure 8. Strategy for assignment of protein NMR signals. ......................................................... 27!Figure 9. An assignment of the 1H-15N HSQC spectrum of N90SA93DETV61-125 ..................... 30!Figure 10. An assigned 1H-15N HSQC spectra for A93D-ETV643-125 ......................................... 32!Figure 11. A 1H-15N HSQC monitored pH titration of A93D-ETV643-125.................................... 35!Figure 12. Overlaid 1H-15N HSQC spectra of A93D-ETV643-125recorded over 4 days................ 37!Figure 13. Reconstructed ESI mass spectrum of A93D-ETV643-125............................................. 38!Figure 14. 1H-15N HSQC spectrum of N90SA93D-ETV643-125.................................................... 40!Figure 15. SSP scores for N90SA93D-ETV61-125 ........................................................................ 43!Figure 16. Secondary structure SSP score for the A93D-ETV643-125 .......................................... 44!Figure 17. A93D-ETV643-125 construct overlaid with A93DK99R-ETV643-125 construct............. 46!Figure 18. 1H-15N HSQC overlay of A93D-ETV643-125 with V112E-ETV643-125 ........................ 47!Figure 19. 1H-15N HSQC overlay of A93DK99R-ETV643-125 with V112E-ETV643-125 ............... 48!Figure 20. A 1H-15N HSQC monitored titration of unlabeled A93D-ETV643-125 into 15N-labeled V112E-ETV643-125................................................................................................................. 50! vii Figure 21. A 1H-15N HSQC monitored titration of unlabeled A93DK99R-ETV643-125 into 15N-labeled V112E-ETV643-125 .................................................................................................... 51!Figure 22. An ITC titration of A93D-ETV643-125 into V112E-ETV643-125 ................................... 54!Figure 23. An ITC titration of A93DK99R-ETV643-125 into V112E-ETV643-125.......................... 55!Figure 24. An ITC titration of A93D-ETV643-125 into V112EK99R-ETV643-125.......................... 56!Figure 25. A cartoon summary of the native binding interface of the ETV6 SAM domain ........ 61!Figure 26. EMS m/z measurement of A93D-ETV643-125.............................................................. 79! viii Abbreviations 2D  two-dimensional 3D  three-dimensional Da  Dalton D2O  deuterium oxide DNA  deoxyribonucleic acid DTT  dithiothreitol EMSA  electrophoretic mobility shift assay ETS  E-twenty-six transformation specific ETV6  ets-variant gene six EN  ETV6-NTRK3 HSQC  heteronuclear single quantum coherence  IPTG  isopropyl-!-D-thiogalactopyranoside ITC  isothermal titration calorimetry MAE  modulator of the activity of Ets MOPS  3-(N-morpholino)propanesulfonic acid NMR  nuclear magnetic resonance NTRK  neurotrophic tyrosine kinase PCR  polymerase chain reaction PNT  pointed PTK  protein tyrosine kinase ppm  parts per million RNA  ribonucleic acid   ix SAM  sterile alpha motif SDS  sodium dodecyl sulfate SUMO  small ubiquitin-like modifier TEL  translocation Ets leukemia Tris  tris(hydroxymethyl)aminomethane WT  wild type  x Acknowledgements This work presented in this thesis would not have been possible without guidance from and support of numerous people. Firstly and foremost I would like to thank my supervisor Dr. Lawrence McIntosh who gave me the opportunity to work on this project. I would also like to thank past and current members of the McIntosh lab for their advice, support, and time. In particular I would like to thank Dr. H. Jerome Coyne for his implementation of protein purification protocols which were the basis for the techniques used in the majority of this thesis, Desmond Lau for his help with learning new techniques and troubleshooting protein precipitation problems, and Dr. Mark Okon for his endless NMR knowledge and patience in teaching me.  Genevieve Desjardins, thank you for the helpful scientific discussions, editing expertise, and ongoing support and friendship over the years. I would also like to thank Dr. Soumya De for his patience and time in teaching me how to run ITC and discuss my results, and Dr. Laura Packer for generosity with her time, helpful editing, and pep-talks.  I would like to thank my family and friends for their support in all of my endeavors. Particularly, Pippa Payne, your outpouring of encouragement and motivating work sessions have been invaluable in the completion of this thesis. My thanks and gratitude to Lyndsey Earl, for supplying meals, pictures of puppies and helpful chats. Lastly my mother, father, and brothers Maxwell and Emmet have been irreplaceable and their support of me through the years this is something I am forever thankful for.  1 Chapter 1 Introduction 1.1 Ets proteins and their significance ETS (E26 transformation specific) proteins play a significant role in cellular differentiation and development. These proteins form a family of eukaryotic DNA-binding transcription factors that direct gene expression by binding to promoters and enhancers and facilitating assembly of transcriptional machinery [23]. ETS members include, among others, PU.1, Ets-1, Fli1, and ETV6 [2]. The biological roles of ETS members vary from organ cell fate determination to neuronal development, oogenesis, and hematopoiesis [2, 3, 5, 15, 23, 37, 38, 47]. Because of the wide variety of functionality of ETS proteins, errors in regulatory pathways of ETS factors can result in a multitude of cellular problems [18]. Understanding the regulation and role of these proteins is of particular interest because of their involvement in oncogenesis. ETS proteins all have a conserved winged helix-turn-helix ETS domain that binds to the major groove of DNA over a region of ~ 9 base pairs with a common 5? GGA(A/T) 3? core [23, 40]. The structural mechanisms for DNA-binding by several ETS domains have been determined from X-ray crystallography and NMR spectroscopy [23]. Although the DNA binding domain of ETS proteins is highly conserved among the 28 members of the ETS family, they have a wide array of cellular functions. This diversity amongst ETS family members arises from numerous routes including additional regulatory domains located elsewhere on the proteins, post-translational modifications, and protein-protein interactions. One such regulatory domain that is found in approximately one third of ETS members is the protein-protein interacting SAM (or PNT) domain.  2 1.2 The SAM domain The SAM (sterile alpha motif) domain is a commonly found helical bundle with diverse functions that generally involve serving as a protein-interaction module in homo-SAM, hetero-SAM, and heterotypic interactions with non-SAM domain containing proteins. Some SAM domains bind even to RNA [18, 25]. Proteins containing SAM domains can dimerize or oligomerize with one another depending on cellular conditions and the structure of the particular SAM domain. The SAM domain is found in over two hundred and fifty regulatory proteins including receptor tyrosine kinases, serine and threonine kinases, transcription factors and adapter proteins [24]. A subset of about one-third of ETS transcription factors, including Ets-1, Ets-2, Erg, Fli-1, GABP! , and ETV6 contain a SAM domain [33].  Because of its role as a protein-interaction module, SAM domains are diverse in both function and regulation. SAM domains are important for the cellular regulation of transcription. One well-documented example of the SAM domain?s role in transcriptional regulation is exemplified by the Sevenless EGF-receptor signaling pathway leading to Drosophila eye development. The pathway involves two ETS factors; Pointed-P2 is an activator of developmental genes in Drosophila, whereas Yan is a repressor [23]. Transcriptional repression by Yan requires higher order structure formation, which is mediated by Yan-SAM polymerization [44]. Mae (modulator of the activity of Ets) has a SAM domain, but not an ETS domain, and aids in Yan depolymerization by binding to its SAM domain [23]. When Mae binds to Yan it exposes a phosphoacceptor site on Yan allowing it to be phosphorylated, by the Rolled kinase (activated by EGF signaling). The resulting phosphorylated Yan is subject to nuclear export and cytoplasmic degradation [30]. Meanwhile, the SAM domain of Pointed-P2 is a docking site for Rolled and its phosphorylation leads to transcriptional activation of many genes  3 including that encoding Mae. As a feedback mechanism, Pointed-P2 is also regulated by Mae. In this case, Mae can heterodimerize with the Pointed-P2 SAM domain, blocking Rolled docking and reducing phosphorylation-dependent transcriptional activation. This example shows that protein partnership and post translational modification of SAM domains are essential for both the activation and repression of a transcriptional pathway.  High resolution structures of many SAM domains have been determined from both X-ray crystallographic and NMR spectroscopic studies (Figure 1) [23]. The core structure of the SAM domain is a four !-helical bundle (denoted as helices H2-H5 for consistency with literature publications) with a short 310 or !-helix (H2?) [23]. The SAM domain structures of ETS family members, Ets1, Pnt-P2, Erg, GABP!, Yan, and ETV6 all show the common !-helical core of the SAM domain with slight variations [11, 47-50, 53]. The interaction surfaces of the SAM domain have been termed the mid-loop (ML) and end-helix (EH). In the crystal structure of the ETV6-SAM domain, the interactions between these two surfaces results in an extended superhelical head-to-tail polymer (Figure 2) [24]. ETV6, Yan, and likely ETV7 are the only ETS proteins with SAM (PNT) domains having exposed ML and EH interfaces necessary for self-association in this head-to-tail fashion. All others, including Ets1 and Pnt-P2, have one or both surfaces blocked by amino acid substitutions or appended helices and are therefore monomeric in solution [23].  4   Figure 1. Structures of several SAM domains. Clockwise from top left: ETS1 (2JV3.pdb), EphB2  (1SGG.pdb), EphA4 (1B0X.pdb), and p73 (1DXS.pdb). Helices are labeled and are colored from N-terminus to C-terminus in blue to red. The common SAM domain core helical structure can be readily seen. H5 H3 H4 H0 H1 H2 H5 H2  H2? H2 H3 H1 H2 H2 H5 H4 H3 H4 H5 H2? H3 ETS1 EphB2 p73 EphA4  5  Figure 2. The structure of the ETV6 SAM domain with a native dimer interface (1LKY.pdb). Mutation of either Ala93 to Asp (A93D; green) or Val112 to Glu (V112E; red) prevents self-association. However, the A93D and V112E mutants can still form a ?heterodimer? with a native interface when added together. Terminal residues (S47 and Q123 respectively) and helices H2-H5 are also labeled.   6 1.3 The ETV6 gene and protein ETV6 (also known as TEL or TEL1 for translocation Ets leukemia) is an ETS family member that plays key roles in embryonic development as well as hematopoietic regulation and is essential for yolk sac angiogenesis [29]. The ETV6 gene consists of 8 exons, with alternative start codons at positions 1 and 43 [12]. Full length ETV6 protein contains 452 amino acids with an N-terminal SAM domain and C-terminal ETS domain. Unlike most ETS factors that activate gene expression, ETV6 is a transcriptional repressor and reported tumor suppressor [32]. Critical to its role as a transcriptional repressor is its ability to self-associate via the SAM domain [12].  SAM domain self-association is hypothesized to allow ETV6 to cooperatively bind adjacent DNA recognition sites as an extended polymer [19]. In contrast, the isolated, monomeric ETV6-ETS domain binds consensus sequence with relatively low affinity (2.8 x 10-8 M) [19]. This low affinity is caused by a dynamic appended inhibitory helix that sterically blocks the DNA-binding interface of the ETS domain and thereby attenuates DNA binding ~50-fold [19]. Previous work by our lab in collaboration with the Graves group (University of Utah), supports a model where ETV6 binds to DNA as a cooperative polymer, thereby compensating for the otherwise low affinity of the inhibited ETS domain [19]. This result was obtained using electrophoretic mobility shift assays (EMSAs) to measure the dissociation kinetics of monomeric versus dimeric forms of ETV6 from DNA. These data showed that ETV6 dimers were more stably bound than monomers on tandem ETS binding sites, supporting the theory that the SAM domain mediated oligomerization of ETV6 would allow overall higher affinity binding as a cooperative polymer [19]. As mentioned previously, the native interface of the ETV6-SAM domain has been determined by crystallographic methods (Figure 2) [24, 55]. The interfaces have a non-polar core  7  with residues Met79, Ala83, Leu86, and Leu87, on the ML surface and Phe67, Leu69, Val112, and Leu106 on the EH surface [55]. Several interdomain salt bridges surround this hydrophobic core including Glu66 to Lys82 ' , Arg95 to Asp101 ' , Asp91 to Arg93 ' , Glu90 to Lys89 ' , and Asp91 to Lys89 '  (with ? denoting the partner protein) [55]. Salt bridges contribute to overall protein structure and stability. These salt-bridge interactions are not necessarily conserved and some mutations can be accommodated at the dimer interface [55].  The polymerization of wild-type ETV6-SAM domain makes in vitro biophysical studies difficult. Fortunately point mutations for the ETV6-SAM domain have been found which substitute in a hydrophobic to charged residue on either the ML or EH surface [23]. These mutations (A93D and V112E ) disrupt polymerization of the SAM domain and are also monomerizing. Mixing a ML -mutant with EH -mutant yields a "hetero -dimer"  that is tightly associated (K d=1.7  nM by surface plasmon resonance [24]) via their respective wild-type interfaces (Figure 3). This dimer is used as a model polymer because it retains one wild-type interaction between the ML and EH surfaces and allows for investigation  of the native binding surface of the SAM-ETV6 domain.  8  Figure 3. Head-to-tail SAM domain polymerization of native ETV6 (left). Solubilizing mutations A93D and V112E are highlighted. A mixture of the two monomerized mutants results in a heterodimer with a native binding interface (right). Based on SAM-ETV6 domain crystal structure, Bowie and co-workers proposed a model whereby monomers self-associate in a head-to-tail fashion with a 65-screw symmetry to form an extended, repressive polymer along DNA [24]. Evidence for this model was obtained by adding soluble monomerized mutants to wild type protein in order to cap the otherwise insoluble polymers [24]. Subsequent electron microscopy showed soluble filaments with a width dimension similar to those of the crystallized helical polymer [24]. This suggested that ETV6 can form a polymeric structure that links many DNA binding elements together and spread repression over a large segment of chromatin [24]. Furthermore, the proposed model shows the C-terminus of the SAM domain pointing outwards away from the polymer axis [24]. The C-terminal ETS domain is therefore also pointed outwards and can help recruit chromatin to toroidally wrap around the polymer [24]. The model is also supported by a 53 ? repeat of the ETV6 polymer, which is the same dimension as a nucleosome core particle [24]. The authors proposed that once nucleated on DNA, only weak-binding affinity for DNA is required for further ETV6 protein association [24]. Thus, the entropic cost of uniting protein and DNA is paid by the strong SAM-SAM interactions [24]. This is consistent with the previously mentioned studies of Dr. Graves showing that co-operative binding to tandem DNA sites by ETV6 overcomes the auto-inhibition of its ETS domain [19].   9  1.4 Receptor tyrosine kinases and their role in cellular regulation Mis-regulation of Neurotrophin-tyrosine kinase receptors (NTRKs) contribute s significantly to neuroblastoma development [31]. TrkC (also called NTRK3) in particular has been shown to be involved in soft tissue cancers [21]. Furthermore, it is of interest to us because of its involvement in an oncogenic chimeric protein, resulting from a chromosomal translocation that fuses the genes encoding a functional SAM domain of ETV6 with the receptor tyrosine kinase domain of NTRK3.  Tyrosine kinases are important regulatory proteins that transfer a phosphate group from ATP to a tyrosine residue of a protein. Receptor tyrosine kinases (RTK s) are a subset of these proteins that also have a transmembrane domain linking an extracellular receptor domain to the intercellular catalytic domain. As such, RTKs are central for signal transduction and cellular regulation. The oncogenic potential of aberrant tyrosine kinase receptors is well documented and thus they are attractive drug targets [28].  Neurotrophin-tyrosine kinase receptors are RTKs which are essential for development and maintenance of the nervous system [57 ]. These proteins generally consist of a ligand binding domain, as well as a kinase domain with a linker region. The crystal structure of the TrkC  kinase domain has been solved in complex with an inhibitor (3V5Q.pdb) [1] and related protein kinase domains TrkA, and TrkB have also been solved by X -ray crystallography (4F0I .pdb and 1HCF .pdb respectively) [4]. The crystal structure of the TrkC  ligand binding domain has also been previously determined (Figure 4, 1WWC.pdb ) [57 ].  10   Figure 4. Structure of the ligand binding domain of TrkC (top, 1WWC.pdb) and the kinase domain of TrkB (bottom, 1HCF.pdb). The kinase domain of TrkC is similar to that of TrkB.  11 1.5 ETV6-NTRK3 fusion gene ETV6 is involved in numerous chromosomal translocations, several of which result in fusion genes encoding chimeric oncoproteins that contribute to the development of leukemia or leukemogenesis [12]. In these chimeric oncoproteins the SAM domain of ETV6 mediates polymerization that is critical for transformation activity. Frequently, the translocations fuse the SAM domain to either a DNA-binding transcription factor such as AML or a protein tyrosine kinase (PTK) such as PDGFR!, ABL, JAK2, ARG, or FGFR3 [12, 29]. These resultant fusion proteins have oncogenic potential and are often associated with myeloid and lymphoid malignancies [60]. Transformation of cells by ETV6 chimeras with PTK's have been shown to require both the active tyrosine kinase domain and an intact SAM oligomerization domain [29]. As previously mentioned, one of these translocation products fuses the N-terminal SAM domain of ETV6 to the C-terminal tyrosine kinase domain of NTRK3 (Figure 5). This chimeric fusion (EN) was first characterized by Dr. Poul Sorensen?s group at the BC Cancer Agency [26]. EN polymerizes via the SAM domain, which results in auto-phosphorylation and constitutive activation of the tyrosine kinase domain [55]. This fusion shows in vivo transformation activity in soft agar colony [29], a technique which will be discussed in detail later. The introduction of the previously mentioned monomerizing mutations A93D and V112E into the SAM domain prevents polymerization and abrogates transformation of cells in soft agar assays [54]. However, replacement of the SAM domain with an inducible dimerization system was shown to not transform cells [54]. Thus, oligomerization, rather than simple dimerization appears to be necessary for cellular transformation and oncogenic activity.   12   Figure 5. Modular structures of wild type ETV6 (top), ETV6-NTRK3 chimera (middle), and wild type NTRK3 (bottom). The EN chimera results from fusion of the ETV6 PNT domain with the NTRK3 protein tyrosine kinase (PTK) domain. Protein domain boundaries are noted for the ligand binding Ig-like C2 domain (cyan), extracellular domain (red), transmembrane domain (orange), SAM domain (green), ETS domain (purple) and protein tyrosine kinase domain (blue).    13 1.6 Sumoylation of ETV6  As mentioned previously, post-translational modification of proteins is an important way of regulating function. Sumoylation of ETV6 was initially reported to occur at a non-consensus Lys99 within the ordered SAM domain [9, 10, 61]. Small ubiquitin-like modifier (SUMO) is a protein that becomes covalently linked to other proteins in order to mediate their function [20]. Sumoylation of proteins is an important post-translational cellular regulatory mechanism and results from several enzymatic steps similar to the ubiquitin E1-E2-E3 cascade in order to occur [20]. SUMO has been shown to help regulate transcription factors [16]. In most cases, sumoylation inhibits transcription, although the mechanism is not fully understood and likely involves the recruitment of additional co-repressors [16]. Sumoylation frequently occurs in unstructured regions of proteins at a consensus modification site ! KXE where !  is a large hydrophobic residue, E is glutamate, X is any residue, and K is the lysine sumoylation site [16]. Several studies have shown that mutation of the non-consensus Lys99 site to an arginine residue affects localization and potentially the ability of the SAM domain to polymerize [45]. Initially this effect was attributed to the inability of the arginine residue to act as a sumoylation site. However a more recent study has shown that Lys11 is the primary sumoylation site in the SAM-ETV6 domain and this post-translational modification was found to inhibit ETV6 repression of gene expression [45]. This study also showed that both monomers and oligomers of ETV6 were sumoylated in vitro, whereas in vivo only oligomers were found to be sumoylated [45]. Although Lys99 is not the primary sumoylation site for the SAM domain of ETV6 is it of interest due to its proximity to the ML/EH interface and the non-transforming phenotypic change that occurs in soft agar assays when it is mutated to an arginine residue [8].  14 1.7 The role of Lys99 at the binding interface In a previous collaborative effort between the Sorensen and McIntosh group, the monomerizing mutations A93D and V112E were found to abrogate cellular transformation by EN [54]. More recently, Naniye Cetinbas, a graduate student in the Sorenson group, observed similar effects while investigating the role of Lys99 [8]. In particular she found that the K99R and K99D mutations reduced high molecular mass complex formation of EN and abrogated its transformation activity. Additionally, soft agar assays were conducted on various constructs of EN. NIH3T3 fibroblasts were used in these assays with an empty vector used as a negative control, and WT-EN was used as a positive control. When the cells are grown in the soft agar, negative controls undergo detachment-induced apoptosis, whereas those cells transformed by WT-EN have been previously shown to be resistant to detachment-induced apoptosis [35, 39]. Several EN constructs were tested, one lacking the SAM domain of the ETV6 portion of EN (!SAM-EN), a kinase dead EN (K380N-EN) and K99R-EN. The !SAM-EN, and K380N-EN were both found to be non-transforming and K99R-EN was shown to have high levels of PARP, a marker for detachment-induced apoptosis. Additionally, these three cell lines showed smaller sized spheroids in the soft agar analysis when compared to cells expressing WT-EN. These results are summarized in Figure 6. She also showed that mutation of Asp101, the intermolecular salt bridge partner of Lys99 to alanine or lysine similarly blocked transformation of NIH3T3 cells by EN, reduced EN tyrosine phosphorylation, inhibited Akt and Mek1/2 signaling downstream of EN, and abolished tumor formation in nude mice. In contrast, mutations of Glu100 and Arg103 residues in the vicinity of the interdomain Lys99-Asp101 salt bridge, had little or no effect of these oncogenic characteristics of EN. Her results underscored the importance of Lys99 and Asp101 for SAM polymerization and EN transformation. Her results  15 led to the hypothesis that these residues contribute to a salt bridge important for SAM domain polymerization and therefore important for transformation. Using several biophysical techniques I sought to test this hypothesis regarding the non-transforming phenotype present in the K99R-EN cell line.   16    Figure 6. A summary of results from collaborators in Sorenson lab; (A) Soft agar colony formation assay shows K99R mutation gives lower colony formation at  (B) Western blot analysis of cleaved PARP to probe detachment-induced cell death. (C) Pictures depicting the spheroid sizes formed by the NIH3T3 cells expressing the indicated constructs. Empty MSCV vector was used as a negative control, KD-EN indicated a kinase-dead version of EN !SAM-EN indicates a version of EN without a SAM domain, and K99R-EN has the single point mutation K99R. (D) NIH3T3 cells stably expressing the indicated constructs were seeded in soft agar. Colony formation was shown to depend on the residue selected for point mutation. Versions of this data have been previously published [8].  17  1.8 Goals and hypothesis The goal of this project was to investigate the role that Lys99 plays in the binding interface of the ETV6 SAM domain. Additionally we sought to determine how a mutation of Lys99 could alter the phenotypic transformation of cells by EN in soft agar assays. We hypothesized that the effect of Lys99 and Asp101 mutations on cell transformation is due to the attenuation of a SAM domain polymerization via disruption of an intermolecular salt bridge. The specific aims of this thesis were therefore to investigate the effect of point mutations on the structure of the isolated ETV6-SAM domain in solution as well as on the thermodynamic parameters underlying polymerization.  Using NM R spectroscopy and isothermal titration calorimetry (ITC ), I found that mutation of Lys99 to arginine does not significantly perturb the structure of the isolated ETV6-SAM domain with the monomerizing A93D substitution. However m ixed dimer formation was weakened 400-fold when the K99R mutation was present in the wi ld-type interface. This quantitatively demonstrated the importance of intermolecular salt bridges in SAM domain polymerization. Furthermore these results support the hypothesis that cellular trans formation by EN is critically dependent upon SAM d omain-mediated self-association, and thus is particularly relevant in terms of oncogenesis.   18 Chapter 2 Materials and methods 2.1 Designing constructs of ETV6 PNT domain  2.1.1 Monomeric N90SA93D-ETV61-125 Initially, the gene encoding a fragment of ETV6 spanning residues 1-125 (Figure 5) was generated by standard PCR methods [27] from the full length EN gene provided by the Sorenson laboratory. Primers used for this PCR reaction can be found in Table 1. This first construct, denoted N90SA93D-ETV61-125, encodes the SAM domain (residues Pro56-Gln123) with the monomizering mutation A93D, preceded by the full N-terminal segment of ETV6. Using introduced EcoRI site and NdeI restriction sites, the PCR product ligated into a pET28a plasmid (Invitrogen). The resulting gene also included an N-terminal His6-tag and a thrombin cleavage site. The ligated plasmid was transformed into Escherichia coli DH5! cells and grown on LB agar plates containing kanamycin (35 mg/L). Plasmids from the resulting colonies were extracted using a GeneJET Plasmid Miniprep Kit (Fermentas) and analyzed by commercial DNA sequencing (GENEWIZ). It was at this time that the unwanted mutation N90S was also discovered. This initial full-length construct is shown in Figure 7 along with the amino acid sequence for the SAM domain of ETV6.  19  Primer Direction Sequence (5? to 3?) EcoRI  CTGAAGCAGAGGAAATGAGAATTCTATA 3? NdeI(1-125)  CTGAAGCAGAGGAAATGAAAGCTTTATA3? HindIII   TATAAAGCTTTCATTTCCTCTGCTTCAG 3? NdeI(43-125)  TATACATATGGAGGAAGACTCGATCCG 3? S90N Forward CAGCAACACGTTTGAAATGAATGGCAAAGATCTCCTGCTGC 3'  Reverse GCAGCAGGAGATCTTTGCCATTCATTTCAAACGTGTTGCTG 3' D101K Forward GAATGAGGAGATCGATAGCGAAATTTCTCTTTGGTCAGCAGCAGGAG   Reverse CTCCTGCTGCTGACCAAAGAGAAATTTCGCTATCGATCTCCTCATTC  N90S into A93D Forward GCAGCAGGAGATCTTTGCCACTCATTTCAAACGTGTTGCTG   Reverse CAGCAACACGTTTGAAATGAGTGGCAAAGATCTCCTGCTGC  K99R into V112E Forward GGCAAAGCTCTCCTGCTGCTGACCCGTGAGGACTTTCGCTATCGATCTCC  Reverse GGAGATCGATAGCGAAAGTCCTCACGGGTCAGCAGCAGGAGAGCTTTGCC K99R into A93D Forward GGCAAAGATCTCCTGCTGCTGACCCGTGAGGACTTTCGCTATCGATCTCC  Reverse GGAGATCGATAGCGAAAGTCCTCACGGGTCAGCAGCAGGAGATCTTTGCC V112E Forward GATCTCCTCATTCAGGCGACGAGCTCTATGAACTCCTTCAGC  Reverse GCTGAAGGAGTTCATAGAGCTCGTCGCCTGAATGAGGAGATC D93A Forward GTTCGAAATGAATGGCAAGGCCCTCCTGCTGCTGACCAAAG  Reverse CTTTGGTCAGCAGCAGGAGGGCCTTGCCATTCATTTCGAAC Table 1. The primers used for PCR are shown. From a full length EN construct, primers EcoR1, and NdeI(1-125) were used to create an N90SA93D-ETV61-125 construct in a pET28a plasmid. Primers S90N (forward and reverse) were used for site directed mutagenesis to remove the N90S point mutation found in this construct. The primer set HindIII and NdeI(43-125) were used to amplify the A93D-ETV643-125 and then ligated into pET28a or pET28-MHL vector.   20  Figure 7. Schematics of the initial ETV6 1-125  and subsequent ETV6 43-125  constructs. The SAM domain is highlighted in green. The solubilizing mutations A93D  and V112E, as well as additional point mutations N90S and K99R , are indicated. Below The wild-type amino acid sequ ence of ETV6 1-125  with the alternative start site M43 in bold and sites of interest N90, A93, K99, D101, V112, colored ( green, red, orange, blue respectively) MSETPAQCSIKQERISYTPPESPVPSYASSTPLHVPVPRALRMEEDSIRLPAHLRLQPIYWSRDDVAQWLKWAENEFSLRPIDSNTFEMNGKALLLLT K EDFRYRSPHSGDVLYELLQHILKQRK  21 2.1.2  ETV643 - 125  constructs  In addition to containing an unwanted N90S mutation, N90SA93D-ETV61-125 also contained a thrombin cleavage site at residues V37-P38-R39|| A40. Due to problems with producing stable soluble protein without a His 6-tag, and given that the residues preceding the SAM domain appeared unstructured in preliminary NMR spectra, a set of shorter ETV643-125 fragments were generated. Residue Met43 is an alternative start site for ETV6 [42] and thus was chosen as the new N-terminus. Similar constructs of this length have been previously studied [55] and thus we felt justified in using a shortened construct. We also explored the introduction of a TEV cleavage system into the clones. In order to efficiently obtain constructs, several cloning strategies were attempted concurrently. The N90S1-125 construct was first corrected back to Asn90 using site directed mutagenesis, and then using NdeI (43-125) and HindIII primer s (Table 1), a shortened version of the SAM domain was PCR amplified from this plasmid. This DNA was then cleaved using the appropriate enzymes  and ligated into a similarly cleaved pETMHL  plasmid. Site directed mutagenesis was then used to introduce the K99R  mutation into the A93D construct. Additionally shortened versions of the V112E point mutant were created using an A93D-ETV643-125-pET28a construct. First the A93D mutation was corrected back to Ala93, and then the V112E mutation was introduced. From this shortened V112E-ETV643-125 pET28a plasmid, a K99R mutation was added using site directed mutagenesis. Additionally a D101K mutation was added into the V112E-ETV643-125 construct. Site directed mutagenesis was also used to introduce N90S mutation back into the A93D-ETV643-125 shortened construct to confirm that a side deamidation reaction was not occurring. This will be discussed later in detail.   22 2.2  P rotein expres sion and purification  The sequence-verified plasmids were introduced into E. coli BL21 (!DE3) cells by either electroporation or heat shock. SOC media (1 mL) was added to freshly transformed cells and they were incubated at 37?C for 1 hr. These cells were then plated on LB agar plates with kanamycin (35 mg/L) and grown overnight at 37?C. Single colonies from these plates were selected and placed in 25 mL LB overnight. These cultures were spun down at 5000 rpm (GSA rotor Sorvall) for 15 minutes, resuspended in 1 mL media and used to inoculate larger volume expression media. The ETV6 constructs were expressed in E. coli BL21 (!DE3) cells grown at 37?C in Luria broth (LB) or minimal M9 media containing 1g/L 15HN4Cl alone or 1 g/L15HN4Cl and 3 g/L13C6-glucose (Sigma Aldrich) [36]. Kanamycin (35mg/L) was included in all media. The cells were induced at OD600 = 0.6 with a final concentration of 1 mM IPTG, grown overnight, and then harvested by centrifugation (GSA rotor, Sorvall) at 5000 rpm for 30 minutes. The cell pellet was resuspended in 50 mL of binding buffer (0.22?m filtered 20 mM imidazole, 50 mM Na2HPO4, 500 mM NaCl, pH 7.5) and either frozen for storage at -80?C or processed immediately. The cell pellet was lysed by passage through a French press twice at 10,000 psi followed by sonication (Branson Sonifier 250, VWR Scientific) at 60% duty cycle for 3x10 minutes. The lysate was spun (SS34 rotor Sorvall) at 15,000 rpm for 1 hour and the resulting supernatent was passed through a 0.8 ?m filter before being loaded on a Ni-NTA column (GE Healthcare) pre-equilibrated with binding buffer. The column was washed with several column volumes of binding buffer and then the ETV6 constructs were eluted with elution buffer (0.22?m filtered 500 mM imidizole, 50 mM Na2HPO4, 400 mM NaCl, pH 7.5). The fractions containing the desired protein were identified by SDS-PAGE gel analysis, pooled and placed in dialysis  23 tubing. For samples studied using ITC, these samples were dialysed into a buffer containing 20 mM MOPS, 50 mM NaCl, 0.5 mM EDTA pH 8.0 overnight. For NMR protein samples the appropriate protease to remove the His6-tag was also added to the dialysis tubing. Constructs with a TEV cleavage site were dialyzed overnight with TEV protease (made in house) at room temperature in 50mM Tris, 50 mM NaCl, pH 8.0, 1mM DTT. Constructs with a thrombin cleavage site were dialyzed overnight in 20 mM Tris pH 8.4, 0.15 M NaCl, 2.5 mM CaCl2 at 4 oC with 1 unit of thrombin (Novagen). Cleavage was stopped the next day with p-aminobenzamidine beads. During preparation of the 13C/15N-labeled N90SA93D-ETV61-125-pET28a construct formation of precipitate during the overnight cleavage of the His6-tag indicated sample instability. In order to collect as much data as possible, this sample was transferred to a 25 mM KCl, 25 mM Na2HPO4, pH 7.0 buffer, concentrated, and 3D NMR data was collected over the course of 4 days. Further purification of this sample was not able to be done in a timely fashion and thus, the His6-tag cleaved and uncleaved portions of the protein were not separated from one another. Additionally this sample was not purified by size exclusion, and likely contained a mixture of species.  For the shortened ETV643-125 constructs, His6-tag cleaved proteins were separated from uncleaved protein by passage through a Ni-NTA column (GE Healthcare) pre-equilibrated with binding buffer. The flow-through was collected and concentrated by ultrafiltration with an Amicon (Ultra-15; 3K) to a volume of 2-5 mL. This concentrated sample was spun to remove any precipitate, and then injected onto a pre-equilibrated Sepharose S75 gel filtration column and eluted with NMR buffer (20 mM MOPS, 50 mM NaCl, 0.5 mM EDTA pH 8.0). Fractions containing desired protein were identified using SDS-PAGE, pooled and concentrated for further  24 analysis. Sample concentration was evaluated by absorbance values at A280 using Beer?s Law based on the predicted molar absorptivity (20950 M-1cm-1) given by the ProtParam program (http://web.expasy.org/protparam/). Mass and purity were confirmed by SDS-PAGE gel. After cleavage with either thrombin or TEV cleavage enzymes, some residual amino acids remain before the wild-type M43 or M1 start sites. Leader sequences of either MGSSHHHHHHSSGLVPRGSHIH (for thrombin cleavage ? pET28a vector) or MHHHHHHSSGRENLYFQGHIH (for TEV cleavage ? pET28MHL) were incorporated into the V112E and A93D variants, respectively. 2.3 NMR spectroscopy ETV6 samples were at 0.25-1.0 mM in the previously defined NMR buffer with an additional 10% v/v D2O added for signal lock. Spectra were recorded at 25?C using 500 MHz Varian Unity, 600 MHz Varian Inova, 500 MHz Bruker Avance III, or 600 MHz Bruker Avance III NMR spectrometers equipped with triple resonance cryogenic probes. Resonance assignments were obtained using standard main-chain correlation experiments, including HNCO, HN(CA)CO, HNCACB, and CBCA(CO)NH [46]. Spectra were analyzed using NMRpipe [13], nmrDraw [13], and Sparky [17]. 1H-15N-HSQC spectra were used to monitor titrations of 15N labeled A93D43-125 conducted over a pH range from 5.5-8.5 using increments of approximately 0.5 units. From this range it was determined that the protein was relatively stable and soluble over a variety of pH conditions. NMR spectra were collected at pH 7.5 in order to minimize signal loss due to hydrogen exchange and to yield good signal-to-noise. Titrations of monomers were observed using 1H-15N-HSQC spectra of 15N-labeled V112E-ETV643-125; these spectra were collected as  25 aliquots of unlabeled A93D-ETV643-125 or A93DK99R-ETV643-125 were added to a final 1:1 equivalency.  2.4 Isothermal titration calorimetry Fractions of His6-tag purified protein were pooled and dialyzed overnight in 4 L of buffer (20 mM MOPS, 50 mM NaCl, 0.5 mM EDTA, pH 8.0). The His6-tag was left on the constructs to expedite experiments. Since previously reported surface plasmon resonance experiments have been done with an intact N-terminal His6-tag [24] we conclude that leaving the tag attached would produce comparable binding data. Proteins were concentrated using an Amicon Ultra-15 3K filter. Due to limited solubility, V112E-ETV643-125 and V112EK99R-ETV643-125 mutants were at 0.019-0.038 mM and used within the ITC cell. A93D-ETV643-125 and A93DK99R-ETV643-125 were concentrated further (0.16-0.4 mM) for use in the ITC syringe.  ITC measurements were run using an ITC200 instrument (GE Healthcare). Experiments consisted of 20-25 injections (1.5-2.0 ? L) of A93D-ETV643-125 or A93DK99R-ETV643-125 (0.16-0.4 mM) into the stirred (1000 rpm) cell. The resulting data were processed using Origin 7.0. Buffer blank titrations were performed in order to determine that there was no thermodynamic contribution due to dilution.   26 Chapter 3 NMR studies of the ETV6 PNT domain 3.1 Protein NMR: a general introduction The use of NMR spectroscopy is a well-established tool for characterizing the structure and dynamics of a protein. The development of heteronuclear NMR experiments, paired with techniques that allowed for uniform 15N and 13C labeling, have led to the ability to fully assign the signals from most nuclei in a protein. The chemical shift information obtained during these experiments allows for structural analysis and the observation of dynamics or conformational changes caused by differing environment or addition of binding substrates.  3.2 An NMR assignment of ETV6 PNT domain The 1H-15N HSQC spectrum is a 2D NMR spectrum that shows the correlated signals between directly bonded 1H and 15N nuclei. The spectrum typically contains the same number of amide signals as the number of non-proline residues in a protein. Additionally, depending upon sample conditions, the 1H-15N signals from Trp, Gln, Asn, and Arg, Lys and His side chains may appear in the 1H-15N HSQC spectrum. Assignment of the 1H-15N HSQC spectrum of a uniformly 13C/15N-labeled protein is typically accomplished using complementary CBCA(CO)NNH and HNCACB spectra (Figure 8).   27  Figure 8. Strategy for assignment of protein NMR signals. Strips plots from HNCACB and CBCACONNH spectra are shown at the amide 15N planes of the indicated adjacent residues (A93D-ETV643-125). The 13C! and 13C"  are seen for each of the experiments. The HNCACB experiment has 13C! and 13C" shift information for both the i and i-1 residues, whereas the CBCACONNH shows only the inter-residue correlations. In addition, the 13C" (green) have opposite phases to the 13C! (red) in the HNCACB spectra, HNCACB F77 S78 L79 HNCACB HNCACB CBCACONNH CBCACONNH CBCACONNH  28 The CBCA(CO)NNH correlate the amide signals of residue i to the 13C! and 13C" of the previous residue i-1. The HNCACB correlates the amide to both the 13C! and 13C" of residues i and i-1. Additionally, in the latter spectrum 13C" signals are of opposite phase to those of the 13C! residues. Combination of these two experiments coupled with knowledge of typical 13C shifts for each amino acid type, the peaks of N90SA93D-ETV61-125 and A93D-ETV643-125 constructs were assigned. The HNCO and HNCACO experiments allow for assignment of carbonyl carbons on the protein backbone and thus helped confirm assignments obtained with the HNCACB and CBCA(CO)NNH spectra and resolve any ambiguities. Using these complementary experiments coupled with knowledge of typical 13C shifts for each amino acid type the amide signals of N90SA93D-ETV61-125 and A93D43-125 constructs were assigned.  3.2.1 An assignment of the backbone residue signals of a full-length construct  Initial constructs were made with a portion of ETV6 (residues 1-125) starting at its natural N-terminus. Although the SAM domain was expected to span residues Pro56-Gln123 we wanted to include the N-terminal sumoylation site, K11. Also, some ETS PNT domains contain a dynamic helix preceding the core helical bundle [30]. Thus we first wished to determine if the ETV6 SAM domain also had this additional helix and to characterize the N-terminal tail containing the sumoylation site Lys11. Three-dimensional NMR experiments were run on a 13C/15N labeled N90SA93D-ETV61-125 sample. Crowding of peaks in the center of the spectra indicated that many residues in this construct had random coil conformations. Unfortunately this also made a full spectral assignment difficult. During the course of this assignment it was discovered that an unwanted point mutation was located at residue N90S. Furthermore the protein was prone to degradation and thus it was  29 difficult to obtain reproducible high quality samples and spectra. Despite issues with sample integrity, the spectra were assigned with certainty to ~2/3 completeness (Figure 9, Table 3).  During the assignment, the unfortunate N90S mutation and a cryptic thrombin site were discovered in the amino acid sequence. The thrombin cleavage site was found at residues V37-P38-R39-A40. Therefore, the N-terminal region was likely partially proteolyzed during the thrombin His6-tag removal. However, some of the protein was believed to be intact as residues in this unstructured tail region were confidentially assigned. These assignments did allow for chemical shift-based secondary structure prediction to be run. Based on this analysis, the SAM domain helical boundaries were confirmed to be similar to those of the X-ray crystallographic structure. No additional N-terminal helices were discovered, and the residues preceding the SAM domain were shown to be disordered. This structure prediction analysis will be discussed later.   30   Figure 9. An assignment of the 1H-15N HSQC spectrum of the N90SA93DETV61-125 construct is shown. Full chemical shift value information can be found in Table 3. Well dispersed signals indicated folded protein.   31 3.2.2 A shortened construct with conformational heterogeneity Ongoing issues with protein stability and sample integrity caused us to a resort to a more stable construct. We decided to remove the unstructured N-terminal residues and continue binding studies with a shortened construct, predicting that the removal of the unstructured tail would result in a more stable sample. The shortened construct (residues 43-125) with the N90S mutation corrected back to wild-type Asn90, was expressed and NMR experiments were run on the resulting 13C/15N labeled A93D-ETV643-125. Unexpectedly, spectra of the shorter constructs (residues 43-125) now showed a doubling of many amide peaks in the 1H-15N HSQC, indicating more than one form of the folded protein in solution. From assignment of the A93D-ETV643-125 (Figure 10) we are able to see that two isoforms of the protein exist in solution. Similar peak intensities suggested that their populations in solution were approximately equal.   32   Figure 10. An assigned 1H-15N HSQC spectra for the shortened A93D-ETV643-125 construct. The crowded center of the spectrum is expanded in the grey inset. Note the spectral overlap and the doubling of many peaks.   33 Conformational exchange can yield distinct NMR behavior depending on its timescale and the chemical shift differences between the resulting conformations. In a simple 2-state equilibrium, fast exchange gives one peak with population-weighted chemical shift of the two forms of a protein. In contrast, two distinct signals arise with slow exchange. These two signals show the separate conformers of a protein, with information about structure from chemical shift values and population from the relative signal intensities.  One common source of multiple amide signals in 1H-15N- HSQC spectra results from the fact that, X-Pro groups can isomerize between cis and trans conformation of their peptide bond. This isomerization can provide a rate limiting kinetic barrier in protein folding [7, 14, 59] and because of its relatively high activation energy (~20 kcal/mol) [51] the interconversion between these two forms in a folded protein usually occurs on the seconds timescale or longer. However, because we observed numerous peaks of approximately equal intensity, this helps rule out the idea that the second set of peaks is caused by proline isomerization. That is, in a disordered polypeptide the cis isomer is usually populated to only ~10%. If either the cis or trans form offer a preferred folding arrangement for the protein, exchange may not occur. Also, if the isomerization has an effect on local but not the global, structure of the protein then only residues near the proline residue would have doubled peaks with similar ppm shifts, whereas those distal to the proline should yield single signals. This effect was not seen thus we concluded the doubling of peaks was not due to proline cis/trans isomerization.  Given the ability of the SAM domain to self-associate it is possible that A93D-ETV643-125 is polymerizing or dimerizing in solution despite the monomerizing mutations introduced at the binding interface. If slow exchange between self-association states were occurring, then the monomeric forms would have sharper lineshapes than the lineshapes of the oligomeric form.  34 However, based on the similar lineshape and signal intensity of the two sets of peaks, such self-association does not appear to be occurring. Furthermore doubled peaks were observed between pH 5.8-8.6 (Figure 11). Along with the fact that N90SA93D-ETV61-125 showed only single peaks, this further argues against self-association occurring.   35   Figure 11. A 1H-15N HSQC monitored pH titration of A93D-ETV643-125. The pH of the sample was raised (pH 8.6, purple) and lowered (pH 5.8, red). Whereas some signals disappeared at higher pH due to rapid exchange with solvent, overall no large rearrangement of signals indicates that the protein structure remains constant over the pH range examined.   36 Another source of the doubled peaks could be issues with purification or degradation. However, over the course of 4 days (Figure 12) the spectra are indicating that it is unlikely that the samples are degrading. Mass spectrometry was performed on the A93D-ETV643-125 sample and only one species was present (Figure 13). This result supports the theory that these doubled peaks are not due to degradation of the protein. The doubling of peaks however could be due to deamination, which causes a mass change of only 1 Da. Deamidation is known to occur in proteins, particularly at asparagine-glycine (N-G) dipeptide to yield an aspartate-glycine (D-G) [43, 52]. The same deamidation reaction can also yield an isoaspartyl residue with its neighboring residue linked via a peptide bond formed with its !-carboxyl. Indeed the Asn90 residue is followed by Gly91, making the site vulnerable to deamidation. Because the longer construct had the unfortunate point N90S mutation we hypothesized that deamidation led to peak doubling in the spectra of A93D-ETV61-125 but not N90SA93D-ETV643-125. If the deamidation led to an isoaspartyl group, characteristic patterns in HNCACB-type spectra would result [56]. Although such patterns were not observed in our spectra, the difficulty in full assignment and the presence of extra unassigned peaks did not allow us to confidently exclude deamidation via this evidence alone.  37    Figure 12. Overlaid 1H-15N HSQC spectra of A93D-ETV643-125 recorded as a function of time (purple to red). The significant overlap over the course of time showed that the sample did not degrade over a period of 4 days while 3D data were being collected. 1111101099887766t2 - 1H  (ppm)130 130125 125120 120115 115110 110t1 - 15N  (ppm) 38  Figure 13. Reconstructed ESI mass spectrum of A93D-ETV643-125 . The observed mass of 12629 Da closely matches the predicted mass of the  construct, eliminating degradation as a source of doubled peaks in its NMR spectra. Deamidation has been seen as a result of protein aging and degradation [43, 52] and is affected strongly by the C-flanking residue to the Asn/Asp-Gly pair in addition to the local flexibility of the region [43]. Deamidation results in both a small change in mass (1 Da) and a change in charge. While the mass difference is difficult to observe, in principle an isoelectric focusing gel could be used to detect if the charge of the protein has changed due to deamidation. However, we did not have ready access to such electrophoresis. Additionally this reaction is accelerated at higher pH. During a titration of the protein over a range of pH values, it was seen that the ratio of the two populations did not significantly change upon raising or lowering the pH. (Figure 11). Also deamidation is effectively irreversible and should proceed to completion rather  39 than approximately equal population of protein species. From Figure 12  we saw unchanging 1 H-15 N HSQC spectra which indicate that deamidation was not occurring to the sample over the course of 4 days.   Using site directed mutagenesis, the point mutation, N90S, was introduced in the shortened construct to eliminate the site of the potential deamidation reaction. A 1 H-15 N HSQC spectrum of the sample was collected (Figure 14 ), and the double peaks were also observed for the N90SA93D-ETV6 43-125  sample. Along with the above results, this allows us to conclude that deamidation of this sample is not occurring at the N90-G91 residues, and thus deamidation is not a source of the doubled peaks in the 1 H-15 N HSQC spectra of the shortened ETV6 43-125  constructs. Because these doubled peaks were not observed in the 1 H-15 N HSQC  spectrum of the N90SA93D-ETV6 1 -125  sample it is possible that the unstructured tail region (residues 1 -42) play a role in selecting one conformer of the SAM-ETV6 domain over another. Determining the exact cause of the multiple forms of the SAM-ETV6 43-125  in solution will require additional studies.   40   Figure 14. 1H-15N HSQC spectrum of N90SA93D-ETV643-125 construct. Again a similar pattern to the rest of the SAM-ETV6 constructs can be seen. This shortened construct (43-125) has a set of doubled peaks, eliminating deamidation of the N90-G91 site as the source of peak doubling in shortened constructs.  11111010998877t2 - 1H  (ppm)130 130125 125120 120115 115110 110t1 - 15N  (ppm) 41  While the peak-doubling observed in the various ETV643-125 1H-15N HSQC spectra made assignment and interpretation of spectra initially difficult it provided insight into the differences between the short and long constructs and we could eliminate several potential sources of the peak-doubling. From the mass-spectroscopy, collection of 1H-15N HSQC over several time-points, and pH titrations we could see that this doubling is not due to degradation of the protein over time. Additionally, the introduction of an N90S point mutation in the shortened construct coupled with the HNCACB spectra of the A93D-SAM-ETV643-125 allow us to conclude that the doubling of peaks is not due to the deamidation of residues N90-G91. Because the doubled peaks were not observed in the 1H-15N HSQC of the N90SA93D-SAM-ETV61-125 it is possible that the unstructured tail region (residues 1-42) play a role in selecting one confomer of the SAM-ETV6 domain over another. We can also conclude that mutiple forms of the SAM-ETV643-125 exist in solution and warrents future study in order to beter understand the SAM domain of ETV6.  3.2.3  Secondary structure analysis  Despite the challenges in interpreting and assigning the NMR spectra of the ETV6 constructs, relatively complete assignments were obtained. Using the SSP algorithm [34], secondary structural elements were predicted for the N90SA93D-ETV61-125 and A93D-ETV643-125 constructs based on the measured chemical shift values of their backbone nuclei (Figure 15, Figure 16). The observed 13C! and 13C" chemical shift differences were compared to those of a random coil polypeptide. Positive values correspond to alpha helices and negative values to beta strands. Despite the difficulty of assigning the double-peaked shortened constructs we were able to run secondary structure propensity on the samples using one set of the assigned amino acid residues.  42 From this approach it is clear that the characteristic SAM domain alpha helix bundle previously found in the ETV6-SAM domain crystal structure [24] is present in solution in both the full and shortened constructs of ETV6. Furthermore, no additional helices (H0 and H1) as found for the SAM domain of Ets1 are present. Rather, residues N-terminal to the SAM domain are disordered with random coil chemical shifts. While SSP scores were only obtained for the A93D variants of the long (1-125) and short (43-125) constructs, when comparing the 1H-15N HSQC?s of the various shortened constructs, we see only small shift perturbations located in areas around the point mutants. This overlap leads us to conclude that these various point mutants of the SAM-ETV6 domain retain the same structure as one another and thus have similar helical boundaries.   43   Figure 15. SSP scores for the N90SA93D-ETV61-125 construct. These values were calculated using chemical shifts from 13C! and 13C" assignments. SSP score (y-axis) is plotted against the residue number (x-axis). Helical boundaries from the crystal structure (1LKY.pdb) are shown for comparison Positive values correspond to !-helices and negative values to "-strands.  !"#$%!&#'%!&#(%&%&#(%&#'%"#$%)% "&% ")% $&% $)% *&% *)% (&% ()% )&% ))% +&% +)% ,&% ,)% '&% ')% -&% -)% "&&% "&)% ""&% "")% "$&% "$)%H1 H2 H3 H4  44   Figure 16. Secondary structure SSP score for the A93D-ETV643-125  construct. SSP score (y-axis) is plotted against the residue number (x-axis). Helical boundaries from the ETV6 SAM domain crystal structure (1LKY.pdb) are shown for comparison. Positive values correspond to !-helices and negative values to "-strands.  !"#$%!&#'%!&#(%&%&#(%&#'%"#$%()% (*% (+% ,$% ,,% ,'% *"% *(% *-% -&% -)% -*% -+% '$% ',% ''% +"% +(% +-% "&&% "&)% "&*% "&+% ""$% "",% ""'% "$"% "$(%H1 H2 H3 H4  45 3.3 Comparing the various monomeric point mutants by NMR  1H-15N HSQC spectra were obtained for 15N-labeled ETV643-125 constructs with the mutations, A93D, A93DK99R, V112E, and N90SA93D. Comparison of these spectra by overlaying the data, allows us to see that while there are subtle changes to the chemical shifts of certain residues, overall there are no major peak shifts (Figure 17, Figure 18, Figure 19). This overlap indicated that the structural core of the ETV6-PNT domain remains unchanged. The subtle changes seen for some residues could be due to small local changes from the addition of point mutations. Because a full assignment requires a 13C/15N-double-labeled sample, these were was not done on each point mutation and the doubled peaks crowd the center region of the spectra, the chemical shift perturbations of specific residues are difficult to accurately determine.   46   Figure 17 . A93D-ETV643-125 construct (red)  overlaid with A93DK99R -ETV643-125 construct (blue). Significant spectral overlap is seen between the two constructs , indicating minimal structural perturbation due to the K99R mutation .  47   Figure 18 . A93D-ETV643 -125 construct (red)  overlaid with V112E-ETV643 -125 construct ( green). Significant overlap is also seen between the two constructs. However several peaks are not seen for the V112E -ETV643 -125 construct as its solubility was  lower than that of any of the A93D -ETV643 -125 constructs  48   Figure 19. A93DK99R -ETV643 -125 construct ( blue) overlaid with V112E -ETV643 -125 construct (green). Again overlap is seen between the two constructs. However several peaks are not seen for the V112E -ETV643 -125 construct as its solubility was significantly lower than that of any of the A93D -ETV643 -125 constructs  49 3.4  An NMR titration of ETV6 SAM  domain monomers  1H-15N HSQC spectra were obtained for the 15N-labeled construct V112E-ETV643-125. To this sample either concentrated unlabeled A93D-ETV643-125 or A93DK99R -ETV643-125 was added in aliquots to a final molar ratio of 1:1  (Figure 20, Figure 21). Prior to the titration s amples were dialyzed  overnight into the identical buffer, and each titration point was monitored with the collection of a 1H-15N HSQC spectra. During the course of these titrations, many amides in 15N-labeled V112E-ETV643-125 showed chemical shifts perturbations. Furthermore, over the course of the titration, these shifts demonstrated that there was a binding interaction between the two SAM domains. Additionally binding occurred in the slow exchange regime as several peaks disappeared and new peaks appeared over the course of the titration. This slow exchange behavior indicated that the two SAM domains bound tightly. However, we were unable to determine the binding constants governing the interaction by NMR methods and sought to characterize the binding event using isothermal titration calorimetry.  50   Figure 20. A titration of unlabeled A93D-ETV643-125 into 15N-labeled V112E-ETV643-125. The superimposed spectra were recorded during addition from red (molar ratio 0:1) to purple (molar ratio 1:1) with peaks visibly shifting and several disappearing with complex formation. The spectral perturbations demonstrate that the two proteins bind in the slow exchange regime. Note that the doubling of peaks is not affected by the binding of the two monomers.   51   Figure 21. A titration of unlabeled A93DK99R -ETV643 -125 into 15N-labeled V112E-ETV643 -125. Similar to the titration of the A93D-ETV643 -125 mutant into the V112E-ETV643 -125, the titration goes from red (0:1 molar ratio) to purple (1:1 molar ratio) with increasing additions of the A93DK99R construct. Again some peaks visibly shift over the titration while others disappear, and presumably reappear elsewhere. These spectral perturbations demonstrate that the interaction between the two domains occurs in the slow exchange regime. Note that the doubling of peaks does not appear to be affected by the titration and binding of the two monomers with one another.  52 Chapter 4 Binding studies of the SAM-ETV6 domain using ITC 4.1 Introduction to ITC Isothermal titration calorimetry (ITC) provides details of the binding energetics governing the interaction between a titrant and an analyte. This technique is widely used to measure biological interactions involving proteins, nuclei acids, lipids, carbohydrates and small molecule metabolites or drugs [22, 41, 58]. The ITC instrument is composed of small coin-shaped sample and reference cells that are enclosed in an adiabatic shield and maintained at the same temperature [58]. A feedback system measures any difference in temperature between the two cells and keeps this difference as close to zero throughout the course of a titration experiment.  For an ITC an experiment, the titrant is placed in the syringe and the analyte within the sample cell. The motorized syringe injects small aliquots of the titrant into the sample cell, which is stirred rapidly to ensure fast mixing. If the two reactants interact, such as forming a non-covalent complex, there is most likely an accompanying release or absorption of heat (exothermic or endothermic reaction, respectively). This release or absorption of heat causes the temperature of the sample cell to transiently change from that of the reference cell. The feedback system either raises or lowers the thermal power required to return the cell to the same temperature as the reference. Integrating the resulting peaks in a power versus time plot gives the total amount of heat associated with each injection and subsequent binding interaction [22]. As the titration continues, this amount of heat progressively decreases in magnitude with the saturation of the reactant in the cell and eventually plateaus at a typically small baseline value associated with dilution effects. The latter can be corrected via control experiments. Commonly, the raw data is re-plotted in terms of heat change per mole of titrant injected versus the molar  53 ratio of titrant to analyte, and fit to an appropriate binding model. This fitting yields directly the enthalpy change (!H) upon binding, as well as the equilibrium association constant (K a) and stoichiometry (n) for the given interaction. [ 22]  From the standard thermodynamic relationships !G = !H - T!S and !Go= -RTlnK a, the accompanying entropy change (!S) can be calculated.  4.2  Mutation of Lys 99 affects binding of monomers  Monomeric samples of ETV6 were titrated by ITC and their thermodynamic constants were measured along with the stoichiometry and binding constant of the interaction (Figure 22, Figure 23, Figure 24 , Table 2). From these experiments we can reach several conclusions regarding the ETV6 SAM domain interactions. From the thermodynamic information, we see that each of the three binding reactions are exothermic. Furthermore, the favorable !S values for these reactions are indicative of hydrophobic interactions between the two monomers. A major contributor to stability of a protein is the hydrophobic effect which brings together apolar residues and allows for the entropically favorable release of caged water [ 6] . This conclusion is supported by the crystal structure, which has shown shared hydrophobic surfaces at the binding interface. The stoichiometries (n) close to 1 allow us to conclude that upon mixing, the monomeric SAM domains form heterodimers.   54   Figure 22. An ITC titration of A93D-ETV643-125 into V112E-ETV643-125. The power required to maintain a constant temperature in the sample cell is plotted against time in the top panel. The heat evolved per mole of A93D-ETV643-125 added is plotted in the lower panel. Fitting yielded the data summarized in Table 2. 0.0 0.5 1.0 1.5 2.0-8.00-6.00-4.00-2.000.00-0.35-0.30-0.25-0.20-0.15-0.10-0.050.000.050 10 20 30 40 50 60Time (min)!"#$%&'"Molar Ratio("#$)*+$,- )+.)/01'"2#02 55  Figure 23. An ITC titration of A93DK99R-ETV643-125 into V112E-ETV643-125. The power required to maintain a constant temperature in the sample cell is plotted against time in the top panel. The heat evolved per mole of A93DK99R-ETV643-125 added is plotted in the lower panel. Fitting yielded the data summarized in Table 2. 0.0 0.5 1.0 1.5 2.0 2.5-1.20-1.00-0.80-0.60-0.40-0.200.00-0.06-0.05-0.04-0.03-0.02-0.010.000.010.020 10 20 30 40 50Time (min)!"#$%&'"Molar Ratio("#$)*+$,- )+.)/01'"2#02 56   Figure 24. An ITC titration of A93D-ETV643-125 into V112EK99R-ETV643-125.The power required to maintain a constant temperature in the sample cell is plotted against time in the top panel. The heat evolved per mole of A93D-ETV643-125 added is plotted in the lower panel. Fitting yielded the data summarized in Table 2. 0.0 0.5 1.0 1.5 2.0-8.00-6.00-4.00-2.000.00-0.50-0.40-0.30-0.20-0.100.000.10 0 10 20 30Time (min)!"#$%&'"Molar Ratio("#$)*+$,- )+.)/01'"2#02 57  Table 2. Binding thermodynamic parameters determination from ITC measurements  Protein in Cell Protein in Syringe n Kd (M-1) !H (kcal/mol)  !S (cal/mol -K) A93D V112E 0.90 ? 0.03  (4.4 ? 2.2) x10 -9 -8 .4 ?  0.2 10 ? 1.0  A93D V112E K99R 0.94 ? 0.05  (6.4 ? 1.6) x10 -9 -7.3 ? 0.3  13 ? 0.8  A93D K99R V112E 0.98 ? 0.19  (1.9 ? 1.2) x10 -6 -2.0 ? 1.0  19 ? 5   Most importantly the ITC experiments  yielded a low dissociation constant Kd (1/K a) value of 4.4 nM for the dimerization of A93D-ETV643-125 and V112E-ETV643-125. These results are entirely consistent with previously reported Surface Plasmon Resonance (SPR) data [24] yielding a dissociation constant of 1.7  ? 0.5  nM for the dimerization of A93D-ETV6 and V112E-ETV6. Our result shows a similarly low K d of 4.4 nM for this pairing. Also, the dimerization of A93D-ETV643-125 and V112EK99R-ETV643-125 has a similar Kd of 6.6 nM. This similarity of this interaction, for which the mutated Lys99 is not at the resulting dimer interface, supports the conclusion that K99R point mutation does not disrupt the structure of the SAM-ETV6 domain.  In striking contrast, the Kd of the interaction of the A93DK99R-ETV643-125 with V112E-ETV643-125 was 1900 nM. Also, !H was substantially smaller in magnitude than observed for the wild-type pairings. Therefore, we conclude that the K99R point mutation dramatically affects the binding thermodynamics of the SAM domain interaction when present at the dimer interface. The results of these ITC experiments show that the K99R point mutation substantially weakens the interaction of the ETV6 SAM domains when present at the binding interface. This diminished binding provides an explanation for the loss of EN -mediated cellular transformation observed by our collaborators (Figure 6).  The participation of residue Lys99 in a salt bridge could be the reason for this large enthalpic change. Salt bridges are bonds that form between oppositely charged residues which are close enough together to experience electrostatic attraction [6]. Salt bridges can contribute to  58 protein structure and specificity in interactions with biomolecules [6]. Electrostatic effects can have a highly variable effect on protein folding and interaction attraction or repulsion of charged residues requires ordering and charges should be desolvated to interact [6].   59 Chapter 5 Concluding remarks and future directions 5.1 Goals of this thesis   The overall goal of this project was to better understand the mechanism by which a K99R mutation affects the self-association of the N-terminal SAM domain of ETV6. Mutation of residue Lys99 in the chimeric EN protein has been shown to interrupt the Ras-dependent transformation that results in aberrant growth (Figure 6) [8] and our hypothesis was that this resulted from disrupting the binding interface of the SAM domain. Consistent with this hypothesis, the K99R point mutant did not disrupt the structure of the SAM domain, and weakened the Kd value for dimerization by two orders of magnitude.  In my work I faced several obstacles in producing samples of soluble concentrated monomeric protein. I first cloned a long construct, N90SA93D-ETV61-125 but difficulties in solubility and longevity of the sample and the discovery of an unfortunate N90S residue made further investigation using constructs of this length problematic. Nevertheless, I was able to assign ~2/3 of the ami no acid residues with certainty. Using these chemical shift assignments we established that this construct in solution has a helical secondary structure with helical boundaries that match those of the published crystal structure [55]. Also, the N-terminal sequence of this construct is intrinsically disordered with no  additional helices such as those found in other ETS-family SAM domains. This disordered tail region also shows that residue Lys11 is conformationally dynamic and accessible for sumoylation. Truncated constructs lacking residues 1-42 were generated and used for the remainder of my studies. 15N-labeled versions of these constructs (A93D-ETV643-125; A93DK99R-ETV643-125; V112E-ETV643-125) of similar length to the previously published crystal structure were successfully purified. The 1H-15N HSQC of these constructs showed similar chemical shifts to  60 one another, as well as N90SA93D -ETV61-125-, indicating similar structures. Unfortuna tely these proteins also showed approximately twice as many peaks as were expected in their 1H -15N HSQC spectra. This was not due to degradation, proline isomerization, or deamidation and remains currently unexplained. The spectra of double-labeled 13C/ 15N-A93D -ETV643-125 were partially assigned. Based on the main chain chemical shift assignments, secondary structure propensity scores were calculated and found to have similar helical boundaries to the crystallized ETV6-SAM domain.  Titrations of unlabeled A93D -ETV643-125 or A93DK99R -ETV643-125 into 15N-labeled V112E-ETV643-125 were conducted and interaction was observed by collection of 1H -15N HSQC  spectra. The binding observed during these titrations was in the slow exchange regime, indicative of a strong interaction between monomers. This interaction was quantified using isothermal titration calorimetry. The binding constants of a pairs of monomers with native interface were determined to be on the same order of magnitude as previously published studies (K d = 4.4 nM versus 1.7 nM). However, when present at the binding interface,  the K99R point mutation was seen to affect the dissociation constant of the SAM domain by over two orders of magnitude.  In contrast, when the mutation is introduced but not at the dimerization interface, tight binding still occurs. Thus the K99R mutation does not disrupt the structure of the SAM domain but does disrupt its intermolecular interactions. A summary of the binding interactions observed during these titrations is shown in Figure 25.   61   Figure 25. A cartoon summary of the native binding interface of the ETV6 SAM domain. A93D (red), V112E (blue), and K99R (orange) are highlighted. Clockwise from top left are: the native polymerization occurring in wild-type protein; a combination of A93D-ETV643-125 and V112E-ETV643-125 monomers with a native interface; a dimer interface with a K99R point mutation perturbation in the pairing A93DK99R-ETV643-125 and V112E-ETV643-125; and a native interface in the A93D-ETV643-125 and V112EK99R-ETV643-125 pairing.  K99 V112E A93D V112E-ETV643-125 + A93D-ETV643-125  K99 V112 A93 Native wild-type polymerization K99 V112E A93D K99R V112EK99R-ETV643-125 + A93D-ETV643-125  K99R V112E A93D K99 V112E-ETV643-125 + A93DK99R-ETV643-125   62 5.2 Future work   The self-association of the SAM domain of ETV6 was studied in the scope of this thesis. The importance of residue Lys 99 was confirmed by our binding studies and our results help explain the mechanisms underlying cellular transformation by the EN oncoprotein. However these results have generated many additional questions. From the long (1 -125) and short (43-125) constructs of ETV6 we saw a doubling of peaks in the 1H-15N HSQC spectra. We showed that this doubling was not due to degradation, deamidation, or proline cis/trans isomerization. Ultimately the source of the doubled peaks has yet to be determined. Perhaps aliquots of the unstructured peptide tail (1-42) could be titrated into the 43-125 constructs and the effect on the number of peaks observed. Additionally, the D101KV112E -ETV643-125 construct was cloned but was not found to be soluble in the same NMR buffer as the other constructs. Determination of soluble conditions for this construct would be of great interest as it forms a salt bridge with the Lys 99 residue at the binding interface and has also been shown to affect phenotype of colony formation in soft agar assays [8 ].  Ideally the ITC titrations should also be performed in reverse. 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Wood, L.D., et al., Small ubiquitin-like modifier conjugation regulates nuclear export of TEL, a putative tumor suppressor. Proc Natl Acad Sci U S A, 2003. 100(6): p. 3257 -62.   68  Appendices Appendix  A Chemical shift assignments  Table 3. A summary of the chemical shift residue assignments for N90SA93D -ETV61-125 in 25 mM KCl, 25 mM Na 2HPO 4, pH 7.0 buffer and recorded at 25oC   Residue 13CO 13C! 13C" 1HN 15N P5 176.8 63.39 32.21 - - A6 178.1 52.81 19.23 8.414 124.1 Q7 176 56.1 29.52 8.381 119.3 C8 176.1 55.65 32.99 - - S9 174.3 58.49 63.8 8.319 116.8 I10 176.1 61.31 38.84 8.117 122.6 K11 176.3 56.34 33.06 8.375 125.6 Q12 175.8 55.64 42.73 8.418 122.3 E13 176.3 - 30.78 8.532 122.9 R14 172.8 - - 8.323 118 I15 176 61.09 38.94 8.233 122.7 S16 173.7 57.92 63.9 8.375 119.9 Y17 175.1 57.93 39.39 8.296 123.4 T18 181.6 59.13 70.06 7.998 121.2 Y27 175.5 57.67 38.73 - - A28 177.4 52.48 19.49 8.154 125.4 S29 174.6 58.29 63.83 8.196 114.9 S30 - - - 8.174 118.7 E45 176.6 57.58 30.34 8.421 119.8 D46 176.4 54.88 38.46 8.42 119.8 S47 174.5 58.73 63.93 8.144 115.1 I48 175.7 61.3 38.46 8.067 122 R49 175.5 55.22 30.63 8.615 126.4 L50 174.8 52.97 36.38 8.198 124.5 P51 177.2 62.35 32.52 - - A52 179.4 55.8 18.57 8.716 124 H53 175.5 57.62 29.03 8.335 112.7 L54 175.8 53.97 42.66 7.09 119.4 P59 - 63.35 37.53 - - Y60 176.7 57.47 39.03 7.922 120.2 W61 178.5 55.62 30.35 8.05 121.4  69   Residue 13CO 13C! 13C" 1HN 15N S62 175.1 57.12 66.11 10.65 125.9 D63 178.2 - - 8.213 120.4 R63 179.4 59.6 30.01 9.153 120.1 D64 178.2 58.2 45.17 8.219 120.4 D65 178 57.75 39.6 7.817 122.1 V66 177.7 67.63 32.38 8.284 120.2 A67 181.2 55.86 18 7.85 121 Q68 179 58.87 28.55 8.229 117.6 W69 174.4 61.57 27.12 8.844 125.9 Y69 176.9 - - 8.84 125.9 L70 176.9 58.26 42.14 8.444 118.6 K71 179 58.17 32.08 7.509 117.4 W72 177.6 61.53 27.92 8.367 122.2 A73 179.1 54.83 17.68 8.84 122.4 E74 178.6 59.41 30.08 8.141 116.6 N75 177.4 55.85 39.66 7.338 114.6 E76 177.8 58.3 28.86 8.575 120.9 F77 174 57.09 37.85 7.69 112.4 S78 174.5 59.12 60.99 7.254 114 L79 177.3 53.12 42.5 8.051 117.9 R80 174 54.72 28.92 8.246 121.6 P81 177.1 63.71 34.9 - - I82 176.1 60.41 40.19 8.5 124.5 D83 177.7 54.2 41.09 8.617 126.3 S84 175.8 61.09 63.06 - - N85 176.4 54.95 37.95 8.772 119.2 T86 173.9 63.76 69.23 7.767 111.4 F87 174.4 57.37 40.25 7.953 120.7 E88 174.1 55.98 28.1 7.994 122.9 M89 172.3 54.58 34.62 8.176 121.8 S90 175.5 57.44 65.27 8.784 112.7 G91 174.6 47.72 - 8.718 108.4 K92 177.8 59.69 32.02 8.163 118.4 D93 179.1 56.72 41.7 7.021 117.6 L94 178.3 57.89 43.76 8.332 125 L95 178.3 56.57 41.88 7.648 115.3 L96 178.3 54.79 42.45 7.331 117.1 L97 178.5 55.43 42.21 7.375 119.5  70   Residue 13CO 13C! 13C" 1HN 15N T98 175.6 60.43 72 9.561 114.4 K99 178.4 61.2 31.91 8.663 122.6 E100 178.9 60.23 28.84 8.283 117.3 D101 179.9 57.73 42.36 7.876 121.1 F102 178.4 - - 8.335 120.4 Y104 177.8 60.48 38.51 - - R105 177 58.24 31.2 7.412 116.5 S106 181.7 54.06 62.26 8.217 110.5 P107 179.3 65.33 31.94 8.16 - H108 177.2 57.46 39.78 8.159 111.5 S109 174.5 58.26 65.24 7.42 111.9 G110 174.1 48.92 - 9.353 111.9 D111 178.1 58.39 40.28 8.648 119.5 V112 178.1 65.82 31.83 7.503 119.3 L113 177.8 58.02 42.64 8.141 119.1 Y114 177.6 62.25 38.08 8.242 118.7 E115 179.6 58.88 29.15 7.833 118.1 L116 178.3 57.93 42.59 8.72 122.3 L117 177.9 57.94 40.8 8.406 120.8 Q118 178.8 58.02 27.69 7.927 115.1 H119 178.1 59.74 32.2 8.137 118.7 I120 178.4 65.32 38.42 8.225 120.6 L121 178.8 57.37 42.29 8.138 119.7 K122 177.2 57.57 32.87 7.557 116.9 Q123 176.3 56.75 29.25 7.792 118.3 R124 175 56.03 30.42 8.002 120.6 K125 181.2 57.76 33.74 7.897 128   71  Table 4. A summary of assignment of chemical shift residues for A93D-ETV643-125. Signals arising from doubled peaks are marked with (*). Recorded in 20 mM MOPS, 50 mM NaCl, 0.5 mM EDTA pH 8.0 buffer at 25oC  Residue 13CO 13C! 13C" 1HN 15N M43 175.6 55.29 33.09 8.275 122.9 E44* 177 - - - - E44 176.8 56.54 30.26 8.458 122 E45* 176.6 - - 8.434 120.7 E45 176.6 57.65 30.14 8.619 122 D46 - 54.86 40.96 8.445 120 D46* - - - 8.24 120.7 S47 - - - 8.153 115.7 S47* - - - 8.116 115.1 P51 177 62.34 32.35 - - P51* 177.1 62.27 32.37 - - A52* 179.4 55.75 18.32 8.706 124.2 A52 179.6 55.85 18.25 8.698 124.7 H53* 175.6 57.67 28.9 8.299 112.8 H53 175.6 57.8 28.77 8.345 113.1 L54* 175.9 54.05 42.66 6.999 119.6 L54 176.2 54.29 42.76 6.885 119 P58* - 63.17 29.99 - - P58 - 63.19 30.01 - - I59 - 63.2 37.39 7.171 113.6 I59* - 63.2 37.39 7.175 113.4 Y60 - - - 7.794 119.7 W61 - - - 8.037 121.5 S62* - - - 10.65 125.5 S62 - - - 10.7 125.5 R63 - - - 9.165 120.3 R63* - - - 9.208 120.5 D65 - - - 7.868 121.8 D65* - - - 7.822 122 V66* - - - 8.134 120.2 V66 - - - 8.188 120.2 Q68 - - - 8.252 117.6 W69* - - - 8.8 126 W69 - - - 8.734 126 L70* - - - 8.446 118.5 L70 - - - 8.383 118.5 K71 - - - 7.465 117.4  72  K71* - - - 7.407 117.4 W72 - - - 8.357 122.3 W72* - - - 8.377 122.5 A73* - - - 8.816 122.5 A73 - - - 8.837 122.7 E74 178.6 59.37 29.88 8.056 116.8 E74* - - - 8.087 116.7 N75 177.4 55.86 39.52 7.319 114.6 E76 177.8 58.21 28.74 8.562 121.1 F77 174.1 57.01 37.79 7.672 112.4 S78 174.6 58.37 60.99 7.236 114 L79 177.3 53.03 42.35 8.046 118 R80 174 54.67 28.71 8.229 121.6 P81 - 63.68 32.18 - - I82 - 60.31 40.01 8.466 124.2 D83 - 54.11 40.99 8.617 126.3 N85 - - - 8.756 119.2 N85* - - - 8.737 119 T86 - - - 7.751 111.3 T86* - - - 7.766 111.7 F87 - - - 7.934 121 M89 - 54.44 34.47 8.2 121.8 N90 - 51.02 38.78 8.531 114.9 G91 - 47.96 - 8.424 105.1 G91* - 47.38 - 8.489 105.5 K92 178.8 59.99 31.75 7.741 119.8 K92* 178.6 59.92 31.57 7.774 120 D93* 179.6 56.96 40.99 7.848 117.8 D93 179.8 56.86 40.47 8.022 117.6 L94 - - - 8.339 125.1 L97 - - - 7.351 117.4 T98 - 60.42 71.99 9.546 114.5 T98* - - - 9.591 114.5 K99 - 61.26 31.78 8.642 122.7 K99? - - - 8.677 123.2 D100 - 60.16 - - - E100 - 60.09 28.68 8.262 117.3 D101 - 60.09 - 7.864 121.1 F102 - 63.58 39.77 - - R103 - 59.08 30.62 8.161 118.7 Y104 - 60.44 38.35 8.127 118.4 R105 - 58.19 31 7.394 116.6  73  R105* - - - 7.366 116.6 S106 - 54.03 62.24 8.213 110.5 P107 - 65.24 31.76 - - H108 - 57.52 31.22 8.053 111.7 P108 - - 31.7 - - S109 - 58.15 65.34 7.367 112 S109* - - - 7.325 112 G110 - 48.85 - 9.323 111.9 D111 178.1 58.34 40.14 8.614 119.5 V112 178.3 65.78 31.65 7.472 119.2 L113 177.7 57.96 42.64 8.147 119.3 Y114 - 62.29 38.03 8.221 118.7 Y114* - - - 8.251 118.8 E115 - 58.74 28.85 - - L116* - 57.81 - 8.655 122.3 L116 - 52.66 42.37 8.584 122.5 L117 178.4 58.01 40.67 8.344 120.8 L117* - - - 8.334 120.5 Q118 178.7 57.87 27.46 7.864 115.1 H119 178.2 60 30.57 8.097 119.5 I120 178.4 65.39 38.23 8.219 120.5 L121 178.8 57.25 42.22 8.198 119.6 L121* - 57.42 42.21 - - K122* 177.5 57.67 32.75 48.46 75.74 K122 177.4 57.62 32.72 7.537 116.9 Q123 176.4 56.58 29.14 7.789 117.8 Q123* 176.5 56.61 29.16 7.839 117.6 R124* 174.9 55.95 30.35 7.944 120.2 R124 175 55.98 30.42 7.962 120.6 K125* 181.2 57.76 33.52 7.818 128 K125 181.3 57.77 33.54 7.846 128  74  Appendix B SSP Scores from NMR assignments Table 5. SSP score values of A93D-ETV61-125  Residue Number SSP Value 5 -0.123 6 -0.261 7 -0.15 8 -0.088 9 -0.119 10 -0.268 11 -0.204 12 -0.305 13 -0.328 14  15 -0.13 16 -0.1 17 -0.1 18  19  20  21  22  23  24  25  26  27 -0.068 28 -0.068 29 -0.068 30  31  32  33  34  35  36  37  38  39  40  41  42  43    Residue Number SSP Value 44  45 0.225 46 0.191 47 0.107 48 0.079 49 -0.179 50  51 0.303 52 0.277 53 0.277 54 0.531 55  56  57  58  59 0.106 60 -0.118 61 0.157 62 0.007 63 0.271 64 0.622 65 0.924 66 0.919 67 1.062 68 1.029 69 1.059 70 1.042 71 1.031 72 0.958 73 0.983 74 0.975 75 0.835 76 0.729 77 0.515 78 0.385 79 0.082 80  81 -0.306 82 0.143   75  Residue Number SSP Value 83 0.244 84 0.399 85 0.396 86 0.493 87 0.122 88 -0.16 89 -0.204 90 0.219 91 0.253 92 0.536 93 0.756 94 0.608 95 0.411 96 0.288 97 0.394 98 0.555 99 0.597 100 0.73 101 0.932 102  103  104 0.561 105 0.661 106  107 0.193 108 0.316 109 0.495 110 0.557 111 0.764 112 0.997 113 0.979 114 0.925 115 1.002 116 1.016 117 0.875 118 0.854 119 0.836 120 0.65 121 0.492 122 0.452 123 0.268 124 0.129 125 0.095  76  Table 6. SSP values for construct A93D-ETV643-125  Residue Number SSP Value 43 0.06 44 0.084 45 0.138 46 0.175 47 0.244 48  49  50  51 0.481 52 0.26 53 0.26 54 0.583 55  56  57  58 0.398 59 0.166 60 -0.035 61 0.185 62 0.141 63 0.242 64 0.556 65 0.846 66 0.725 67  68  69  70  71  72  73  74 0.911 75 0.769 76 0.759 77 0.515 78 0.365 79 0.043 80  81 -0.348 82 -0.287  Residue Number SSP Value 83 -0.287 84  85  86  87  88  89 -0.108 90 0.415 91 0.581 92 1.014 93 1.155 94  95  96  97  98 0.877 99 0.945 100 0.852 101 0.992 102 0.92 103 0.73 104 0.653 105 0.699 106  107 0.241 108 0.354 109 0.516 110 0.606 111 0.767 112 1.011 113 0.983 114 0.803 115 0.861 116 0.868 117 0.773 118 0.773 119 0.893 120 0.74 121 0.628 122 0.532  77  123 0.319 124 0.174 125 0.113  78 Appendix C  ITC titration results  Table 7 . ITC results for i ndividual experiments  Protein in Cell Protein in Syringe n Ka (M-1) !H (kcal/mol) !S (cal/mol-K) V112E A93D 0.948 1.15x108 -8055 9.9 V112E A93D 0.888 3.19x108 -8395 10.8 V112E A93D 0.879 1.44x108 -8559 8.6 V112E A93D 0.874 3.32x108 -8440 10.7 V112E A93DK99R 0.937 9.35x105 -1155 23.4  V112E A93DK99R 0.728  6.22x105  -1326 22.1  V112E A93DK99R 1.140  3.39x105  -2332 17.5  V112E A93DK99R 1.100 2.04x105  -3271 13.3 V112EK99R A93D 0.897 1.15x108 -7078 13.1 V112EK99R A93D 0.938 1.91x108 -7326 13.3 V112EK99R A93D 0.989  1.61x108 -7638  11.9  79  Appendix D EMS results  Figure 26. EMS m/z measurement of A93D-ETV643-125  

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