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Cell surface iron-complex binding and transport by Staphylococcus aureus Grigg, Jason Christopher 2010

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     CELL SURFACE IRON-COMPLEX BINDING AND TRANSPORT BY STAPHYLOCOCCUS AUREUS   by  Jason Christopher Grigg  B.MSc., The University of Western Ontario, 2005      A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY   in   The Faculty of Graduate Studies   (Microbiology and Immunology)   THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)     October 2010    © Jason Christopher Grigg, 2010   ii Abstract  Iron uptake systems are paramount to the survival of many organisms. Pathogenic bacteria are faced with the especially daunting task of acquiring essential iron within their host environment. Staphylococcus aureus is a Gram-positive bacterial pathogen and one of the most common causes of bacterial infections in hospitals. In addition, multi-drug resistant S. aureus isolates are emerging and now constitute the majority of isolated strains from clinical settings. The prevalence of S. aureus is attributed, in part, to its ability to specifically use most host iron sources for growth. S. aureus uses high affinity uptake systems for many different forms of iron in the human body with the source preference varying through the time course of infection and the tissues infected. To gain insight into iron binding and import by S. aureus, surface receptors from the iron surface determinant (Isd) heme uptake system and the staphyloferrin A siderophore uptake systems (unfortunately named heme transfer system (Hts)) were studied. The systems use distinct methods for ligand import. In the Isd system, heme is received and relayed through cell wall anchored proteins (including IsdA) to the substrate binding protein (IsdE) for import through the permease. Crystal structures of IsdA and IsdE in complex with heme, in concert with in vitro heme transfer kinetics contributed to the development of a heme transfer model for NEAT domains. In contrast to heme uptake, staphyloferrin A is bound directly at the substrate binding protein (HtsA). HtsA and IsdE are homologous membrane anchored binding proteins and both receive and deliver the iron-complex to the permease. Crystal structures and ligand affinity measurements of IsdE and HtsA reveal distinct mechanisms for ligand reception and specificity. Furthermore, crystal structures of open and closed conformations of HtsA highlight unique structural changes proposed to enable discrimination by the permease of ligand-bound   iii and -free receptor. These studies provide insight into iron import in S. aureus, which have contributed to the development of models for heme and siderophore transport from the cell surface to the permease.                                     iv Preface  The majority of work presented in this thesis is drawn from published literature with corresponding publications listed by chapter number. The body of work was made possible through ongoing collaborations, primarily with Dr. David Heinrich’s lab at the University of Western Ontario. Since the collaboration provides crucial support to the findings, all materials from the publications are included and the relative contributions to the work are outlined below.  Chapter 1: Grigg JC, Ukpabi G, Gaudin CFM, Murphy MEP. 2009. Structural Biology of Heme Binding in the Staphylococcus aureus Isd system. J Inorg Biochem (104), 341-348.  The introduction is partially drawn from a review article written by several members of Dr. Michael Murphy’s lab. I wrote the first draft of the abstract, introduction, biology, cell wall anchored surface receptors and membrane transporter sections. G. Ukpabi wrote the Isd heme degrading enzymes section and C. Gaudin wrote the related Isd Systems and Summary sections. The manuscript was edited by Dr. Michael Murphy. Only sections that were my responsibility in the review are used in the thesis. The manuscript has been expanded upon to provide a more complete background.   Chapter 3: Grigg JC, Vermeiren CL, Heinrichs DE, Murphy ME. 2007. Heme recognition by a Staphylococcus aureus NEAT domain. Mol Microbiol 63(1), 139-149.  Chapter 3 was largely derived from the manuscript in which C. Vermeiren and I shared first authorship. I cloned the expression vector, crystallized the protein, determined the structure of IsdA and completed the bioinformatic analysis. C. Vermeiren was responsible for the growth assays and mutagenesis work. C. Vermeiren and I wrote the first draft of our respective sections   v of the methods and results and I wrote the balance of the discussion. Drs. D. Heinrichs and M. Murphy edited the manuscript.   Chapter 4: Grigg JC, Mao CX, Arrieta AL, Murphy ME. Iron coordinating tyrosine is a key determinant of heme transfer by the Staphylococcus aureus Isd NEAT domain. In preparation.  Chapter 4 is a draft of a manuscript that will be submitted. Dr. M. Murphy and I developed the original hypothesis and experimental design. Dr. Federico Rosell provided helpful discussion on stopped-flow spectroscopy and Dr. Rahul Singh and Antonio Ruzzini trained me to use the stopped-flow spectrometer in Dr. L. Eltis’ lab. As an undergraduate in the lab under my co- supervision, C. Mao purified and crystallized CoPPIX-bound IsdA and the wild-type IsdA used for the dithionite-reduced structure. A. Arrieta cloned the IsdBN2 expression system. I performed the remaining experimental work and wrote the first draft of the manuscript. Dr. M. Murphy edited the manuscript.   Chapter 5: Grigg JC, Vermeiren CL, Heinrichs DE, Murphy ME. 2007. Heme coordination by Staphylococcus aureus IsdE. J Biol Chem 282 (39), 28815-22  I did the cloning, protein expression and structure determination as well as bioinformatic analysis. C. Vermeiren performed all iron growth assays with S. aureus, purified the GST-tagged IsdE variants for spectroscopic analysis, and wrote the corresponding experimental methods and results sections of the manuscript. I wrote the first draft for the balance of the manuscript. Dr. David Heinrichs and Dr. M. Murphy edited the manuscript.     vi Chapter 6: Beasley FC, Vines ED, Grigg JC, Zheng Q, Liu S, Lajoie GA, Murphy ME, Heinrichs DE. 2009. Characterization of staphyloferrin A biosynthetic and transport mutants in Staphylococcus aureus. Mol Microbiol 72(4), 947-63  Grigg JC, Cooper JD, Cheung J, Heinrichs DE, Murphy MEP. 2010. The Staphylococcus aureus siderophore receptor HtsA undergoes localized conformational changes to enclose staphyloferrin A in an arginine-rich binding pocket. J Biol Chem. 285(15), 11162-71  Chapter 6 is largely derived from two papers. “Characterization of staphyloferrin A biosynthetic and transport mutants in Staphylococcus aureus” was primarily the work of Dr. David Heinrichs’ group. I contributed the crystal structure of apo-HtsA as well as writing the first draft of the structure description and analysis. The apo structure was a component of a directed studies undergraduate research project carried out by Q. Zheng under my co- supervision. Q. Zheng purified and crystallized the protein. I collected the data and together we determined the structure of apo-HtsA. Since the apo-structure is an important part of the thesis discussion and only a minor part of the manuscript was my contribution, the respective part has been merged with the second manuscript. The second manuscript included in this work is entitled “The Staphylococcus aureus siderophore receptor HtsA undergoes localized conformational changes to enclose staphyloferrin A in an arginine-rich binding pocket”. The manuscript describes two structures of siderophore- bound HtsA. The initial experimental design was proposed by Dr. D. Heinrichs and Dr. M. Murphy. J. Cooper and J. Cheung purified and provided staphyloferrin A for my co- crystallization experiments, provided the fluorescence titration data, and wrote the corresponding methods and results sections of the paper. I determined and analyzed the two crystal structures and provided bioinformatic analysis. I wrote the first draft of the balance of the manuscript and Dr. D. Heinrichs and Dr. M. Murphy edited the work.   vii   Chapter 7: Grigg JC, Ukpabi G, Gaudin CFM, Murphy MEP. 2009. Structural biology of heme binding in the Staphylococcus aureus Isd system. J Inorg Biochem (104), 341-348.  Portions of the overview and future directions section were drawn form the same review article used in the introduction. I wrote the first draft of the abstract, introduction, biology, cell wall anchored surface receptors and membrane transporter sections. G. Ukpabi wrote the Isd heme degrading enzymes section and C. Gaudin wrote the related Isd Systems and Summary sections. The manuscript was edited by Dr. Michael Murphy. Only sections that were my responsibility in the review are used in the thesis.   viii Table of Contents Abstract ........................................................................................................................................................................ ii	
  Preface.......................................................................................................................................................................... iv	
  Table	
  of	
  Contents………………………………………………………………………………………………………….vii	
  List	
  of	
  Tables ............................................................................................................................................................. xi	
  List	
  of	
  Figures...........................................................................................................................................................xii	
  List	
  of	
  Symbols,	
  Abbreviations	
  and	
  Nomenclature.................................................................................xiv	
  Acknowledgements ............................................................................................................................................ xvii	
  	
  Chapter	
  1.	
  Introduction ......................................................................................................................................... 1	
   1.1.	
  Staphylococcus	
  aureus ................................................................................................................................ 1	
  1.2.	
  Iron	
  in	
  the	
  human	
  host .............................................................................................................................. 2	
  1.3.	
  Iron	
  uptake	
  in	
  Gram-­‐negative	
  bacteria .............................................................................................. 4	
  1.4.	
  Iron	
  uptake	
  in	
  Gram-­‐positive	
  bacteria ............................................................................................ 10	
  1.5.	
  S.	
  aureus	
  iron	
  uptake	
  systems ............................................................................................................. 11	
  1.5.1.	
  The	
  S.	
  aureus	
  heme	
  uptake	
  system............................................................................................ 12	
  1.5.2.	
  Siderophore	
  uptake	
  systems........................................................................................................ 16	
  1.6.	
  Objectives	
  of	
  this	
  thesis.......................................................................................................................... 20	
  	
  Chapter	
  2.	
  Methods............................................................................................................................................... 22	
  2.1.	
  S.	
  aureus	
  growth	
  conditions ................................................................................................................. 22	
  2.2.	
  Construction	
  of	
  S.	
  aureus	
  mutant	
  strains........................................................................................ 22	
  2.2.1.	
  Construction	
  of	
  S.	
  aureus	
  isdA::Km............................................................................................ 22	
  2.2.2.	
  Construction	
  of	
  S.	
  aureus	
  isdE::Km............................................................................................ 23	
  2.2.3.	
  Site-­‐directed	
  mutagenesis	
  of	
  S.	
  aureus	
  isdE	
  for	
  in	
  vivo	
  assays....................................... 23	
  2.3.	
  Heme	
  plate	
  bioassays.............................................................................................................................. 24	
  2.4.	
  Heme-­‐dependent	
  bacterial	
  growth	
  studies	
  in	
  liquid	
  culture................................................. 24	
  2.5.	
  Peroxidase	
  staining.................................................................................................................................. 24	
  2.6.	
  Recombinant	
  protein	
  expression	
  systems	
  for	
  non-­‐crystallographic	
  use......................... 25	
  2.6.1.	
  GST-­‐IsdA	
  site	
  directed	
  mutagenesis	
  and	
  expression	
  for	
  heme	
  scavenging	
  from	
  E.	
   coli........................................................................................................................................................................ 25	
  2.6.2.	
  Cloning	
  and	
  IsdE	
  protein	
  expression	
  for	
  heme	
  scavenging	
  from	
  E.	
  coli ................... 26	
  2.6.3.	
  Purification	
  of	
  staphyloferrin	
  A	
  biosynthetic	
  enzymes.................................................... 27	
  2.7.	
  UV/Visible	
  absorption	
  spectroscopic	
  characterization	
  of	
  heme	
  binding	
  from	
  the	
  cytoplasm	
  of	
  E.	
  coli ........................................................................................................................................... 27	
  2.8.	
  Staphyloferrin	
  A	
  purification	
  from	
  culture	
  supernatants ....................................................... 28	
  2.9.	
  Staphyloferrin	
  A	
  in	
  vitro	
  synthesis.................................................................................................... 29	
  2.10.	
  Determination	
  of	
  ferric-­‐staphyloferrin	
  A	
  concentration ...................................................... 29	
  2.11.	
  Fluorescence	
  spectroscopy	
  siderophore	
  titrations................................................................. 30	
  2.12.	
  Cloning,	
  expression	
  and	
  purification	
  for	
  structure	
  determination .................................. 30	
  2.12.1.	
  Expression	
  of	
  IsdAN	
  and	
  IsdAN	
  variants ............................................................................... 31	
  2.12.2.	
  Protein	
  expression	
  of	
  IsdE	
  for	
  structure	
  determination............................................... 32	
  2.12.3.	
  Cloning	
  and	
  protein	
  expression	
  of	
  HtsA	
  for	
  structure	
  determination .................... 32	
  2.13.	
  Crystallization	
  and	
  structure	
  determination ............................................................................. 33	
     ix 2.13.1.	
  IsdA	
  NEAT	
  domain	
  structure	
  determination ..................................................................... 33	
  2.13.2.	
  IsdA	
  variant	
  structure	
  determination ................................................................................... 34	
  2.13.3.	
  IsdE	
  structure	
  determination ................................................................................................... 36	
  2.13.4.	
  Apo-­‐	
  and	
  ferric-­‐staphyloferrin	
  A-­‐bound	
  HtsA	
  structure	
  determination................ 36	
  2.13.5.	
  Protein	
  structure	
  figures............................................................................................................. 39	
  2.14.	
  Determination	
  of	
  heme	
  transfer	
  to	
  apomyoglobin.................................................................. 39	
  2.15.	
  Kinetics	
  of	
  heme	
  transfer.................................................................................................................... 39	
  2.16.	
  Bioinformatic	
  analysis ......................................................................................................................... 40	
  2.16.1.	
  Multiple	
  sequence	
  alignment	
  of	
  NEAT	
  domains	
  from	
  S.	
  aureus ................................ 40	
  2.16.2.	
  Multiple	
  sequence	
  alignment	
  of	
  IsdE	
  homologues .......................................................... 40	
  2.16.3.	
  Multiple	
  sequence	
  alignments	
  of	
  HtsA	
  homologues ....................................................... 41	
  	
  Chapter	
  3.	
  Structure	
  and	
  heme	
  recognition	
  by	
  the	
  IsdA	
  NEAT	
  domain ........................................ 42	
  3.1.	
  Introduction ................................................................................................................................................ 42	
  3.2.	
  Results ........................................................................................................................................................... 43	
  3.2.1.	
  Acquisition	
  of	
  heme-­‐iron	
  by	
  S.	
  aureus	
  is	
  enhanced	
  by	
  IsdA ........................................... 43	
  3.2.2.	
  The	
  IsdA	
  NEAT	
  domain	
  binds	
  heme ......................................................................................... 46	
  3.2.3.	
  The	
  IsdA	
  NEAT	
  domain	
  structure	
  reveals	
  heme-­‐iron	
  co-­‐ordination	
  by	
  a	
  conserved	
  tyrosine.............................................................................................................................................................. 46	
  3.2.4.	
  IsdA	
  point	
  mutants	
  show	
  that	
  Tyr166	
  and	
  Tyr170	
  are	
  essential	
  for	
  heme	
  binding............................................................................................................................................................................... 50	
  3.2.5.	
  NEAT	
  domain	
  sequence-­‐structure	
  alignments	
  reveal	
  that	
  the	
  tyrosine	
  ligand	
  is	
  a	
  prognosticator	
  of	
  heme	
  binding ............................................................................................................. 52	
  3.3.	
  Discussion .................................................................................................................................................... 53	
  	
  Chapter	
  4.	
  Heme	
  transfer	
  to	
  and	
  from	
  IsdA............................................................................................... 58	
  4.1.	
  Introduction ................................................................................................................................................ 58	
  4.2.	
  Results ........................................................................................................................................................... 59	
  4.2.1.	
  Structures	
  of	
  IsdAN	
  variants ......................................................................................................... 61	
  4.2.2.	
  Structures	
  of	
  IsdAN	
  in	
  complex	
  with	
  protoporphyrin	
  IX	
  containing	
  altered	
  metal	
  centers................................................................................................................................................................ 64	
  4.2.3.	
  Rates	
  of	
  heme	
  transfer	
  to	
  myoglobin ....................................................................................... 66	
  4.2.4.	
  IsdBN2	
  to	
  IsdAN	
  heme	
  transfer	
  rates ......................................................................................... 69	
  4.2.5.	
  IsdAN	
  to	
  IsdCN	
  heme	
  transfer	
  rates ........................................................................................... 72	
  4.3.	
  Discussion .................................................................................................................................................... 74	
  	
  Chapter	
  5.	
  Heme	
  recognition	
  by	
  IsdE ........................................................................................................... 82	
  5.1.	
  Introduction ................................................................................................................................................ 82	
  5.2.	
  Results ........................................................................................................................................................... 83	
  5.2.1.	
  Overall	
  Protein	
  Structure .............................................................................................................. 83	
  5.2.2.	
  Heme	
  Binding ..................................................................................................................................... 85	
  5.2.3.	
  Multiple	
  Sequence	
  Alignments.................................................................................................... 88	
  5.2.4.	
  Contribution	
  of	
  Residues	
  to	
  Heme	
  Binding	
  and	
  IsdE-­‐mediated	
  Heme	
  Transport	
  in	
   vivo....................................................................................................................................................................... 91	
  5.3.	
  Discussion .................................................................................................................................................... 94	
     x 	
  Chapter	
  6.	
  Staphyloferrin	
  A	
  recognition	
  by	
  HtsA..................................................................................101	
  6.1.	
  Introduction ..............................................................................................................................................101	
  6.2.	
  Results .........................................................................................................................................................102	
  6.1.1.	
  Affinity	
  of	
  HtsA	
  for	
  staphyloferrin	
  A.......................................................................................102	
  6.1.2.	
  The	
  crystal	
  structure	
  of	
  apo-­‐HtsA	
  identifies	
  a	
  positively	
  charged	
  binding	
  pocket.............................................................................................................................................................................103	
  6.1.3.	
  Crystal	
  structures	
  of	
  open	
  and	
  closed	
  FeSA-­‐HtsA.............................................................106	
  6.1.4.	
  Structure	
  of	
  Staphyloferrin	
  A.....................................................................................................109	
  6.1.5.	
  Siderophore	
  bound	
  in	
  the	
  open	
  conformation	
  of	
  HtsA ..................................................112	
  6.1.6.	
  Siderophore	
  bound	
  in	
  the	
  closed	
  conformation	
  of	
  HtsA................................................114	
  6.1.7.	
  Multiple	
  sequence	
  alignments...................................................................................................115	
  6.3.	
  Discussion ..................................................................................................................................................117	
  	
  Chapter	
  7.	
  Overview	
  and	
  future	
  directions..............................................................................................124	
  7.1.	
  Heme	
  recognition	
  by	
  Gram-­‐positive	
  cell	
  wall	
  anchored	
  receptors...................................124	
  7.2.	
  A	
  mechanism	
  for	
  heme	
  transfer	
  through	
  the	
  cell	
  wall ...........................................................130	
  7.3.	
  Ligand	
  reception	
  by	
  the	
  substrate	
  binding	
  protein .................................................................132	
  7.4.	
  Substrate	
  binding	
  protein	
  docking	
  on	
  the	
  permease..............................................................135	
  7.5.	
  Future	
  work...............................................................................................................................................139	
  	
  References ..............................................................................................................................................................144	
      xi List of Tables  Table 2-1. S. aureus strains........................................................................................................... 22	
   Table 2-2. E. coli expression vectors for non-crystallographic analysis. ..................................... 25	
   Table 2-3. Plasmids for recombinant protein used in crystallography and heme transfer experiments. ........................................................................................................................... 31	
   Table 3-1. Data collection and refinement statistics for apo and holo IsdA NEAT domains....... 47	
   Table 4-1. Data Collection and Refinement Statistics. ................................................................. 60	
   Table 4-2. IsdAN heme release rates ............................................................................................. 67	
   Table 4-3. Heme transfer kinetics to and from IsdAN variants..................................................... 70	
   Table 5-1. Data collection and refinement statistics for the IsdE-heme complex. ....................... 84	
   Table 6-1. Data collection and refinement statistics for the HtsA structures. ............................ 104	
   Table 6-2. FeSA-HtsA bond distances (Å). ................................................................................ 113	
   Table 6-3. Dissociation constants (Kd) for receptor-ferric-siderophore complexes. .................. 121	
       xii List of Figures  Figure 1-1. Overview of iron uptake system architecture in Gram-negative (left) and Gram- positive bacteria (right). ........................................................................................................... 4	
   Figure 1-2. Schematic of E. coli ferric hydroxamate uptake (fhu) system. .................................... 6	
   Figure 1-3. BtuCD-F crystal structure. ........................................................................................... 8	
   Figure 1-4. Schematic representation of the Isd system heme transport components. ................. 13	
   Figure 1-5. Schematic representation of Isd surface proteins....................................................... 15	
   Figure 1-6. Schematic of siderophore transporters in S. aureus. .................................................. 17	
   Figure 1-7. S. aureus siderophores. .............................................................................................. 19	
   Figure 3-1. Peroxidase activity in S. aureus cell wall extracts. .................................................... 44	
   Figure 3-2. IsdA enhances S. aureus growth on hemin as a sole source of iron. ......................... 45	
   Figure 3-3. The overall structure of the IsdA NEAT domain heme complex. ............................. 48	
   Figure 3-4. The IsdA NEAT domain crystal structures. ............................................................... 49	
   Figure 3-5. Stereo view of the heme site including the residues of the heme-binding pocket illustrated in Fig. 2A............................................................................................................... 50	
   Figure 3-6. Electronic spectra of IsdA variants. ........................................................................... 51	
   Figure 3-7. Multiple sequence alignment of NEAT domains from S. aureus. ............................. 53	
   Figure 4-1. Structure of IsdAN-heme. ........................................................................................... 61	
   Figure 4-2. IsdAN variants. ........................................................................................................... 63	
   Figure 4-3. The structures of IsdAN bound to protoporphyrin IX with altered metal centers. ..... 65	
   Figure 4-4. IsdAN heme binding and transfer ............................................................................... 68	
   Figure 4-5. Heme transfer rates between IsdBN2 and IsdAN variants. .......................................... 71	
   Figure 4-6. Heme transfer rates between IsdAN variants and IsdCN. ........................................... 73	
   Figure 4-7. Model of NEAT domain heme transfer. .................................................................... 78	
   Figure 4-8. Crystal contacts between NEAT domain heme interfaces. ........................................ 80	
   Figure 4-9. Isd NEAT domain protein-protein complexes predicted by ClusPro (174,175)........ 81	
   Figure 5-1. The overall structure of the IsdE-heme complex. ...................................................... 85	
   Figure 5-2. Heme binding and surface structure of IsdE. ............................................................. 87	
   Figure 5-3. Multiple sequence alignment of IsdE homologues. ................................................... 90	
   Figure 5-4. Conservation of Pro residues within the N-terminal domain of IsdE. ....................... 91	
   Figure 5-5. Heme binding and transport by point mutants of IsdE. ............................................. 92	
   Figure 5-6. Heme binding by mutants of IsdE.............................................................................. 93	
   Figure 5-7. Superposition of IsdE and BtuF. ................................................................................ 98	
   Figure 6-1. Saturation curve of the binding of FeSA to HtsA. ................................................... 103	
   Figure 6-2. Apo-HtsA crystal structure. ..................................................................................... 105	
   Figure 6-3. The overall structure of the HtsA-staphyloferrin A complex. ................................. 108	
   Figure 6-4. The structure and chirality of staphyloferrin A........................................................ 110	
   Figure 6-5. Staphyloferrin A models in omit difference (Fo-Fc) electron density...................... 111	
   Figure 6-6. Staphyloferrin A in the HtsA binding pocket. ......................................................... 113	
   Figure 6-7. HtsA sequence alignments. ...................................................................................... 116	
   Figure 7-1. NEAT Domain fold.................................................................................................. 126	
   Figure 7-2. Heme binding by IsdA, IsdC and IsdH. ................................................................... 128	
   Figure 7-3. Shp binding domain shares the NEAT domain fold. ............................................... 129	
   Figure 7-4. Proposed models for heme transfer between S. aureus NEAT domains. ................ 131	
     xiii Figure 7-5. Overlay of IsdE-heme and HtsA-FeSA.................................................................... 133	
   Figure 7-6. Models ABC transporters in S. aureus..................................................................... 137	
   Figure 7-7. FeSA binding induces a decrease in inter-glutamate spacing in HtsA. ................... 138	
                                           xiv  List of Symbols, Abbreviations and Nomenclature   6xHis Poly-histidine affinity purification tag AAS Atomic absorption spectroscopy Å Angstrom (1 Å = 0.1 nm) ATP Adenosine triphosphate ABC Transporter ATP-binding cassette transporter B-factor Crystallographic thermal factor CLS Canadian Light Source CoPPIX Protoporphyrin IX with a central Co3+ EDDHA Ethylenediamine-di(o-hydroxyphenylacetic acid) FeSA Ferric-bound staphyloferrin A FeSB Ferric-bound staphyloferrin B Fhu Ferric hydroxamate uptake GST Glutathione S-transferase Hb Hemoglobin Hemin Protoporphyrin IX with a central Fe3+ Heme Protoporphyrin IX with a central Fe2+ Hp Haptoglobin Hp-Hb Haptoglobin-hemoglobin complex IPTG Isopropyl β-D-thiogalactopyranoside Isd Iron surface determinant   xv IsdAN IsdA NEAT domain (residues 62-184) IsdBN2 IsdB C-terminal most NEAT domain (residues 341-458 IsdCN IsdC NEAT domain (residues 22-152) IsdHN1 IsdH N-terminal-most NEAT domain (86-229) IsdHN3 IsdH C-terminal-most NEAT domain (539-664) Km Kanamycin LB Luria-Bertani MRSA Methicillin resistant Staphylococcus aureus NEAT Near iron transport PBP Periplasmic binding protein PBS Phosphate buffered saline PEG Polyethylene glycol PPIX Protoporphyrin IX r.m.s.d Root mean square deviation SA Staphyloferrin A SB Staphyloferrin B SeMet Selenomethionine SSRL Stanford Synchrotron Radiation Lightsource S. aureus N315 Staphylococcus aureus strain N315 S. aureus Newman Staphylococcus aureus strain Newman SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis TMS Tris minimal succinate   xvi TSB Typtic soy broth     xvii Acknowledgements  I acknowledge funding from the Natural Sciences and Engineering Research Council Canada Graduate Scholarship and the Michael Smith Foundation for Health Research Junior Graduate Trainee Award. The projects were funded by Canadian Institute of Health Research grants held by Michael EP Murphy. I thank Michael EP Murphy, who did a wonderful job as a supervisor and always made time to provide a training environment where I could develop important skills beyond basic lab techniques. My committee members Drs. JT Beatty, L Eltis and R Fernandez were extremely helpful throughout my studies and their advice and guidance was appreciated. The past and present members of the Murphy Lab have been supportive and helpful and the atmosphere was enjoyable. Importantly, I thank the collaborators on most of my work, Dr. David Heinrichs and members of his lab, especially Dr. Christie Vermeiren, Dr. Enrique Vines, Federico Beasley, John Cooper and Johnson Cheung. They are skilled microbiologists/biochemists that have driven my work and provided an efficient collaborative approach to rapidly understand biological function. Finally, I thank my family for their unwavering emotional support through my many years of academic training, especially Andrea, who has been patient, understanding and extremely helpful through this degree.     1 Chapter 1. Introduction  1.1. Staphylococcus aureus  S. aureus is a member of the family Micrococcaceae, which includes species of staphylococci and micrococci, among others (1). These genera share the general feature of being Gram- positive, catalase-positive cocci (2). The staphylococci are further divided into numerous, diverse species primary residing on the skin and mucosal surfaces of mammals, as part of the normal flora and a few are found free living in soils (2). Of the many staphylococci, only a few are capable of causing disease in humans, but Staphylococcus aureus is by far the most devastating and widespread infectious staphylococci. S. aureus is normally a commensal, which colonizes the epithelial surfaces of ~ 20 % of the human population at any given time (3). However, S. aureus can cause significant disease when it invades defects in the skin surface or mucosal barriers, and accesses the deeper tissues and bloodstream (4). In hospitals, S. aureus is one of the most commonly acquired bacterial infections and is responsible for ~ 20% of nosocomial bloodstream infections (5). The disease outcome of an S. aureus infection is determined by the infection site and immune status of the host, ranging from minor skin wounds, pimples and boils to more severe infection including endocarditis, osteomyelitis, and toxic shock (1). The prevalence of S. aureus infections is compounded by the emergence of multi-drug resistant strains. Since the inception of penicillin in clinical use, drug-resistance in human pathogens has become widespread. Methicillin resistant staphylococci were first described in 1961 (6), and since then, the prevalence of methicillin resistant S. aureus (MRSA) has rapidly increased to account for greater than 60 % of hospital onset S. aureus infections in intensive care units in the United States (7). Each year in the United States, MRSA strains alone cause an estimated 94 360 invasive infections resulting in ~18 650 deaths (8). Though the incidence of   2 MRSA in Canada is lower than the United States, it still represents a significant financial burden with annual costs for treatment and control of MRSA in Canadian hospitals estimated to be ~$129 million (9). The drug of last resort for treating S. aureus infection is vancomycin; however, vancomycin resistant S. aureus strains are increasingly isolated in care facilities (10). Clearly, novel targets need to be identified to curb the emergence of highly resistant S. aureus (11). The pathogenic success of S. aureus compared to the other staphylococci is due to its ability to survive within many different hostile host environments. The S. aureus surface is coated by a capsule and the thick cell wall is laden with other protective components to impede immune recognition (12). Numerous surface adhesins allow S. aureus to adhere to almost any surface in the human body (13). Aside from avoiding host immune attack S. aureus secretes numerous proteases, lipases and lytic toxins that exacerbate tissue and immune damage (14). Amid the battle for infection and establishment, a steady supply of nutrients must be garnered from host tissues for S. aureus growth; this is accomplished through tissue damage and high affinity uptake systems. Iron is likely one of the most important limiting nutrients and as a result, S. aureus expends significant resources to acquire iron.  1.2. Iron in the human host  Iron is the fourth most abundant metal in the earth’s crust and is an essential component of nearly every biological system (15). As a cofactor in heme moieties, it functions in electron transport as well as having enzymatic roles in diverse processes from general metabolism to neutralizing oxidative damage (15,16). Iron is poorly soluble at physiological pH and in oxidizing environments, where it is primarily found in hydroxide precipitates which are   3 unavailable to most organisms. Since free iron is not typically available, organisms need high affinity systems to acquire and store iron (17,18). In humans and other mammalian host systems, restricting the access of iron is a common strategy to limit pathogenic growth. The human body contains  ~ 4 g of iron, but free iron concentrations are maintained at levels orders of magnitude below those required to support bacterial growth (19). Approximately 75% of the intracellular iron pools are heme–iron, predominantly found in the oxygen transporter, hemoglobin (Hb), while other heme-iron sources include myoglobin and heme-containing enzymes (19). The other prominent intracellular iron supply is the labile iron compartment that stores chelatable Fe(III) iron that can also be mobilized for use by pathogens (20,21). Extracellular iron levels are minimized by sequestering Fe(III) in transferrin and lactoferrin, and extracellular heme is quickly removed by circulating heme scavenging proteins, such as hemopexin and albumin. Free hemoglobin can be detrimental and is scavenged by haptoglobin, and ultimately both heme-hemopexin and hemoglobin-haptoglobin are recycled in the liver (22). Clearly, the ability to use host iron sources to overcome growth-limiting iron offers an advantage in an otherwise hostile environment. Bacteria use several different strategies to acquire iron and these are often broadly grouped into Gram-negative and Gram-positive uptake systems (Figure 1-1).    4  Figure 1-1. Overview of iron uptake system architecture in Gram-negative (left) and Gram- positive bacteria (right). Proteins or small molecules carrying out similar functions are similarly coloured. Secreted iron- chelators (red), siderophores or hemophores, are analogous in both groups of bacteria. Gram- negative bacteria receive complexes in outer membrane receptors (orange) and use energy from the TonB-ExbB-ExbD complex (tan) to drive import of the molecule. Gram-positive bacteria on the other hand receive small iron bound chelators, such as ferric-siderophores directly at the cytoplasmic membrane, while protein-bound sources are bound at the cell wall. The two groups converge at the homologous substrate binding protein (green), differing in that the Gram-positive equivalent is tethered to the membrane. From the binding protein, substrate is imported through the permease (blue) by energy derived by the ATPase (purple). Protein components that are variably present in uptake systems are coloured grey. Tails from protein components represent anchors to either the outer membrane or the cell wall.  1.3. Iron uptake in Gram-negative bacteria  The physiological differences between Gram-positive and Gram-negative bacteria provide unique challenges to iron uptake. In Gram-negative bacteria iron complexes must traverse the   5 outer membrane, periplasmic space and the inner membrane (Figure 1-1). Additionally, the diverse iron sources necessitate different uptake mechanisms. Gram-negative iron uptake systems are more completely understood than their Gram-positive counterparts and will serve as the prime introduction to strategies for iron uptake. In Gram-negative bacteria, small soluble nutrients, such as Fe(II), can diffuse through outer membrane porins and gain access to inner membrane transporters (23). Fe(II)-uptake is important in E. coli, Helicobacter pylori, and several other bacteria that primarily colonize sites in the acidic, and anaerobic or microaerophillic digestive tract, where Fe(II) is likely a predominant form of iron (23-25). The best described inner membrane Fe(II) transporter consists of two components, FeoA and FeoB (feoC is additionally present in the γ-proteobacteria), that import iron from the periplasm by an ATP/GTP driven mechanism (26,27). Many bacterial pathogens reside at near neutral pH and often in aerobic environments, where Fe(III) is the predominant form of iron and porins are no longer a viable means of passage through the outer membrane. The poor solubility of iron in these environments, necessitates that Fe(III) be removed from host iron sources by direct methods, such as hemoglobin, transferrin and lactoferrin binding, or indirect methods, such as siderophore or hemophore iron scavenging and subsequent reception (18). Transport across the outer membrane is an energy-driven process. Whether direct or indirect iron sources, energy for import is derived from the inner membrane proton gradient and transmitted to the outer memebrane receptor via TonB, leading to the classification of this broad class of importers as TonB-dependent transporters. The ferric hydroxamate uptake (Fhu) system from E. coli is one of the most studied TonB-dependent import systems and can serve as a model, so it will be introduced here as a typical Gram-negative iron importer (Figure 1-2).    6  Figure 1-2. Schematic of E. coli ferric hydroxamate uptake (fhu) system.    In this system, E. coli imports exogenously synthesized hydroxamate-type siderophores, such as the fungal siderophore ferrichrome or ferricrocin (28). The iron complex is received by the outer membrane receptor, FhuA, composed of a β-barrel with 22 membrane-spanning sheets, well-defined extracellular loops and an N-terminal plug domain that gates the periplasmic face of the β-barrel (29-31). Ligand is bound within the receptor barrel, where it makes contact with surface loops as well as regions within the plug domain (30,31). In several TonB-dependent receptors, ligand binding induces unwinding of a switch helix on the periplasmic face that appears to facilitate TonB binding. However, recent studies demonstrate the same switch helix in FhuA is constitutively disordered, suggesting additional levels of control (32). Regardless of the conformational change upon siderophore binding, TonB make extensive contacts with the   7 periplasmic face of FhuA (33). The TonB-ExbB-ExbD complex is located at the inner membrane and TonB traverses the periplasmic space to couple energy from the proton motive force across the inner membrane to opening of FhuA. Several competing models have been proposed for the mechanism of energy transmission (for a review of the energy coupling models see (34)). The FhuA-TonB interaction allows TonB, and thus the proton motive force, to drive FhuA conformational change in the plug domain to open the transporter and facilitate import of the ligand for delivery into the periplasm (33). In the periplasm, the substrate binding protein, FhuD receives the siderophore and delivers it to the inner membrane ABC transporter. Recent evidence indicates FhuD is localized to FhuA through interaction with TonB. The localization assists ligand delivery through the integral membrane protein directly to the FhuD (35). Typically, substrate binding proteins shuttle their ligand across the periplasm to inner membrane ABC transporters, preferentially docking when ligand bound (36). Crystal structures and molecular dynamics simulations of FhuD suggest the discrimination between ligand-bound and -free states is finely tuned (37,38). While the structure of the Fhu ABC transporter structure has not been determined, the well-studied E. coli vitamin B12 transporter is homologous and can serve as a model for docking and transport at the inner membrane (36). The ferric hydroxamate-bound FhuD conformation allows salt bridges to form between the apex of each binding protein domain and the permease (Figure 1-3) (36,39).   8  Figure 1-3. BtuCD-F crystal structure. The complex of BtuC (orange), BtuD (cyan) and BtuF (blue) is shown as a cartoon embedded within the cell membrane (PDB ID: 2QI9; (39)). Docking of BtuF on BtuC is mediated by salt bridges between two Glu and Arg residues on the substrate binding protein and permease, respectively. Insets show magnified image of salt bridge with sidechains shown as sticks and oxygen and nitrogen atoms coloured in red and blue, respectively (39).   ATP hydrolysis by the cytoplasmic ATPase components drives transport of the Fe(III)- siderophore through the FhuB homodimeric permease into the cytoplasm. In the cytoplasm, siderophore iron must be released from the complex for use. Depending on the siderophore, this is accomplished by one of two known mechanisms. In the Fhu system, FhuF reduces Fe(III) to Fe(II) bound to ferrichrome (40) or alternatively, E. coli uses the protein, Fes, to hydrolyze enterobactin (41). Either strategy decreases iron affinity by the siderophore to facilitate iron dissociation. Most iron-complexes imported into the cell enter through specialized transporters and while the identity of the transporters differs, the general architecture is   9 maintained. Therefore, the Fhu system represents a good model system for iron uptake in Gram- negative bacteria. Specialized systems for direct iron import involve the binding of host iron sources through specialized, high affinity receptors in the outer membrane. Receptors have been identified for haptoglobin, hemopexin, hemoglobin, transferrin and lactoferrin (42-45). The Neisserial TbpB/TbpA duo is a well-studied model for transferrin and lactoferrin uptake systems (42). The lipid anchored surface protein, TbpB, is not absolutely required for transferrin-iron uptake, but does enhance the rate of import by the integral membrane protein, TbpA, where Fe(III) is stripped from the host protein and imported (46-48). Heme proteins can also bind directly to integral membrane receptors in the presence and absence of accessory receptors. For example, the hemoglobin-haptoglobin receptor from meningococci HpuB (44,49) employs an accessory membrane anchored protein HpuA and the broad substrate heme receptor from Yersinia enterocolitica, HemR, functions alone (45,50). Again, the receptor is then able to strip heme from the host protein for transport. Regardless of the surface reception system, whether directly at the outer membrane transporter or by an accessory receptor anchored on the extracellular side of the outer membrane, the integral membrane transporter is largely conserved in both structure and mechanism for the many iron sources (51). In the cytoplasm, different iron sources have different fates. Fe(III) can be used directly for iron metabolic functions, but must be stored until use to avoid toxicity (52). Alternatively, heme has three potential fates all of which have been shown to occur in E. coli. It can simply be reused in heme proteins or the iron can be removed by either porphyrin ring degradation by the heme oxygenase, ChuS in O157:H7 strains (53), or removed from the porphyrin without ring cleavage by the recently discovered deferrochelatase, YfeX and EfeB (54).   10 Indirect iron uptake systems, which include siderophores and hemophores, are analogous to direct systems, differing in that the ligand received at the surface is produced by the bacteria and chelates iron from the host prior to reception. Siderophores are low molecular weight, high affinity iron chelators that are synthesized by organisms and exported to scavenge Fe(III), with subsequent reception of the Fe(III)-loaded siderophore at the outer membrane. Hemophores are functionally analogous to siderophores in that they are synthesized by bacteria and secreted into the environment to scavenge heme (55). However, hemophores are typically protein molecules, such as HasA from Serratia marcescens or HxuA from Haemophilus influenzae, which extract heme from hemoglobin and hemopexin, respectively (55-57). Once ligand-bound, siderophores or hemophores are received directly by a TonB-dependent transporter that functions analogously to the direct reception systems, transporting extracted heme or intact siderophores. Generally, iron uptake in Gram-negative bacteria is accomplished by TonB-dependent transport systems. Due to the complex nature of traversing the outer and inner membrane, these transporters often contain additional components relative to their equivalent Gram-positive transporters.  1.4. Iron uptake in Gram-positive bacteria  Iron uptake systems in Gram-positive bacteria are not as well understood as their Gram- negative counterparts despite the fact that many of these systems are comprised of fewer components. Gram-positive bacteria lack an outer membrane and typically have much thicker, more complex cell wall structures (58). Fe(III) chelates including siderophores can gain direct access to the cell membrane by diffusion through the cell wall, removing the need for surface receptors beyond those at the inner membrane. A ferric hydroxamate uptake (Fhu) system was discovered in S. aureus with similarities to the E. coli system. However, unlike the Gram-   11 negative counterpart, Fe(III)-hydroxamates can diffuse to the cytoplasmic membrane, and the system contains two paralogous substrate binding proteins, FhuD1 and FhuD2, a homodimeric permease, FhuB and an ATPase, FhuC (59,60). Though FhuD1 and FhuD2 are functionally equivalent to E. coli FhuD, the S. aureus proteins are lipidated at the N-termini since there is no outer membrane to contain them. From the anchored position, a flexible linker enables the ligand bound receptor to interact with the permease for ATP driven import analogously to the E. coli strategy. Interestingly, FhuD2 is the outermost receptor and binds siderophores with a much lower dissociation constant (~20 nM) in contrast to the comparatively weak binding to E. coli FhuD (~1 µM) (61,62). Though siderophores readily traverse the cell wall in Gram-positive bacteria, large or hydrophobic ligands require reception at the cell wall. In these systems, the pathway becomes more complex and often comprises specialized receptors that span the cell wall or receptors covalently attached to the cell wall. In either case, the surface exposed receptors bind the iron- protein complex, remove the iron and pass it to the lipid anchored substrate binding protein for transport through the ABC transporter and processing in the cytoplasm analogously to the Gram- negative systems. Though the general framework in many cases has been identified, the mechanisms of reception and transport are poorly understood.  1.5. S. aureus iron uptake systems  S. aureus possesses the ability to use most human iron sources for growth. It has been shown to grow on heme, hemoglobin, hemoglobin-haptoglobin, transferrin, and ferric or ferrous iron (63-65). Though the protease complement may enhance its growth on protein-iron sources, S. aureus has been shown to possess specific uptake systems to extract iron from several host   12 protein sources. Since Gram-positive iron uptake systems in general are poorly understood, the work in this thesis focuses on systems in S. aureus as both a model Gram-positive organism as well as a significant human pathogen.  1.5.1. The S. aureus heme uptake system  The primary S. aureus heme-uptake system is termed the iron-responsive surface determinant (Isd) system (63,66,67). Since identification in 2002, a large amount of work has been done to characterize the structure and function of component of the Isd system. The S. aureus genome encodes nine proteins directly involved in heme uptake by the Isd system and sortase B (SrtB) (Figure 1-4). IsdA, IsdB, IsdC and IsdH (also referred to as HarA) are cell wall anchored surface receptors (63-65,68). The Gram-positive bacterial cell wall is a dynamic structure with diverse functions, from imparting rigidity to providing scaffolding for surface proteins and interactions with host factors (69). The cell wall is typically 15–30 nm thick (58) and is a substantial barrier for large ligands such as proteins to reach the cell membrane. Thus, cell wall anchored transporter proteins may be used by the cell if passive diffusion through the cell wall is not sufficient. Since Gram- positive bacteria lack an outer membrane, surface proteins are often covalently anchored to the cell wall by the action of sortase enzymes which recognize a C-terminal anchor signal and covalently anchor the protein to the peptidoglycan cross-bridges (70). SortaseB (SrtB) is expressed in one of the transcriptional units of the system and IsdC appears to be the lone SrtB target in S. aureus, with the remainder of the surface receptors anchored by sortase A (SrtA) (63). The Isd system provides a relay system to move heme by anchoring several proteins at varying depths in the cell wall. As determined by protease susceptibility, IsdB and IsdH are   13 completely exposed to the environment, while IsdA is partially exposed and IsdC is buried within the cell wall (63). The localization of the proteins led to the proposed mechanism of transfer from the outer proteins, inward (71). The proposed topology is supported by in vitro heme transfer experiments that demonstrated preferential heme transfer directionally from hemoglobin to IsdB or IsdH, then to IsdA followed by IsdC and IsdE (Figure 1-4) (72-74).    Figure 1-4. Schematic representation of the Isd system heme transport components. Heme transport and iron liberation is accomplished by the coordinated effort of nine Isd proteins. IsdA, IsdB, IsdC, and IsdH (green) are covalently anchored to the cell wall. IsdE and IsdF (blue) are the substrate binding protein and permease components of an ABC transporter, respectively. IsdE is shown in the heme-bound state prior to complexation with IsdF for transport. IsdD (blue) is a membrane protein of unknown function. IsdG and IsdI are cytoplasmic heme-degrading enzymes. The ligand preferences for each member are illustrated as Hm (heme), metHb (methemoglobin) and Hp (Haptoglobin–hemoglobin). For simplicity, IsdB and IsdH are shown interacting with only one protein ligand, but in fact, IsdB also binds Hp–Hb and IsdH also binds metHb. The predominant heme transfer path in the Isd system is represented by arrows.      14 IsdE and IsdF comprise the substrate binding protein and permease components of an ABC transporter, respectively (75). IsdD is a predicted membrane protein of unknown function, yet has been suggested to interact with IsdE and IsdF as part of the membrane transporter (71). However, obvious homologues of IsdD are absent in Isd systems of other bacteria. Once in the cytoplasm, heme has two potential fates: either direct incorporation into host proteins, or ring degradation and iron liberation (67). IsdG and IsdI are paralogous cytoplasmic heme oxygenases that liberate the central iron atom for use by the organism (76,77). The IsdG family of heme oxygenases are structurally unique among heme oxygenases and heme is bound in a largely ruffled conformation in the binding pocket (78). In addition to the unique structural fold, the degradation product of IsdG is distinct from typical heme oxygenases with cleavage at the heme β− or δ-meso carbons as opposed to the α-meso carbon that is cleaved in bilirubin formation (79). In S. aureus, all cell wall anchored Isd proteins, IsdA, IsdB, IsdC and IsdH, contain one to three copies of a conserved near iron-transport (NEAT) domain (80). The ~120 residue domain acquired its name since its predicted secondary structure was similar to that of proteins in the genomic neighbourhood of putative iron-compound ABC transporters in Gram-positive bacteria, including Streptococcus, Bacillus, Listeria and Clostridium species (80). A schematic representation of the NEAT domains of IsdA, IsdB, IsdC and IsdH is presented in Figure 1-5. Each Isd surface protein encodes, at minimum, a secretion signal, a sortase anchoring signal and a NEAT domain. The NEAT domains are often flanked by regions of approximately 15–70 charged amino acids with low sequence complexity and a high likelihood of disorder as determined by the program, Disopred (81,82). Recombinant expression of individual NEAT   15 domains has enabled their structural and functional analysis. Currently, several structures of NEAT domains are described in the literature.    Figure 1-5. Schematic representation of Isd surface proteins. Secretion signals are represented by a white box at the N-terminus of each protein. NEAT domains are indicated as IsdX-Ny, where “X” indicates the unique protein designation, N indicates the NEAT domain and “y” indicates the order of the NEAT domain numbered from the N-terminus of the protein. Heme binding NEAT domains are indicated by an asterisk following the identifier. For each of the proteins the sortase recognition sequence is indicated near the C- terminus.   The Isd system is required for maximal S. aureus growth on heme as the sole source of iron and for full virulence in several models of pathogenesis. In the original characterization of the system as a heme transporter, genetic inactivation of IsdA, IsdF or either SrtA or SrtB led to decreased association of heme with S. aureus cells (63). Subsequent work demonstrated that inactivating IsdA, IsdG or IsdI impaired growth in iron-chelated minimal media with heme as an iron source (63,83,84). Furthermore, inactivating IsdH and IsdB also led to decreased growth on Hb as an iron source (85). Inactivating any one of IsdA, IsdB, IsdC or IsdG and IsdI in S. aureus strain Newman resulted in reduction of organisms in heart, kidney and spleen abscess models of infection (84-86). Interestingly, a recent study demonstrated high levels of IsdA expression in S. aureus isolated from the murine heart and liver, and high levels of IsdB in the heart but not the liver (87). Furthermore, inactivating IsdA impairs S. aureus growth in the heart and liver, while   16 inactivating IsdB impaired infection of heart tissue alone (84,87). Whereas IsdG and IsdI both contribute to bacterial growth in the heart, only IsdG is central for infection in kidneys (84). Clearly, the Isd system is expressed during infection and plays a role in the pathogenic potential of S. aureus. In order to establish progressive infection, S. aureus must successfully colonize multiple host environments. Several studies demonstrate that IsdA interacts with host components in addition to heme and heme proteins. It adheres to many serum and extracellular matrix proteins, inhibits S. aureus killing by binding lactoferrin, and in particular, promotes adherence to human corneocyte envelope proteins (involved in nasal colonization) (64,68,88,89). In addition, IsdA promotes resistance to host innate defenses and survival on human skin by altering the hydrophobicity of the cell surface (90). Furthermore, IsdA promotes binding to nasal epithelial cells, promoting rat nasal colonization (88,91,92). These multifactorial actions of IsdA make this component important to S. aureus in many stages of colonization and infection. Although antibiotics are increasingly ineffective against S. aureus infection, the human body mounts a considerable humoral immune response against Isd components (91,93). IsdA and IsdB have both been successfully used alone or in multi-component vaccine trials, preventing infection in a murine abscess model (94,95) and a rat nasal carriage model (91). Though there has been some question about the possibility of developing an efficacious S. aureus vaccine, several multi-component vaccines which include Isd system components have demonstrated promise (96).   1.5.2. Siderophore uptake systems  S. aureus has the ability to use many siderophores. Despite lacking detectable levels of hydroxamate siderophores in cell culture or identification of biosynthetic pathways in genomic   17 sequences, S. aureus possesses a generalized, high affinity Fhu uptake pathway introduced in Section 1.4 (Figure 1-6) (60,97). The pathway most likely utilizes exogenously produced siderophores, such as ferrichrome, in a parasitic mechanism. No structures of the S. aureus FhuD-siderophore complexes are available, but biochemical studies and growth assays suggest ferric-siderophore binding is mediated, at least partially, by Tyr57, Tyr191, Trp197 and Glu202, while Glu97 and Glu231 are required for docking to the permease for ferric-siderophore import (61,62). Deletion of the FhuCBG components of the system renders S. aureus severely impaired in growth on hydroxamates as a sole iron source, but only moderately impaired in growth in a mouse kidney abscess infection model, where Fe(III)-hydroxamates are not likely to be a significant source of iron (60,98).   Figure 1-6. Schematic of siderophore transporters in S. aureus. ABC transporters for known S. aureus siderophore transporters are shown schematically. For simplicity, surface structures such as the cell wall and capsule are excluded. Substrate binding proteins (HtsA, FhuD1, FhuD2 and SirA) are drawn with lipid anchors embedded in the cell membrane. The heterodimeric permeases (HtsBC, FhuBG and SirBC) are drawn embedded in the membrane in a lighter shade of the same colour as the receptor. FhuC is the functional ATPase for all three importers and is shown as a blue spherical dimer in the cytoplasm. The type of siderophore transported is listed in brackets in the receptor label. SA-staphyloferrin A, SB- staphyloferrin B.   18    S. aureus synthesize at least two siderophores: staphyloferrin A (SA) and staphyloferrin B (SB). SA and SB are both α-hydroxycarboxylate-type siderophores that have been purified and their chemical structures determined (99-101) (Figure 1-7). Recently, the biosynthetic and export loci for both siderophores were identified. The biosynthetic pathways were reconstituted in vitro (102-105) and shown to be important for growth in iron-restricted media and in mouse models of infection (102,106). The biosynthetic genes are arranged in operons and, in both cases, a divergently transcribed iron-regulated ABC transporter (HtsABC for SA and SirABC for SB, Figure 1-6) is present and was shown to be essential for the use of the respective siderophore by S. aureus (102,107). Interestingly, the SA biosynthetic operon is found in many staphylococci, including coagulase negative strains, such as S. epidermidis and S. haemolyticus (102). Alternatively, SB biosynthetic genes have only been found in genomes of S. aureus, but not in all other sequenced staphylococci. However, the SB biosynthetic operon has been identified in the plant pathogen Ralstonia solanacearum and the soil bacterium R. metallodurans (103,106).        19   Figure 1-7. S. aureus siderophores. (A) staphyloferrin A. (B) staphyloferrin B.   SA is synthesized and exported through the action of four proteins, SfaABCD (102,104). SfaC is likely a racemase that converts L-ornithine into D-ornithine. SfaD then condenses D- ornithine and citric acid into a δ-citryl-D-ornithine intermediate. The intermediate is further condensed with another citric acid molecule by SfaB to form staphyloferrin A. SfaD and SfaB are both ATP requiring enzymes that have been used in vitro to synthesize SA (102,104). The SA and SB ABC transport operons contain a lipidated substrate binding protein (HtsA or SirA, respectively), and a heterodimeric permease (HtsBC or SirBC, respectively) (Figure 1-6). Interestingly, like the Isd system, both siderophore transport operons lack an ATPase component for the transporter. Recent work demonstrated that FhuC likely works as an indiscriminate ATPase with several iron uptake systems (98). Despite the well-defined biological basis for SA and SB synthesis and import in S. aureus, the mechanisms of reception, specificity and import are poorly understood.     20 1.6. Objectives of this thesis  S. aureus is a devastating pathogen throughout the world and the dramatic emergence of drug resistant isolates highlights that few effective therapeutics remain to treat invasive forms of infection. Iron is absolutely required by S. aureus, so it must use iron uptake systems that are highly tailored to specific iron sources encountered in the human body. To address the hypothesis, surface receptors from the Isd heme uptake system and the Hts staphyloferrin A uptake systems were studied to examine their contributions to growth and their binding and transport mechanisms. A strong collaboration was initiated with Dr. David Heinrichs at the University of Western Ontario to tackle the objectives from an interdisciplinary approach, through the use of in vivo iron source growth assays, in vitro biochemical assays, and x-ray crystallography. Herein, we demonstrate that the cell wall anchored protein, IsdA, and substrate binding protein, IsdE, contribute to the growth of S. aureus using heme as a sole iron source. Furthermore, we show that heme binding in IsdA is isolated to the conserved NEAT domain, and by defining the IsdA-heme ternary complex by x-ray crystallography, the binding mode of heme by IsdA and other NEAT domain containing proteins is defined. Next, to gain insight into heme movement through the cell surface receptors, insight gained from the wild-type IsdA-heme structure was used to make site-directed variants of residues implicated in heme binding. The variants were analyzed by x-ray crystallography to examine individual residues contributions to binding and by stopped-flow spectroscopy to examine contributions to heme transfer into and out of IsdA. From these experiments, a model for heme transfer between NEAT domains was developed whereby heme is passed from IsdB to IsdA and then to IsdC.   21 Since heme must be transported across the cell wall whereas siderophores diffuse through it, the mechanism of reception by the substrate binding proteins, IsdE and the staphyloferrin A binding protein, HtsA, were investigated. Complexes of the receptors with their respective ligands were characterized both biochemically and by x-ray crystallography. Although the receptors bind their ligand in similar locations within the binding pocket, the mode of binding and affinities are highly ligand-specific. For instance, ligands such as SA that are encountered in low concentration and must diffuse to the substrate binding protein likely require high affinity, specialized binding. Alternatively, ligands that are funneled through the cell wall to the membrane can function equivalently with modest binding affinities due to the effective increase in local concentration of ligand by the cell wall anchored receptors. Finally, the structures provide models for docking of the receptor on the permease based on a homologous receptor permease complex crystal structure. In particular, the HtsA structure was determined in open and closed conformations that revealed small shifts in conserved docking residues that could enable discrimination between apo and holo receptors. The work detailed in this thesis characterizes fundamental aspects of heme and siderophore binding and transport in S. aureus, which have contributed to the development of molecular models for transport.               22 Chapter 2. Methods   2.1. S. aureus growth conditions  Tris-minimal succinate medium (TMS) was used for iron restricted growth, prepared as previously described (108). Residual free iron was chelated from TMS medium using either 200 µM 2,2’ dipyridyl or 10 µM ethylenediamine-di-(o-hydroxyphenyl)acetic acid (EDDHA). Tryptic soy broth (TSB) (Difco) was used for routine culture of S. aureus at 37ºC. When appropriate, the antibiotics tetracycline (4 µg/ml) and chloramphenicol (5 µg/ml) were included in the growth media.   2.2. Construction of S. aureus mutant strains  S. aureus strains described in this work are summarized in Table 2-1. Table 2-1. S. aureus strains. S. aureus strain Description Reference Newman Wild-type S. aureus (109) RN4220 Cloning strain, receives E. coli plasmids (110) RN6390 ΔisdA Previously described ΔisdA mutant strain (64) H734 S. aureus strain Newman with ΔisdA Chapter 3 H734 + pJT35 H734 with plasmid copy of isdA Chapter 3 H834 S. aureus strain Newman with ΔisdE Chapter 5 H834 + pCLVEc H834 with plasmid copy of isdE wild-type Chapter 5 H834 + pCLVEc-M78A H834 with plasmid copy of isdE M78A Chapter 5 H834 + pCLVEc-H229A H834 with plasmid copy of isdE H229A Chapter 5 H834 + pCLVEc- M78A/H229A H834 with plasmid copy of isdE M78A/H229A Chapter 5  2.2.1. Construction of S. aureus isdA::Km  S. aureus RN6390 carrying an insertionally-inactivated isdA gene has been described previously (64). This strain was used to generate the S. aureus Newman strain H734 via transduction using   23 phage 80α (108). Similarly, the multicopy plasmid encoding isdA (64), pJT35, was transduced into H734.   2.2.2. Construction of S. aureus isdE::Km  Allelic replacement, using methodologies described previously (111), was used to generate a non-polar insertion mutation in the chromosomal copy of isdE; the kanamycin cassette, from plasmid pDG782 (112), was inserted into a unique EcoRI site present within the isdE coding region. The Newman isdE mutant was named strain H834. To complement the chromosomal isdE mutation, a DNA fragment containing isdE was PCR-amplified from the S. aureus Newman chromosome and cloned into pAW8 to generate pCLVEc. Plasmid pCLVEc was introduced into H834, via S. aureus RN4220, by standard methodologies (98).   2.2.3. Site-directed mutagenesis of S. aureus isdE for in vivo assays  Site directed mutagenesis was used to alter residues in the IsdE-heme binding pocket. Specifically, site-directed mutagenesis was performed using the QuikChange PCR Kit (Invitrogen), with Pfu Turbo polymerase and pGST–IsdE or pCLVEc as a template. The PCR products were incubated with DpnI (Roche) for 45 min to degrade template DNA, and transformed into E. coli ER2566. Mutations were confirmed by sequencing. Constructs generated using pCLVEc as the template were introduced, via electroporation, into S. aureus RN4220 (110) and subsequently transduced to H834 (ΔisdE) using phage 80α. The constructs were isolated from H834 and sequenced to ensure that mutations had not been introduced during the transformation and transduction procedures.    24 2.3. Heme plate bioassays  S. aureus Newman strains, cultured overnight in TMS broth, were diluted into TMS broth containing 200 µM 2,2’ dipyridyl and grown to an optical density at 600 nm of 0.3-0.5. Cells were washed three times in sterile saline and added to cooled TMS agar containing EDDHA to a final concentration of 105 cfu/ml. Equal volumes of the agar were poured into sterile Petri plates. Sterile paper discs saturated with hemin (10 µg) were placed on the agar plates and incubated for 72 hrs at 37ºC at which point the diameter of growth around the paper discs was recorded.   2.4. Heme-dependent bacterial growth studies in liquid culture  S. aureus cultures were pre-grown, from single colony, overnight in TSB. The cells were washed with saline, and 107 CFU of each strain was inoculated into TMS medium containing 10 µM EDDHA with or without either 50 µM FeCl3 or 5 µg/mL hemin. Cultures (300 µL) were incubated at 37°C with continuous shaking and bacterial growth was monitored every 30 minutes over 20 hours using a Bioscreen C (MTX Lab Systems). Growth curves were plotted using Sigma Plot 2000.   2.5. Peroxidase staining  S. aureus cell-wall extracts were prepared as previously described (113). Briefly, cells were grown overnight in TMS broth containing 2,2’ dipyridyl.  Cells were washed with 0.9% saline and incubated in cell-wall digestion buffer for 2 hrs at 37ºC. Protoplasts were removed by centrifugation. The remaining cell-wall fraction was divided into two samples and either hemin (Sigma), dissolved in 0.1 N NaOH, to a final concentration of 0.1µg/ml, or the equivalent volume of 0.1 N NaOH was added to each sample. The samples were incubated for 1 hr at 37ºC.   25 Cell-wall fractions were separated by SDS-PAGE and stained with either coomassie brilliant blue R-250 (Sigma) or 3,3’5,5’ tetramethylbenzidine as previously described (114).   2.6. Recombinant protein expression systems for non-crystallographic use  E. coli expression vectors and their products for non-crystallographic uses are shown in Table 2-2. Table 2-2. E. coli expression vectors for non-crystallographic analysis. Plasmid Product (application) Reference pGEX-2T® TEV GST expression control GE Healthcare pGST-IsdA GST-IsdA (heme scavenging from E. coli) Chapter 3 pGST-IsdA H83A GST-IsdA H83A (heme scavenging from E. coli) Chapter 3 pGST-IsdA Y87A GST-IsdA Y87A (heme scavenging from E. coli) Chapter 3 pGST-IsdA     Y101A/Y102A GST-IsdA Y101A/Y102A (heme scavenging from E. coli) Chapter 3 pGST-IsdA Y150A GST-IsdA Y150A (heme scavenging from E. coli) Chapter 3 pGST-IsdA Y166A GST-IsdA Y166A (heme scavenging from E. coli) Chapter 3 pGST-IsdA Y170A GST-IsdA Y170A (heme scavenging from E. coli) Chapter 3 pGST-IsdE GST-IsdE wild-type (heme scavenging from E. coli) Chapter 5 pGST-IsdE M78A GST-IsdE M78A (heme scavenging from E. coli) Chapter 5 pGST-IsdE H229A GST-IsdE H229A (heme scavenging from E. coli) Chapter 5 pGST-IsdE     M78A/H229A GST-IsdE M78A/H229A (heme scavenging from E. coli) Chapter 5 pET28a-sfaB 6xHis-SfaB (staphyloferrin A in vitro synthesis) Chapter 6 pET28a-sfaD 6xHis-SfaD (staphyloferrin A in vitro synthesis) Chapter 6    2.6.1. GST-IsdA site directed mutagenesis and expression for heme scavenging from E. coli  The portion of the isdA gene corresponding to amino acids 48 - 316 (omitting codons for the signal peptide and the cell wall anchoring motifs) was cloned into the GST fusion vector pGEX- 2T TEV (Amersham Biosciences). Site-directed mutagenesis of isdA was performed using the QuikChange PCR Kit (Invitrogen), with Pfu Turbo polymerase and pGST-IsdA as a template. The PCR products were immediately DpnI (Roche) treated for 45 min to degrade template DNA,   26 and transformed into E. coli ER2566. Mutations were confirmed by sequencing at the Robarts Research Institute DNA Sequencing Facility (London, Ontario). GST-IsdA was expressed from E. coli ER2566 grown in Luria–Bertani (LB) broth (Difco) supplemented with 100 µg/ml ampicillin at 37°C to an OD of 0.8. Isopropyl β-D-thiogalactopyranoside (IPTG) (0.4 mM) was added and cultures were grown for 20 h at room temperature. GST-IsdA was purified using a GSTPrep column (Amersham Biosciences). GST-IsdA was eluted from the column with 10 mM reduced glutathione, 100 mM NaCl, and 50 mM Tris–Cl, pH 9.0 and dialyzed into phosphate buffered saline (PBS).  2.6.2. Cloning and IsdE protein expression for heme scavenging from E. coli  The majority of the isdE gene, corresponding to amino acids 21–292 (excluding the signal sequence), was cloned into the GST fusion vector pGEX-2T TEV (GE Healthcare) to generate pGST-IsdE. Overexpression of GST-tagged IsdE in E. coli ER2566 (protease-deficient) was achieved by growing plasmid-containing cultures in Luria–Bertani broth (Difco), containing 100 µg/ml ampicillin, at 37°C to an optical density at 600 nm of approximately 0.8. IPTG (0.4 mM) was added and cultures were grown for a further 20 h at room temperature. Bacterial cells were pelleted, resuspended in phosphate-buffered saline (PBS), and lysed in a French pressure cell. Insoluble material was removed by centrifugation at 100,000 g for 20 min. GST-IsdE fusions were purified by passage of cell lysates across a 20 ml GSTPrep column (GE Healthcare). GST- IsdE was eluted from the column with 10 mM reduced glutathione, 100 mM NaCl, and 50 mM Tris–Cl, pH 9.0.     27 2.6.3. Purification of staphyloferrin A biosynthetic enzymes  Recombinant SfaB and SfaD (staphyloferrin A synthetases) were required to produce staphyloferrin A in vitro. The sfaB and sfaD coding regions were amplified from S. aureus Newman genomic DNA and cloned into pET28a(+) for overexpression in E. coli BL21(DE3). Cultures were grown in LB broth (Difco) supplemented with 30 µg/mL kanamycin at 37°C to an optical density of approximately 0.9. IPTG (500 µM) was added and cultures were incubated an additional 18 hours at room temperature with shaking. Cells were harvested by centrifugation at 15000 g and resuspended in binding buffer consisting of 50 mM HEPES, pH 7.4, 500 mM NaCl, 10 mM imidazole and passed through a French pressure cell at 1500 psi. Cell lysate was centrifuged at 15000 g to remove unbroken cells and debris before the supernatant was subjected to additional centrifugation at 150000 g for 60 minutes to precipitate insolubles.  The soluble fraction was then applied to a 1 ml HisTrap nickel affinity column (GE Healthcare) equilibrated with binding buffer and the 6xHis-tagged proteins were eluted with a gradient of 0-80% elution buffer over 20 column volumes (elution buffer consisted of 50 mM HEPES buffer (pH 7.4), 500 mM NaCl, 500 mM imidazole). Proteins were then dialyzed into 50 mM HEPES (pH 7.4), 150 mM NaCl and 10% glycerol at 4 °C. Protein purity was confirmed using sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-Page) and then frozen (at -80°C) and stored as 100 µL aliquots. The protein yields for SfaB and SfaD were 6.8 mg/L and 25 mg/L of culture, respectively.   2.7. UV/Visible absorption spectroscopic characterization of heme binding from the cytoplasm of E. coli  GST-IsdA and GST-IsdE proteins were purified as described above. Wild type and point mutant proteins were purified as expressed from E. coli and relative heme binding was assessed   28 based upon the ability of the proteins, all expressed in an equivalent fashion, to scavenge and retain association with heme derived from the cytoplasm of E. coli. Proteins were adjusted to an equivalent concentration and electronic spectra were recorded using a Cary 500 spectrophotometer (Varian Inc.) with a 1 cm path length and 1 mL quartz cuvettes. All recordings were taken at room temperature.   2.8. Staphyloferrin A purification from culture supernatants  S. aureus strains were grown with aeration in TMS broth containing 0.1 mM EDDHA for 40 h at 37°C. Cells were removed by centrifugation and supernatants were lyophilized. Dried supernatant was extracted with methanol (one-fifth the original supernatant volume), passed through Whatman No. 1 filter paper to remove insoluble material, and rotary evaporated. Material was solubilized in water to 5% of the original supernatant volume. To purify the siderophore present in supernatant concentrates, material was passed across an amberlite IRA- 402 (Fluka) anion exchange column. The column was washed extensively with water before elution with 3 M formic acid. Bioactive fractions were subjected to a desalting step using a C18 cartridge (Applied Separations, Allentown, PA) prior to LC-MS/MS analysis. For use in crystallization, FeCl3 was added to concentrated siderophore extracts to a final concentration of 5 mM, added in 3-fold excess to recombinant HtsA and incubated at room temperature for ~30 minutes. Protein solutions were then passed over a Sephadex G-50 (GE Healthcare) column and concentrated to 25 mg/ml.      29 2.9. Staphyloferrin A in vitro synthesis  Using recombinant SfaB and SfaD, staphyloferrin A biosynthesis reactions were set up as previously described (104). Briefly, 5 µM SfaB and 5 µM SfaD were combined with 1 mM sodium citrate, 1mM D-ornithine, 5 mM ATP, 0.5 M MgCl and 50 mM HEPES pH 7.3 and incubated for 12 hours. The staphyloferrin A reaction was centrifuged in an Amicon Ultra-0.5 10k filter column (Millipore) at 14000 g for 15 minutes to remove enzymes. The filtrate was then supplemented with 3 mM FeCl3 and centrifuged at 18000 g to remove precipitate. 50 µL of the solution was then injected onto a Waters xTerra C18 reversed-phase 5 µm column (150 mm x 2.1 mm) on a Beckman System Gold HPLC equipped with a photodiode array detector. Samples were run at 0.2 mL/min using a step gradient as previously described (104). Solvent A was 10 mM tetrabutylammonium phosphate pH 7.3 in HPLC grade water (Fisher Chemicals) and solvent B was 100% acetonitrile (Fisher chemicals). Data was analyzed using the 32 Karat Software version 8.0 system and peaks were monitored at 340 nm. The peak corresponding to FeSA eluted at 17 min and was collected. Collected fractions were then vacuum centrifuged to dryness, resuspended in deionized water and the concentration of iron determined using atomic absorption spectroscopy (see below) before use in fluorescence titration experiments with HtsA.   2.10. Determination of ferric-staphyloferrin A concentration  Atomic absorption spectrometry was used to determine the concentration of iron in HPLC- purified FeSA samples. The concentration of iron was used to determine the concentration of staphyloferrin A by assuming a 1:1 molar ratio in the FeSA complex. Samples were diluted in 1 M nitric acid before being drawn by an SPS 5 sample preparation system (autosampler) into a Varian AA240 atomic absorption spectrometer (AAS). Absorbance was detected by a Fe/Mn   30 hallow cathode lamp which emits at 248.3 nm specific for iron detection. Absorbance data were analyzed and compared to a linear calibration curve based on known iron standards in ppm. Iron standards were diluted in 1 M nitric acid from an AAS certified 1000 ppm ± 1% stock (Fisher). Calibration standards were separately analyzed first before iron-siderophore samples.   2.11. Fluorescence spectroscopy siderophore titrations  Fluorescence titration experiments were performed at room temperature using recombinant HtsA (15 nM) in 50 mM HEPES (pH 7.4) in a Fluorolog-3 spectrofluorometer (ISA instruments). The excitation and emission slits were set at 2.1 and 6.3 nm, respectively. The excitation and emission wavelengths were set at 280 and 334 nm, respectively. Titration experiments were performed on two separate occasions, each time in triplicate, and the values reported are an average of all data sets. Dissociation constants (Kd) and relevant parameters were calculated by fitting the fluorescence titration data for FeSA (across a concentration range between 0.22 nM and 226 nM of ligand) to a one-site binding model accounting for ligand depletion. Data were analyzed by non-linear regression analysis using the solver tool add-in from Microsoft Excel software, as described previously (61,62).   2.12. Cloning, expression and purification for structure determination  All protein expressed for crystallization was cloned into pET28a(+) (Novagen). The clones were designed to incorporate a thrombin cleavable, N-terminal 6xHis-tag. Details of the individual clones are summarized in Table 2-3.    31 Table 2-3. Plasmids for recombinant protein used in crystallography and heme transfer experiments. Plasmid Product (S. aureus N315 residue #)  Application Chapter pET28a(+) N-terminal 6xHis tag Cloning vector Novagen pET28a-isdAN 6xHis-IsdAN (62-184) structure, kinetics 3 pET28a-isdBN2 6xHis-IsdBN2 (341-458) kinetics 4 pET28a-isdCN 6xHis-IsdCN (22-152) kinetics 4 pET28a-isdAN K75A 6xHis-IsdAN K75A (62-184) structure 4 pET28a-isdAN H83A 6xHis-IsdAN H83A (62-184) structure, kinetics 4 pET28a-isdAN Y166A 6xHis-IsdAN Y166A (62-184) structure, kinetics 4 pET28a-isdAN Y166F 6xHis-IsdAN Y166F (62-184) kinetics 4 pET28a-isdAN Y170F 6xHis-IsdAN Y170F (62-184) kinetics 4 pET28a-isdE 6xHis-IsdE (32-289) structure 5 pET28a-htsA 6xHis-HtsA (38-327) structure 6  2.12.1. Expression of IsdAN and IsdAN variants  The	
  domains	
  were	
  expressed	
  in	
  E.	
  coli	
  BL21	
  grown	
  in	
  2YT	
  media	
  (Difco)	
  supplemented	
  with	
  25	
  µg/ml	
  kanamycin.	
  Cultures	
  were	
  grown	
  at	
  30°C	
  to	
  an	
  optical	
  density	
  of	
  0.8.	
  IPTG	
  (0.5	
  mM)	
  was	
  added	
  and	
  cells	
  were	
  incubated	
  for	
  16	
  hours	
  at	
  25°C.	
  The	
  6xHis-­‐tagged	
  domain	
  was	
  purified	
  using	
  a	
  Chelating	
  Sepharose	
  Fast	
  Flow	
  Ni2+	
  column	
  (GE	
  Healthcare)	
  and	
  dialyzed	
  against	
  50	
  mM	
  HEPES,	
  pH	
  7.2.	
  The	
  6xHis-­‐tag	
  was	
  removed	
  by	
  thrombin	
  digestion.	
  Apo	
  protein	
  was	
  isolated	
  using	
  a	
  Source	
  S	
  column	
  (GE	
  Healthcare)	
  and	
  dialyzed	
  against	
  20	
  mM	
  Tris,	
  pH	
  8.0.	
  Holo	
  protein	
  was	
  generated	
  as	
  previously	
  described,	
  using	
  20	
  mM	
  Tris,	
  pH	
  8	
  as	
  Buffer	
  A	
  (115).	
  Selenomethionine	
  labelled	
  protein	
  was	
  expressed	
  as	
  described	
  previously	
  (116)	
  and	
  purified	
  as	
  above.	
  All UV/visible absorbance spectroscopy readings of protein or heme concentrations and heme release experiments were performed using a Cary 50 UV-Vis spectrophotometer (Varian). Heme concentrations were determined using the pyridine hemochrome assay with the extinction coefficient of 191.5 mM-1cm-1 at 418 nm (117).      32 2.12.2. Protein expression of IsdE for structure determination  The IsdE coding region (G32-K289), excluding the signal sequence and 11 N-terminal amino acids following the Cys lipidation site and three C-terminal amino acids was expressed from E. coli BL21 (DE3). This construct was designed to optimize crystallization based on an analysis using the DISOPRED2 disorder prediction server (81,82,118). Cultures containing the expression vector were grown at 30°C to an optical density at 600 nm of approximately 0.8, followed by induction with 0.5 mM IPTG and growth overnight at 25°C. Cells were resuspended in 20 mM Tris (pH 8), 200 mM NaCl and lysed at 4°C using an Emulsi Flex-C5 homogenizer (Avestin). 6xHis-IsdE was purified using a Ni-Sepharose High Performance column (GE Healthcare) and dialysed against 50 mM Tris (pH 8), 100 mM NaCl prior to thrombin digestion to remove the His6 purification tag. Cleaved protein was dialysed into 50 mM HEPES (pH 7.5) for Source S  column (GE Healthcare) purification, followed by dialysis into 20 mM Tris (pH 8) and reconstitution with hemin as previously described (119). Selenomethionine (SeMet) labelled IsdE was produced as previously described (116) and purified similarly to native IsdE.   2.12.3. Cloning and protein expression of HtsA for structure determination  The HtsA expression construct was designed to exclude the N-terminal secretion signal and lipidation site (residues 1-20) and 17 additional N-terminal residues (21-37) that were omitted from the design because of predicted disorder (81,82,118). pET28a-htsA (to express residues 38- 327) was transferred into E. coli strain ER2566. Cultures were grown in 2YT media at 30°C to an optical density of approximately 0.8. At this point the culture temperature was shifted to 25°C and protein expression was induced with 0.4 mM IPTG. Induced cells were incubated for an additional 16 hours. Cells were resuspended in 50 mM Tris, pH 8.0, 100 mM NaCl and disrupted   33 using an EmulsiFlex-C5 homogenizer (Avestin). 6xHis-tagged protein was purified using a His- Trap HP column (GE Healthcare) in the same resuspension buffer and eluted with a 0-500 mM imidazole gradient. 6xHis-HtsA was dialyzed into 50 mM HEPES, pH 7.8 and the 6xHis-tag was cleaved by thrombin digestion (1:500 mass ratio HtsA:thrombin). HtsA was further purified by cation exchange chromatography (Source 15S, GE Healthcare) in 50 mM HEPES, pH 7.8 and eluted with a 0-500 mM NaCl gradient. Protein samples were dialyzed into 20 mM Tris, pH 8.0 for crystallization. Selenomethionine labelled HtsA was produced by methods previously described (120) and purified similarly to native HtsA.   2.13. Crystallization and structure determination  2.13.1. IsdA NEAT domain structure determination  Crystals were grown at 19 ◦C by hanging drop vapour diffusion. Drops contained 1 µl of 25 mg/ml protein and 1 µl of reservoir solution. The apo-protein reservoir contained 0.1 M CHES, pH 9.5, 30% polyethylene glycol (PEG) 4000. Crystals were transferred to mother liquor supplemented to 15% glycerol and flash frozen in liquid nitrogen. The reservoir for holo-protein contained 0.1 M MES, pH 6.5, 0.2 M ammonium sulphate, 30% PEG 6000. Crystals were transferred into mother liquor supplemented to 20% glycerol and immersed in liquid nitrogen. Heme content of holo crystals was examined by dissolving crystals in water and analyzing them by electronic spectroscopy using a Varian Cary Bio50 UV spectrophotometer and an 80 µl quartz cuvettes with a 1 cm path length. X-ray diffraction data were collected at the Stanford Synchrotron Radiation Lightsource (SSRL) (Menlo Park, CA) at 100 K on beamline 1-5. Multiple wavelength anomalous diffraction data was collected for SeMet labelled apo-protein at the selenium peak (0.97879 Å) and   34 inflection (0.97927 Å) wavelengths. Holo IsdA NEAT domain data sets were collected at a wavelength of 0.97944 Å. Data was processed and scaled with HKL2000 (121). Apo and holo IsdA NEAT domain crystals are not isomorphous and had two and four molecules in the asymmetric unit, respectively. Initial phases and a preliminary model of the apo structure were determined using Solve (122) and Resolve (123,124). Manual construction of the molecule was done using the program O (125) and refined with Refmac5 (126) from the CCP4 program suite (Collaborative Computational Project, 1994). Molecular replacement with the apo form as a search model was used to solve the holo structure using the program MolRep from CCP4 (Collaborative Computational Project, 1994) and refined as above. For the apo structure, chain A was used for figures and structural comparisons since chain B contained a CHES buffer molecule bound in the heme pocket altering the conformation of neighbouring residues. Chain A from the holo structure was used for figures and analysis since chains C and D form a crystal contact at their respective heme binding pockets. Chain A and B molecular contacts are removed from the heme binding site (not shown).  2.13.2. IsdA variant structure determination  Only wild-type IsdAN produced diffraction quality crystals in hanging drop plates with 0.1 M MES pH 6.5, 0.2 M ammonium sulfate, 30 % PEG-6000, as previously reported, with four molecules in the asymmetric unit (83). IsdAN H83A was optimized in a similar condition containing 0.1 M Bis Tris, pH 5.5, 0.2 M ammonium sulfate, 25 % PEG 3350 and formed with two molecules in the asymmetric unit. IsdAN K75A was optimized in 0.1 M citrate pH 3.5 with 2-2.2 M ammonium sulfate and IsdAN Y166A crystals were optimized in 0.1 M HEPES pH 7.5, 2.0 M Ammonium sulphate, crystallizing in different forms with two and three molecules in the   35 asymmetric unit, respectively. IsdAN wt-Cobalt(III)-protoporphyrin IX (CoPPIX) crystals were optimized in 0.1 M citrate pH 3.5, 1.5 M ammonium sulphate. The crystals were isomorphous to the H83A crystals with two molecules in the asymmetric unit. Crystals from PEG containing conditions were frozen in mother liquor supplemented with 16 % glycerol, whereas crystals from ammonium sulfate conditions were frozen supplemented with 30 % glycerol. In both cases crystals were immersed in cryoprotectant for ~ 10 seconds prior to flash freezing in liquid nitrogen. To attain the IsdAN reduced structure, mother liquor was supplemented with 10 mM dithionite and crystals were soaked for ~10 minutes prior to flash freezing.  Data	
  were	
  collected	
  at	
  the	
  Canadian	
  Light	
  Source	
  on	
  beamline	
  08ID-­‐1	
  (IsdAN	
  reduced,	
  IsdAN-­‐CoPPIX,	
  IsdAN	
  Y166A)	
  and	
  the	
  Stanford	
  Synchrotron	
  Radiation	
  Lightsource	
  on	
  beamline	
  7-­‐1	
  (IsdAN	
  K75A,	
  IsdAN	
  H83A).	
  Data	
  were	
  processed	
  using	
  HKL2000	
  (127)	
  or	
  iMosflm	
  (128).	
  Phases	
  were	
  determined	
  with	
  the	
  program	
  MolRep	
  (129)	
  using	
  a	
  single	
  protein	
  chain	
  of	
  the	
  previously	
  determined	
  IsdAN	
  crystal	
  structure	
  (PDB	
  ID:	
  2itf),	
  with	
  heme	
  omitted	
  and	
  several	
  heme	
  binding	
  residues	
  (Lys75,	
  Ser82,	
  His83,	
  Tyr166	
  and	
  Tyr170)	
  substituted	
  with	
  Alanine.	
  Refmac	
  5	
  (130)	
  from	
  the	
  CCP4	
  program	
  suite	
  (131)	
  and	
  Coot	
  (132)	
  were	
  used	
  to	
  refine	
  and	
  modify	
  the	
  structures.	
  Density	
  for	
  heme	
  was	
  clearly	
  defined	
  in	
  all	
  structures	
  by	
  a	
  prominent	
  iron	
  peak	
  and	
  heme	
  was	
  modelled	
  into	
  the	
  binding	
  site	
  prior	
  to	
  water	
  addition.	
  Waters	
  were	
  added	
  with	
  ArpWaters	
  (133)	
  or	
  Coot	
  (132).	
  Since	
  all	
  unit	
  cells	
  contain	
  multiple	
  molecules,	
  comparative	
  measurements	
  are	
  made	
  using	
  chain	
  A	
  unless	
  stated	
  otherwise.	
  	
       36 2.13.3. IsdE structure determination  Heme-bound IsdE crystals were grown by hanging-drop vapour diffusion at room temperature. The well solution contained 50 mM MES (pH 5.5), 0.2 M ammonium acetate, 28% PEG 4000. Drops were made from 1 mL of 30 mg/mL protein solution and 1 mL well solution. Crystals were briefly immersed in well solution supplemented to 16 % glycerol prior to immersion in liquid nitrogen. X-ray diffraction data were collected at the SSRL at 100 K on beam lines 11-1 and 9-2 for the SeMet and native crystals, respectively. Single wavelength anomalous diffraction data were collected at a wavelength of 0.978894 Å. Native crystal data was collected at a wavelength of 1.0 Å. Data were processed using HKL2000 (134). Crystals grew in the space group P43212 with one IsdE molecule in the asymmetric unit. The programs Solve (122) and Resolve (123,124) were used to obtain phases from the nine identified selenium sites and to build a preliminary model. The phase solution had an initial figure of merit of 0.37 that was improved to 0.62 by density modification. The structure was manually constructed using Coot (132) and refined using TLS parameters (135,136) with Refmac5 (126) from the CCP4 program suite (137). All analysis and figures were generated from the native IsdE structure. The refined structure contains residues Gly32-Lys289 and 246 water molecules. The Ramachandran plot reveals 92 % of residues are in the most favoured conformation and none are in the disallowed regions.   2.13.4. Apo- and ferric-staphyloferrin A-bound HtsA structure determination  Apo- and holo HtsA crystals were grown by hanging drop vapour diffusion at room temperature. Apo crystal well solutions contained 0.1 M HEPES (pH 6.8) and 24–30% Jeffamine ED-2001. Hanging drops were made from 1 ml of a 25 mg/ml protein solution and 1 ml of well   37 solution. Crystals were flash frozen in liquid nitrogen after brief immersion in well solution supplemented with 15% glycerol. HtsA was exposed to ferrated concentrated spent culture supernatant from S. aureus grown under iron restriction to form the HtsA-staphyloferrin A complex. Two different conditions yielded diffraction quality holo-protein crystals. Crystal form 1 was grown in hanging drop plates by microseeding with apo-HtsA. The crystals formed in 0.1 M HEPES, pH 7.0, 24% Jeffamine ED-2001. The crystals were frozen in the same buffer with 26% Jeffamine ED-2001 and 15% glycerol. Crystal form 2 grew in sitting drop plates containing 1:1 ratio of protein sample to well solution (0.05 M zinc acetate, 20 % PEG 3350). The crystal was frozen in well solution supplemented with 20% ethylene glycol. Single-wavelength anomalous diffraction data for selenomethionine-labelled protein crystals was collected at the SSRL on beam line 7-1. The data were processed and scaled using Mosflm (128) and SCALA (138). Crystals grew in the space group P21 with one molecule in the asymmetric unit. Phases were determined using Solve (139) and Resolve (140,141) with an initial figure of merit of 0.40 that was improved to 0.80 with density modification. An initial model was built using ARP/wARP (142). Native protein crystal X-ray diffraction data were collected at the CLS on beam line 08ID-1 and was processed and scaled using HKL2000  (127). For both structures, manual building and refinement was completed using Coot (132) and Refmac5 (130), respectively. X-ray data for holo crystal form 1 were collected at the SSRL on beamline 9-2 at 1.00 Å wavelength. Data were processed to 1.3 Å resolution using HKL2000 (134). The protein crystallized in the P21 space group with one molecule in the asymmetric unit. The siderophore- bound HtsA structure was solved by molecular replacement using MolRep (143) from the CCP4 program suite (137) with the previously described apo-HtsA structure (PDB entry: 3EIW) as the   38 search model (102). The structure was edited using COOT (132) and refined with Refmac5 (126). X-ray data for holo crystal form 2 were collected at the CLS on beam line 08ID-1 using a wavelength of 0.97934 Å. Data were processed to 2.2 Å using Mosflm (144) and Scala (138). Indexing suggested an apparent space group of C2221. The structure was solved by molecular replacement, refined and edited as described for crystal form 1. However, poor statistics, namely high values and large discrepancies between Rwork and Rfree values despite good density maps in addition to near identical values for a and c cell dimensions suggested the space group was P21 (a = 52.28, b = 148.60, c = 52.27, β = 117.1°) with twinning. Several cases of twinning in P21 by the operator l, -k, h have been recently described (145-148). The revised solution in P21 contained two molecules in the asymmetric unit and was refined using Refmac with amplitude based twin refinement (126). A twinning fraction of 0.49 (twinning operator l, -k, h) dramatically improved Rwork (0.21 to 0.18) and Rfree (0.29 to 0.24). The model was further refined with TLS parameters (149,150) to a final Rwork and Rfree of 17.0 and 22.8, respectively. Staphyloferrin A coordinates were generated using the program Sketcher from the CCP4 Program Suite (131). The central iron was identified in the electron density by a large peak in the difference map. FeSA was modelled into the structures after fitting the protein backbone, prior to adding waters. FeSA was modelled into the closed structure at full occupancy and has an average B-factor of 28.5 Å2. The open conformation was modelled with FeSA at 0.70 occupancy, as determined to minimize density peaks in an Fo-Fc map at the iron atom. FeSA refined with an average B-factor of 34.6 Å2.    39 2.13.5. Protein structure figures  All protein structure figures were prepared and rendered using the program PyMol (151). Formatting varies and is described in the respective figure legends.  2.14. Determination of heme transfer to apomyoglobin  Apo myoglobin was prepared from horse heart myoglobin (Sigma) as previously described (152). Holo-IsdA variants (2 µM) were incubated with 50 µM apomyoglobin and spectra (250- 650 nm) were recorded for 2-30 hours, variant depending. All transfers were done in triplicate. Absorption differences could be fit to a single exponential equation using Graphpad Prism software to determine a first order rate constant. The concentration of apomyoglobin was varied to ensure the rate is first order with respect to IsdAN.  2.15. Kinetics of heme transfer  Absorption spectra (300-700 nm) were collected using a SX.18MV stopped-flow reaction analyzer (Applied Photophysics Ltd., Leatherhead, UK) equipped with a photodiode array detector. All experiments were performed at 25°C. To determine the wavelength of maximal absorbance change, 2 µM holo-protein was mixed with 20 µM apo-protein and difference spectra were calculated between the initial reading and 100 subsequent time points logarithmically distributed over 10 seconds. For most reactions, absorbance increased maximally at ~375.9 nm and decreased maximally at ~414 nm. For calculation of the observed rate (kobs), the SX.18MV monochrometer was used to monitor absorbance at 375.9 nm and confirmed at 414 nm. For all transfer pairs, 2 µM holo-protein was mixed with 20 µM apo-protein and single wavelength data was collected for > 10 half-lives (between 0.1 and 5 seconds, variant depending). The mean and standard error of the mean for kobs values were obtained from three to five independent reactions.   40 Change in absorbance data were fit to single or double phase exponential equations, as appropriate, using Graphpad Prism software.   2.16. Bioinformatic analysis  2.16.1. Multiple sequence alignment of NEAT domains from S. aureus  Multiple sequence alignments of NEAT domains from several Gram-positive organisms were generated. NEAT domains were identified in the Pfam database (153) Alignments were generated in ClustalX (154) using a BLOSUM matrix with gap opening and extension penalties of 15 and 2, respectively. Alignments were edited using BioEdit (155). The alignment of S. aureus NEAT domains was extracted and viewed in BioEdit (155). In the designation for each of the domains, the number before the dash identifies the NEAT domain number in order from the N-terminus of the protein, and the number after the dash indicates the total number of NEAT domains present in the protein. Positions shaded in dark grey and light grey are identical or similar, respectively, in at least 85% of the aligned S. aureus sequences. Residues identified by asterisk are those that contribute contacts to the heme group. The primary accession numbers are as follows: SauIsdA (Q7A655), SauIsdB (Q7A656), SauIsdC (Q7A654), SauHarA (Q99TD3). The figure was generated in BioEdit .   2.16.2. Multiple sequence alignment of IsdE homologues  Homologous sequences with 28 – 60% identity to IsdE were identified using BLAST (156), aligned using ClustalX (154), and manually edited using BioEdit (155). Accession numbers are as follows: SauIsdE (BAB42229), SpyHtsA (NP_269807), SeHtsA (ABI79312), Lmonocy   41 (CAD00262), Banthra (NP_846991), Bclausi (YP_176916), Bhalodu (NP_244163), Lbrevis (YP_794873), Cperfri (YP_697547), Ctetani (NP_781828), and Cnovyi (YP_877527).   2.16.3. Multiple sequence alignments of HtsA homologues  A BLAST (156) search of the NCBI non-redundant protein database identified many homologous proteins. The top 100 hits (E values < 7 x 10-31) were exported from NCBI and filtered to remove sequences above 80 % identity. The 34 remaining sequences were aligned using the program T-coffee (157,158). The alignment was manually adjusted and visualized using Jalview (159).   42 Chapter 3. Structure and heme recognition by the IsdA NEAT domain  3.1. Introduction  Heme is the most abundant iron source in the human body and can be used by pathogenic bacteria as an iron source (19). To decipher the heme uptake pathway in S. aureus, the first step in heme reception at the surface receptors was studied by a combination of in vivo iron growth assays and biochemically by absorption spectroscopy and x-ray crystallography. At the time of publication of material detailed in this chapter, the cell wall anchored Isd proteins from S. aureus (IsdA, IsdB, IsdC and IsdH) were known to bind heme and facilitate growth with heme as an iron source (63,67). Furthermore, hemoglobin and hemoglobin/haptoglobin binding had been demonstrated for IsdB and IsdH, respectively (65,80,160) and conserved Near iron transport (NEAT) domains were identified in the four cell wall anchored proteins (Figure 1-5) (80). In this study, we show that overexpression of IsdA does indeed enhance the growth of S. aureus and mutants lacking isdA demonstrate a growth defect in media containing heme as a sole source of iron. We further show that, when expressed in E. coli, both full-length IsdA and the isolated NEAT domain are capable of binding heme. To elucidate the basis of heme binding by the IsdA NEAT domain, the domain was crystallized. Crystal structures of the IsdA NEAT domain and of a heme complex reveal a ligand binding cleft and allow the identification of conserved amino acid residues that make specific interactions with bound heme.            43 3.2. Results  3.2.1. Acquisition of heme-iron by S. aureus is enhanced by IsdA  Previously, IsdA has been shown to bind heme (63,161) and Vermeiren et al. (161) demonstrated that one molecule of IsdA binds one heme molecule. However, to date, the role of IsdA in heme-iron acquisition by S. aureus is poorly understood. To assess the affect of isdA on the biology of S. aureus with respect to the ability of this bacterium to utilize heme as a sole source of iron, the isdA gene was insertionally inactivated with a tetracycline resistance cassette in the chromosome of S. aureus Newman to create strain H734. When cell wall protein fractions were taken from S. aureus cultures and stained for peroxidase activity to identify heme proteins, wild-type cells (strain Newman) incubated with heme show clear staining of a band corresponding to IsdA that is not present in H734 (isdA–) cells (Figure 3-1). This band is restored in H734 containing plasmid pJT35 which expresses a cloned isdA gene (Figure 3-1). Two different bioassays were performed to assess the significance of IsdA in the acquisition of heme as a source of iron. In a plate assay, there was no observable growth defect in H734 relative to Newman (Figure 3-2A). Notably, however, H734 expressing isdA from plasmid pJT35 showed a 40% growth enhancement on heme as a sole source of iron. In an alternative liquid culture assay, we demonstrated that while there was no difference in growth rate or yield between strain Newman and strain H734 (Newman isdA::tet) in iron-replete or iron-restricted chemically defined media (TMS), there was a significant difference in the growth rate and yield between the two strains when heme was provided as a sole source of iron (Figure 3-2B). This effect is due to the specific lack of isdA expression in the H734 strain because introduction of pJT35 (which contains only isdA) into H734 complemented the growth defect (Figure 3-2B).    44     Figure 3-1. Peroxidase activity in S. aureus cell wall extracts. Cell wall proteins from S. aureus Newman (lanes 1 and 2), S. aureus Newman isdA mutant (lanes 3 and 4) and S. aureus Newman isdA mutant complemented with pJT35 (lanes 5 and 6) were incubated with (lanes 2, 4, 6) or without heme (lanes 1, 3, 5) prior to electrophoresis. Gels were then stained with coomassie Brilliant Blue R-250 (A) or 3,3',5,5'-tetramethyl benzidine (TMBZ) (B). The TMBZ-stained bands indicate the presence of heme-dependent peroxidase activity.      45   Figure 3-2. IsdA enhances S. aureus growth on hemin as a sole source of iron. (A) Plate bioassays were used to measure growth on hemin as a sole source of iron for S. aureus Newman, H734 (isdA::tet) and H734 containing a multicopy plasmid-expressing isdA. The asterisk indicates a statistically significant change in growth promotion compared with Newman or H734 (P < 0.001 as determined by Student's t-test). Solid horizontal line denotes the diameter of the paper disk that contained the hemin. (B) Liquid culture assays were used to compare the growth of S. aureus strains Newman (squares), H734 (circles) and H734 containing pJT35 (triangles) in TMS media containing 10 µM EDDHA with 50 µM FeCl3 (grey fill), 5 µg/ml hemin (black fill) or no further additions (no fill). Data points represent the mean of three replicates and error bars represent standard deviations.     46 3.2.2. The IsdA NEAT domain binds heme  The presence of a NEAT domain in IsdA, and in other heme-binding proteins such as IsdB and IsdC (63,160,162), suggests that a function of some NEAT domains is in heme binding. Therefore, we expressed only the IsdA NEAT domain in E. coli and found that, indeed, it alone was capable of binding heme as judged by UV-visible spectroscopy at 280 and 407 nm. An absorbance ratio of 280 nm to 407 nm indicated that the NEAT domain, as expressed from E. coli, was approximately two-thirds saturated with heme (data not shown). Partial heme occupancy is typical for heme transport proteins expressed intracellularly in E. coli (162-164). To ensure sample homogeneity for protein crystallization, apo and holo protein were separated by anion exchange chromatography. The apo protein was then crystallized as purified and after reconstitution with heme. Reconstituted IsdA resembles the holo protein fraction as purified from E. coli, except for a greater ratio of the heme Soret (407 nm) to 280 nm absorption (data not shown), indicating greater substrate saturation.  3.2.3. The IsdA NEAT domain structure reveals heme-iron co-ordination by a conserved tyrosine  The IsdA NEAT domain apo structure was solved to 1.6 Å resolution by selenomethionine labelling and multi-wavelength anomalous dispersion phasing (165), whereas the IsdA NEAT holo structure was solved to 1.9 Å resolution by molecular replacement. Data collection and refinement statistics are shown in Table 3-1. The IsdA NEAT domain forms an Ig-like fold composed of seven β-stands forming two sheets of a β-sandwich. However, an additional eighth β-strand is present which results in the N- and C-termini being located in close proximity (Figure 3-3). The recently described fold of the IsdH/HarA NEAT domain 1 (19% sequence identity to the IsdA NEAT domain) is similar except for residues forming an additional N-terminal strand   47 on one β-sheet such that the N and C-termini are on opposite ends of the molecule (160). Although no significant sequence similarity is present, a Dali search (166) reveals that the IsdA NEAT domain fold is similar to that of the clathrin adapter appendage superfamily (2.9 Å r.m.s.d. for 94 aligned Cα atoms to PDB entry 1GYU). The NEAT domain fold is unique with respect to heme-binding proteins.  Table 3-1. Data collection and refinement statistics for apo and holo IsdA NEAT domains.  apo IsdANEAT holo IsdANEAT Data collection* Resolution Range (Å) 45-1.6 (1.64-1.60) 50-1.9 (1.95-1.90) Space group P21 P21 Unit cell dimensions (Å) a=44.5, b=58.3, a=56.02, b=58.6,  c=45.2, β=95.4° c=96.03, β=93.0° Unique Reflections 28462 45710 Completeness (%) 100 (100) 98.3 (96.2) Average I/σI  21.7 (4.5) 15.7 (3.6) Redundancy  5.1 (3.7) 5.5 (3.0) Rmerge   0.07 (0.26) 0.09 (0.29)  Refinement Rwork (Rfree) 0.166 (0.208) 0.171 (0.213) No. of water molecules 398 498 Average B-values (Å2) 16.9 19.5 r.m.s.d bond length (Å) 0.013 0.013 Ramachandran plot, residues In most-favourable region (%) 89.6 90.8 In disallowed regions (%) 0.0 0.0 * Values for the highest resolution shell are shown in parenthesis     48  Figure 3-3. The overall structure of the IsdA NEAT domain heme complex. Heme carbon, nitrogen and iron atoms are shown in red, blue and orange respectively.    The NEAT domain β-sandwich is splayed open at the end opposite the chain termini, forming a pocket between one of the β-sheets and a loop formed from residues Ser82 to Tyr87 . Heme binding in this pocket does not cause significant displacement of the backbone (0.53 Å r.m.s.d.) or side-chain atoms of most residues in the IsdA NEAT domain (Figure 3-4D). Only His83 and Met84 appear to undergo significant conformational changes (Figure 3-4D). The side-chain of His83 rotates ~135° about χ1 to vacate the pocket and form a hydrogen bond to one of the heme propionates. Met84 also shifts to accommodate the heme by moving deeper into the binding pocket (SD displacement of ~4.7 Å). The heme group is bound in the hydrophobic pocket in two equally occupied orientations related to one another by a 180° rotation about the α,γ-meso axis. In both orientations the propionate groups overlap and are facing out towards the solvent. Approximately 278 Å2 (~35%) of heme surface area is exposed to solvent (as determined with AREAIMOL (131)). The propionates form hydrogen bonds with residues Lys75 (2.9 Å), Ser82 (2.6 Å) and His83 (2.9 Å) (Figure 3-4A and Figure 3-5).   49    Figure 3-4. The IsdA NEAT domain crystal structures. A. Secondary structure of the NEAT domain with the heme and selected amino acid residues drawn as sticks. Oxygen and nitrogen atoms are red and blue respectively. The heme carbon atoms are in red and the carbons of amino acid side-chains are shown in yellow. B. Electrostatic potential surface representation of the domain in the same orientations as (A). Positive potentials are indicated in blue and negative potentials are in red. Heme (green) is in the binding pocket. C. Superposition of the backbone of the apo (yellow) and holo (magenta) structures of the IsdA NEAT domain. Heme is shown in sticks within the binding pocket. D. Superposition of residues in the heme-binding pocket of both the apo (yellow) and holo (magenta) protein are drawn in sticks and are labelled. Nitrogen (blue), oxygen and sulphur (orange) atoms are indicated by colour in displayed side-chains.    50   Figure 3-5. Stereo view of the heme site including the residues of the heme-binding pocket illustrated in Fig. 2A. The electron density represented in grey is a 2Fo–Fc map contoured at 1.0 σ. Carbon atoms of the heme and the amino acid residues are shown in red and yellow respectively. Nitrogen, oxygen and iron atoms are blue, red and magenta respectively.   In full agreement with magnetic circular dichroism analyses of full-length IsdA in complex with heme (161), the crystal structure of the NEAT domain–heme complex reveals that the iron is five-coordinate, with the axial ligand (2.1 Å) provided by the phenolic oxygen of conserved Tyr166 (Figure 3-4A and Figure 3-5). The iron atom is displaced from the heme nitrogen plane towards the coordinating oxygen by 0.4 Å and the angle between the phenol ring and the ligand bond is ∼130°. Tyr166, in turn, forms a hydrogen bond (2.6 Å) to Tyr170 OH (Figure 3-5). His83 is positioned on the opposite side of the tetrapyrrole plane to Tyr170; however, the imidazole ring is coplanar to the heme and does not serve as a second axial ligand. Several hydrophobic residues that comprise the binding pocket, namely Met84, Tyr87, Phe112, Trp113, Val157, Ile159, Val161 and Tyr170, contact the tetrapyrrole rings of the heme.  3.2.4. IsdA point mutants show that Tyr166 and Tyr170 are essential for heme binding  Spectroscopic studies of full-length (excluding signal peptide and C-terminal sorting sequences) IsdA reveal a strong absorbance at 407 nm (A407 nm/A280 nm ratio = 0.629), as well as   51 signals in the visible region at 503 nm, 538 nm and 625 nm, indicative of heme binding (Figure 3-6). Alanine scanning mutagenesis of GST–IsdA was performed to monitor the significance of IsdA residues for heme binding (Figure 3-6). Mutation of the conserved heme-iron coordinating residue, Tyr166, to alanine resulted in almost complete abolition of absorption at 407 nm (A407/A280 ratio = 0.20) indicating that this residue is essential for heme binding. Mutation of Tyr170 to Ala (A407/A280 ratio = 0.23) also significantly affects heme binding while mutation of Tyr87 to Ala (A407/A280 ratio = 0.40) diminishes, but does not abolish, heme binding. These latter two residues are situated in the heme-binding pocket and provide hydrophobic interactions in addition to the H-bond between Tyr170 and Tyr166. That His83 is not involved in coordinating heme-iron is aptly demonstrated by the result that a His83 to Ala mutation does not at all diminish heme binding (A407/A280 ratio = 0.68). As magnetic circular dichroism of IsdA identified a tyrosyl residue as the axial ligand (161), alanine substitution of all remaining NEAT domain tyrosines (Tyr101, Tyr102 and Tyr150) was performed. None of these tyrosines are found within the heme-binding pocket in the crystal structure and none of these substitutions affected the absorbance spectra of IsdA compared with wild-type protein.  Figure 3-6. Electronic spectra of IsdA variants. Wild-type and alanine-substitution mutants of GST–IsdA fusion proteins expressed and isolated from E. coli.   52   3.2.5. NEAT domain sequence-structure alignments reveal that the tyrosine ligand is a prognosticator of heme binding  Heme is recognized in a deep, largely hydrophobic pocket formed by a single IsdA NEAT domain. A multiple sequence alignment of a representative set of 43 NEAT domains is given in Fig. S1 and the seven NEAT domains found in S. aureus (one in IsdA, two in IsdB, one in IsdC and three in IsdH/HarA) are presented in Figure 3-7. Three residues in IsdA that make key heme interactions, Ser82, Tyr166 and Tyr170, are generally conserved in a large number of NEAT domains (not shown) but are present in only four out of the seven S. aureus NEAT domains (Figure 3-7). Furthermore, the residues that form the hydrophobic heme-binding pocket in IsdA are either conserved or replaced with similar hydrophobic amino acids in most NEAT domains. In S. aureus NEAT domains with a Tyr residues aligned at positions 166 and 170, heme pocket residues at positions 157, 159 and 161 are either valine or isoleucine and residue 87 is either tyrosine or phenylalanine (Figure 3-7). Pilpa et al. (160) identified conserved tyrosine and histidine residues (potential heme-iron ligands) in S. aureus NEAT domains known to bind heme that were absent in those NEAT domains shown not to bind heme, such as the N-terminal NEAT domain in IsdH/HarA. Thus, Tyr52, Tyr132, His134 and Tyr136 in IsdC, which correspond to Tyr87, Tyr166, His168 and Tyr170 in IsdA, were speculated to be potential heme-iron ligands (160). Now, based on a distinct pattern of conservation of amino acid residues observed to interact with the heme in the IsdA structure, we predict that the single NEAT domain of IsdC, and the C-terminal NEAT domains of IsdB and IsdH/HarA bind heme and co-ordinate the heme- iron by the residue analogous to Tyr166 in IsdA. The other S. aureus NEAT domains either do not bind heme, or bind heme in a completely different manner.    53  Figure 3-7. Multiple sequence alignment of NEAT domains from S. aureus. The alignment was extracted from an alignment of NEAT domains from several Gram-positive organisms (Fig. S1). In the designation for each of the domains, the number before the dash identifies the NEAT domain number in order from the N-terminus of the protein, and the number after the dash indicates the total number of NEAT domains present in the protein. Positions shaded in dark grey and light grey are identical or similar, respectively, in at least 85% of the aligned S. aureus sequences. Residues identified by asterisk are those that contribute contacts to the heme group. The primary accession numbers are as follows: SauIsdA (Q7A655), SauIsdB (Q7A656), SauIsdC (Q7A654), SauHarA (Q99TD3). The figure was generated in BioEdit (155).    3.3. Discussion  Heme binding and uptake are effective means for pathogens to acquire growth limiting iron from their host and indeed overexpression of IsdA does enhance growth (Figure 3-2A) and growth in liquid culture of H734 (isdA–) is reduced significantly when heme is provided as a sole iron source (Figure 3-2B). In contrast, plate bioassays suggest that redundancy exists either within the cell surface Isd system or with other heme transport systems such as Hts (67) which are able to compensate for the loss of isdA, at least in vitro. The plate bioassay showing that overexpression of IsdA in S. aureus enhances heme-iron acquisition suggests that IsdA may function as a cell surface reservoir for heme. It should be noted, however, that although hemin is   54 used commonly in in vitro bioassays to probe the involvement of heme-iron transport proteins, S. aureus would rarely, if ever, encounter free heme within the host. More likely, IsdA would encounter heme complexed to proteins such as hemopexin and, in that context, IsdA may be an essential component of the heme-iron acquisition pathway. The surface potential of the IsdA NEAT domain reveals two large electropositive regions, one located near the polypeptide chain termini, and the other on the same face of the molecule, behind the heme-binding pocket (Figure 3-4). Alignments reveal several conserved residues, Lys75, Lys100, Arg140, Lys156 and His158 (not shown), contributing to these positively charged regions (Figure 3-4A). Interestingly, hemopexin, a proposed IsdA substrate (71), has two dominant negatively charged surfaces near the tunnel entrance of each β-propeller domain (167). The location of the complementary charge surfaces on hemopexin and IsdA, should such an interaction occur, suggests that host protein binding and heme transfer may require more than one IsdA molecule. Non-specific, electrostatic interactions may help to explain the ability of IsdA to interact with several different host proteins (68). Alternatively, the positive surfaces on the IsdA NEAT domain may assist in the orientation of the protein with respect to the negatively charged cell wall or provide an interaction surface for other components of the Isd heme uptake system that convey the heme to the membrane transport complex. In contrast, analysis of the solution structure of the IsdH/HarA N-terminal NEAT domain reveals a large surface with a negative potential (160). This surface is suggested to be involved in the binding of hemoglobin by IsdH/HarA in a 2:1 stoichiometry (160). Heme-iron bound by a single axial tyrosine ligand is uncommon in heme proteins but is an emerging characteristic of heme transporters, including serum albumin, ShuT from Shigella dysenteriae and ChaN from Campylobacter jejuni (115,163,168). The binding of heme to the   55 hemophore from Serratia marcescens, HasA, and the heme chaperone, CcmE, involved in cytochrome c maturation are also similar to IsdA; however, in these instances, an additional histidine residue is coordinated to the iron (169,170). Similar to the IsdA NEAT domain, the multifunctional catalases co-ordinate heme by a single tyrosine. Moreover, and also like the IsdA NEAT domain, a histidine is present on the opposite side of heme in catalase, but does not co- ordinate to the iron (171). No catalase activity could be detected for the IsdA NEAT domain and this is likely because His83 blocks the sixth coordination position of the heme iron (Figure 3-5). That His83 is not a ligand to heme-iron in the IsdA crystal structure, as observed in HasA and CcmE, is further supported by the fact that heme binding is not diminished in the His83 to Ala mutant (Figure 3-6). In fact, the observed slight increase in heme loading of the mutant relative to wild-type IsdA might be explained by a less constricted heme-binding pocket in the mutant protein. Moreover, histidine is not found aligned at position 83 in any other NEAT domain from S. aureus, nor is this position conserved among NEAT domains in general (Figure 3-7). In contrast, Tyr170 is generally conserved in NEAT domain sequences. Tyr170 may play a similar role to His83 in HasA, where it forms a hydrogen bond to the heme-iron coordinating Tyr75 (169). In HasA, protonation of His83 is proposed to alter the affinity of Tyr75 for heme (169). Similarly, the hydrogen bond observed between Tyr170 and Tyr166 in the IsdA NEAT domain crystal structure may control heme affinity by altering the pKa of the phenolate iron ligand. Indeed, the Tyr170 to Ala IsdA mutant protein exhibited reduced heme loading as isolated from E. coli (Figure 3-6). In general, bound heme is more solvent exposed in heme transport proteins than is typical of non-transport heme proteins. For heme bound to IsdA, 35% of the surface area is exposed to the solvent. The solvent exposure of heme atoms observed in the structures of HasA (169), ChaN   56 (115), HemS (164) and hemopexin (167) ranges from 18% to 26%. In contrast, the exposure to solvent of heme cofactors of enzymes such as myoglobin and cytochrome c is typically less than 15%. Exposure to a high dielectric solvent such as water is correlated with a lowering of the reduction potential of heme (172) and the reduction potential of HasA is unusually low (−550 mV) (173). Therefore, high solvent exposure of the heme group in heme transport proteins likely aids in stabilization of the ferric (Fe3+) state of heme iron. The ferric oxidation state of heme is less reactive with oxygen and may facilitate release of the heme group. That the N-terminal NEAT domain in IsdH/HarA does not bind heme (160) is supported by the striking differences observed between what is the heme-binding pocket in IsdA NEAT structure and the equivalent region in the IsdH/HarA N-terminal NEAT domain solution structure. Structural alignment of the IsdA and IsdH/HarA NEAT domain structures reveals that Tyr166, which coordinates to heme-iron in IsdA, is replaced by Glu in IsdH/HarA. Tyr170 is conserved; however, conformational differences in the main chain result in directing the phenol group into the heme pocket in the location of the pyrrole ring observed in the IsdA crystal structure. In the solution structure of the IsdH/HarA NEAT domain, the loop between β-strand 1b and β2 (residues Gln124 to Ser130) is disordered (160). The corresponding region of IsdA, consisting of residues Lys81 to Tyr87, is highly ordered in all six molecules in the asymmetric units of the apo and holo structures (B-factors < 20 Å2). This loop in IsdA interacts directly with the heme propionates (Ser82, His83) as well as providing hydrophobic contacts to the heme (Met84, Tyr87). Residues 81–87 are not involved in crystal contact and this loop is also anchored to the hydrophobic core in both the apo and holo structures by residue Met84. Thus, for heme to bind to IsdH/HarA N-terminal NEAT domain analogous to that observed for IsdA would require substantial conformational rearrangement. Significantly, no large-scale   57 conformational change is observed between the apo and holo IsdA NEAT domain structures (Figure 3-4C). IsdA NEAT domain residues Tyr166 and Tyr170 are critical for heme-iron co-ordination and these residues are conserved in IsdC and in the C-terminal NEAT domains of IsdB and IsdH/HarA. Therefore, we anticipate that each of the four cell wall-anchored S. aureus Isd proteins has at least one NEAT domain that binds heme. The sequence variation among Isd NEAT domains, including positions associated with heme binding, may serve to differentiate these proteins with respect to the recognition of unique heme proteins and non-heme proteins. Given the differences in the 'heme pocket' in the N-terminal IsdH/HarA NEAT domain and the IsdA NEAT domain, coupled with observed differences in binding substrates, additional NEAT domain structures and co-crystal structures will undoubtedly shed more light upon the biological role that the individual domains play and will initiate more detailed studies on the working hypothesis that these domains are involved in transfer of heme across the envelope of Gram- positive bacteria.     58 Chapter 4. Heme transfer to and from IsdA  4.1. Introduction  Heme is bound at the S. aureus surface by cell wall anchored Isd proteins and moved through the ABC transporter for use as an iron source (63). To gain insight into the mechanism of heme transfer between Isd surface proteins, the heme binding and transfer properties of several IsdA NEAT domain site-directed mutants were examined by a combination of stopped-flow spectroscopy and x-ray crystallography. In S. aureus, the heme transfer pathway between Isd surface proteins was defined in vitro for transfer between pure full-length recombinant Isd proteins by visible spectroscopy (72,74), and between their respective NEAT domains by mass spectrometry (73). Both techniques led to the same conclusion: heme is rapidly transferred from hemoglobin through the cell wall anchored proteins, from IsdB to IsdA to IsdC, with transfer to the substrate binding protein component of the ABC transporter occurring at appreciable rates from only IsdC (72-74), although the molecular mechanism of transfer was unclear. In this study, site directed mutagenesis and heme analogs were used to examine the role of individual binding pocket residues, and the metal bound by the porphyrin in heme binding and transfer. Through combination of x-ray crystallography and measurement of heme transfer kinetics, a model for heme transfer is proposed where the heme-iron ligand, Tyr166 of IsdA, is required for direct transfer into and out of IsdA.         59 4.2. Results  The crystal structures of several IsdAN (residues 62-184) variants (IsdAN K75A, IsdAN H83A, and IsdAN Y166A) as well as a reduced wild-type IsdAN and a CoPPIX-bound wild-type structure were determined to 2.05 Å resolution or better. All the structures refine with reasonable geometry (> 90 % of residues in most favoured and < 0.5 % of residues in disallowed regions of a Ramachandran plot as determined using Procheck (174)). See Table 4-1 for structure refinement statistics. Interstingly, heme is bound in all variant structures with minimal disorder as shown by clear electron density and average atomic B-factors < 30 Å2. Furthermore, no single variant resulted in a significant alteration to the NEAT domain backbone conformation with r.m.s.d values for all main chain atoms of < 0.51 Å when compared to the wild-type heme-bound IsdAN structure (PDB ID: 2itf) (83). Notably, the r.m.s.d variations were on the same order as values among the four molecules in the wild-type IsdAN-heme asymmetric unit (~ 0.5 Å).              60 Table 4-1. Data Collection and Refinement Statistics.  IsdAN wt, reduced IsdAN + CoPPIX IsdAN K75A  IsdAN H83A  IsdAN Y166A Data Collection1  Resolution Range (Å)   Space group  Unit cell dimension (Å)     Unique reflections  Completeness (%)  Redundancy  Average I/σI  Rmerge Refinement  Rwork (Rfree)  No. protein molecules  No. water molecules  r.m.s.d bond length (Å)  Average B-values (Å2)  Ramachandran plot (%)     In most-favourable     In disallowed  40 – 2.0 (2.08 – 2.00) P21 a = 56.0, b = 58.6, c = 97.8, β = 93.0° 46318 99.6 (97.2) 3.6 (3.1) 16.5 (3.12) 0.08 (0.337)  0.195(0.246) 4 449 0.013 29.2  90.4 0.2  45 – 1.95 (2.02-1.95) P21 a = 52.5, b = 51.8, c = 55.8, β = 92.0° 20389 97.7 (95.5) 3.5 (3.0) 14.5 (4.5) 0.08 (0.261)  0.182(0.232) 2 341 0.013 17.7  90.2 0.0  50 – 1.35 (1.40-1.35) P21 a = 35.6, b = 76.6, c = 51.7, β = 90.1° 61032 97.3 (80.4) 3.5 (3.5) 16.0 (3.7) 0.07 (0.255)  0.158 (0.185) 2 225 0.013 18.8  91.0 0.0  50 – 1.25 (1.32-1.25) P21 a = 52.5, b = 51.8, c = 55.7, β = 91.9° 80959 97.2 (87.0) 3.4 (2.5) 9.9 (5.0) 0.046 (0.141)  0.139 (0.166) 2 250 0.013 12.9  91.6 0.0  45 - 2.05 (2.13 – 2.05) P212121 a = 61.3, b = 95.8, c = 96.8  34625 99.9 (99.9) 6.6 (6.4) 27.4 (5.6) 0.07 (0.344)  0.188 (0.232) 3 409 0.013 28.5  92.2 0.3 1Data collection values in parentheses represent data for the highest resolution shell   Heme binding in wild-type IsdAN is mediated by direct interactions with several binding pocket residues (Figure	
  4-­‐1A and B) (83). Heme-iron is five-coordinate with a single protein iron ligand, the Tyr166 phenolate, which in turn forms a H-bond with the Tyr170 phenol in an interaction thought to stabilize the Tyr166-iron bond (Figure	
  4-­‐1B). Tyr170 is oriented to allow π stacking interactions with a heme tetrapyrrole ring. Tyr166 and Tyr170 are both located in a β- strand on the proximal side of the heme pocket. Several hydrophobic residues line the base of the binding pocket and a loop crosses the distal side of the heme face (Figure	
  4-­‐1A). Three distal loop residues form H-bonds with the heme propionates. Lys75 and Ser82 form H-bonds with the more buried propionate group, and His83 forms a H-bond to the more solvent exposed   61 propionate (83). Furthermore, the His83 imidazole ring is located ~3.4 Å from heme-iron, and though it does not form an iron ligand, it occludes access to the sixth coordinate position. For the sake of clarity, all following discussion will refer to the Tyr166 face of heme pocket as the proximal side and the His83 side as the distal side regardless of changes in heme-iron coordination.  Figure 4-1. Structure of IsdAN-heme. (A) Overall structure of IsdAN –heme complex (pdb ID: 2itf) shown as a cartoon colored from blue (N-terminus) to red (C-terminus). Heme and selected sidechains are shown as sticks with carbon colored red and yellow, respectively. Oxygen, nitrogen and iron are colored red, blue and orange, respectively. Hydrogen and metal ligand bonds are indicated by dashed lines. (B) Close up of the wild-type binding pocket (pdb ID: 2itf). Residues are shown as in A.  4.2.1. Structures of IsdAN variants  To gain insight into the roles of K75, His83 and Tyr166 in mediating heme binding in the NEAT domain pocket, the crystal structures of three variants were determined. Mutating the heme-iron ligand, Tyr166 to Ala results in heme-iron coordination through His83. The His83 imidazole ring is rotated ~ 90° about χ2, such that Nε coordinates the iron atom (~2.0 Å) (Figure	
     62 4-­‐2A and B). Heme is oriented in the pocket similarly to that in the heme-bound wild-type structure (PDB ID: 2itf). However, the Y166A substitution results in an exposed sixth-coordinate position on the proximal side of the heme, resulting in two distinct conformers in the asymmetric unit. In chain A, a water molecule is bound in the sixth-coordinate position of heme-iron (Figure	
  4-­‐2A). Alternatively, in chains B and C, this water is replaced by His168 Nε (2.1 Å) (Figure	
  4-­‐2B). The side chain of His168 is rotated into the binding pocket relative to chain A, causing a local disruption at residues 166 and 170 of the proximal β-strand. The flexibility imparted by the Tyr-Ala replacement likely enables the alternative conformation of His168 into the pocket. For this reason, an IsdAN Y166F variant was generated for analysis of a more sterically similar replacement, though a x-ray crystal structure is not yet available.   63  Figure 4-2. IsdAN variants. (A) Y166A chain A binding pocket. Coloring is as in Figure 4-1B. The water molecule is represented by a blue sphere. Hydrogen and metal ligand bonds are indicated by dashed lines. (B) Y166A chain B binding pocket. (C) H83A binding pocket. (D) K75A binding pocket.   The loop that forms the distal side of the binding pocket was probed by determining crystal structures of K75A and H83A to resolutions of 1.25 and 1.35 Å, respectively. The IsdAN H83A variant removes the His83-heme propionate H-bond, resulting in an alternative conformation for the propionate group (Figure	
  4-­‐2C). Nonetheless, the conformation of the distal loop is   64 otherwise unchanged, likely due to interactions between the remaining heme binding residues in the loop. Though the H83A substitution renders the sixth coordinate position of heme-iron more solvent accessible, the position remains unoccupied and the heme iron is five-coordinate (Figure	
  4-­‐2C). In the wild-type structure, Lys75 forms a H-bond to the more buried propionate. However, despite the K75A substitution, the propionate remains well ordered and the second H- bond to Ser82 is maintained (Figure	
  4-­‐2D). Additionally, the conformation of the residues that comprise the distal loop are unchanged such that of the variants characterized, the IsdAN K75A variant most closely resembles the wild-type structure. The apparent lack of effect of K75A on IsdAN fold is not surprising in retrospect, since sequence alignments reveal that position 75 is not conserved (83).  4.2.2. Structures of IsdAN in complex with protoporphyrin IX containing altered metal centers  The contribution of the ferric iron to heme binding was investigated by examining the structures of Co(III) and Fe(II) protoporphyrins in complex with IsdAN. Structures of non-ferric metalloporphyrins provide insight into the specificity of the system for ferric-heme. Replacement of Fe(III) by Co(III) results in six-coordinate metal centre with His83 (2.1 Å) and Tyr166 (2.4 Å) as ligands (Figure	
  4-­‐3A). The His83 imidazole rotates ~ 90° about χ2 relative to the wild-type IsdAN heme-bound structure such that Nε coordinates Co(III) (Figure	
  4-­‐3A). The pocket opens slightly to accommodate six-coordinate binding, highlighted by shifts away from the porphyrin ring of ~ 0.9 Å shift of Tyr166 Cα and ~ 1.1 Å shift of His83 Cα relative to the heme-bound IsdAN structure. As in the IsdAN H83A structure, the more exposed propionate group loses its lone protein H-bond and adopts an altered propionate conformation. The remainder of the CoPPIX environment is similar to that of the heme-bound structure.   65   Figure 4-3. The structures of IsdAN bound to protoporphyrin IX with altered metal centers. (A) The IsdAN-CoPPIX crystal structure. Coloring is as in Figure	
  4-­‐1B, but Co(III) is shown as an orange sphere in the porphyrin ring instead of iron. (B) Reduced heme-bound structure.   The IsdAN-Fe(II)heme crystal structure was determined by growing IsdAN-heme crystals as previously described (83) and reducing the central iron by soaking the crystal in cryoprotectant supplemented with 10 mM dithionite for ~ 10 minutes prior to freezing. The crystals undergo an obvious red shift in the visible spectra from typical red-brown of IsdA-heme to a bright pink-red, suggesting heme reduction has taken place. In the crystal structure, central Fe(II) is five- coordinate, by His83 Nδ (2.2 Å), from the distal side (Figure	
  4-­‐3B). The His83 imidazole ring is rotated ~ 90° about χ2 bringing it from parallel to perpendicular to the tetrapyrrole plane. However, in comparison to His83 coordination in the IsdAN Y166A and IsdAN-CoPPIX structures through the His83 Nε atom, the 90° about χ2 occurs in the opposite direction such that Nδ coordinates Fe(II). The rotation of His83 side chain disrupts the H-bond between His83 Nε and the more exposed heme propionate, resulting in an altered propionate conformation with elevated B-factors. On the distal side of the heme pocket, though the Fe(II)heme tetrapyrrole ring   66 is essentially unmoved in the pocket, the Tyr166 OH-Fe(II) distance is stretched to ~ 3.0 Å. Otherwise, the heme contacts are maintained in the Fe(II)heme-bound structure in Chain A. Of the four molecules in the asymmetric unit, Chains A and B undergo Y166 to H83 coordination change, whereas chains C and D more closely resemble the Fe(III)heme structure. However, chains C and D form a direct contact involving atoms of the heme that likely hinders the change in iron coordination without disruption of the crystal lattice.  4.2.3. Rates of heme transfer to myoglobin  For each of the IsdAN variants constructed, the rate of heme transfer to apomyoglobin was determined as an estimate of the heme off rate. The use of apomyoglobin as a heme chelator has become a standard method for measuring off-rates (163). Since previous work demonstrated IsdA does not directly interact with myoglobin (85) and the rates are first order with respect to IsdAN and independent of the apomyoglobin concentration, the observed rate of spectral change is assumed to be direct measure of the IsdAN heme off-rate. The concentration of native and variant IsdAN- heme complex were determined by assaying heme concentration using the pyridine hemochrome assay (117). Liu M. et al determined the off-rate from full length IsdA to be 0.95 h-1 (72), whereas the IsdAN construct used in our studies has an off-rate of 0.60 h-1 (Table	
  4-­‐2). Although our rate is slightly slower, the data support the previous hypothesis that the NEAT domain is the lone functional heme-binding region in IsdA (161,175). Although the crystal structure of the Y170F variant has not yet been determined, the variant is included in all kinetic analysis since it is hypothesized to mediate the strength of the Tyr166-Fe(III) bond. Off- rates from all variants are also slow, and were monitored for > 3.5 half-lives (from 2-15 hours depending on the variant). For all the variants, the rates of absorbance change at 408 nm could be   67 fit to a single exponential assumed to define the off-rate (Table	
  4-­‐2, Figure	
  4-­‐4A). Relative to the off-rate for wild-type IsdAN, the H83A and Y170F variants have decreased rates of heme release by ~3-fold. By contrast, the Y166A and Y166F variants have increased off-rates of 9 and 6-fold, respectively.  Table 4-2. IsdAN heme release rates IsdAN variant k-1 ± SE (h-1)  Fold-change Wild-type 0.60 ± 0.04  1 H83A 0.18 ± 0.01 0.3 Y166A 5.2 ± 0.3 8.6 Y166F 3.9 ± 0.3 6.4 Y170F 0.16 ± 0.02 0.3     68  Figure 4-4. IsdAN heme binding and transfer (A) Absorbance change over time in mixture of 2 µM holoIsdAN H83A with 50 µM apomyoglobin. The black curve is a single exponential fit of the mean and standard error from three independent transfer reactions. A plot of residuals is inset on the same timescale. (B) Sample spectra for transfer from 2 µM holoIsdBN2 to 20 µM apoIsdAN. Maximal spectral change occurs at ~376 nm. ApoIsdAN was used in 10-fold excess to maintain pseudo-first order  reaction conditions. (C) Data from part B plotted as change in absorbance at 375.9 nm against time with standard error from 5 replicates shown as black bars. A single exponential fit of the data is shown as a black line. A plot of the residuals is inset.   69   4.2.4. IsdBN2 to IsdAN heme transfer rates  To investigate the role of key heme-iron coordinating residues, His83, Tyr166 and Tyr170 in heme transfer from IsdB to IsdA, in vitro heme transfer reactions were monitored by stopped- flow spectroscopy for wild-type and variants at these positions. The transfer rates from the heme binding NEAT domain of IsdB (IsdBN2) (2µM) to apo-IsdAN (20 µM) was determined as a baseline for comparison to the individual variants. The maximal change in absorbance occurred at 375.9 nm and the rate of absorbance change could be fit to a single exponential with an observed rate (kobs) of  ~ 57 s-1 (Table	
  4-­‐3 and Figure	
  4-­‐4B and 4C). The observed rate for transfer between these NEAT domains is in line with the previously determined kobs of ~115 s-1 for heme transfer from full-length holo-IsdB (3 µM) to full-length apo-IsdA (30 µM) (74). Since transfers between full-length proteins were performed in 20 mM Tris pH 8.0 at 22°C (74) and the NEAT domain heme transfer reactions were in 50 mM Tris, pH 8.0, 100 mM NaCl at 25°C, these values are not directly comparable. However, the similarity in the kobs for transfer and the fact that kobs is much greater than (~500-fold) the rate of heme release from IsdBN2 (0.11 s-1; C. Gaudin & M. Murphy, unpublished data) indicates that the NEAT domains alone are sufficient for efficient transfer between IsdB and IsdA. The kobs for transfer between 2 µM donor and 20 µM acceptor for the point variants are summarized in Table 3 (See Figure	
  4-­‐5 for additional kinetic traces).  The kobs increased from holo-IsdBN2 to apo-IsdAN for the H83A (2.2-fold) and Y170F (5.6-fold) IsdAN variants, relative to wild-type IsdAN. In contrast, the Y166A and Y166F IsdAN variants decreased the rate of heme transfer from IsdBN2 to IsdAN by 100 and 33-fold, respectively (Table	
  4-­‐3).    70   Table 4-3. Heme transfer kinetics to and from IsdAN variants. IsdA variant kobs ± SE (s-1) holoIsdBN2- IsdAN      Wild-type       58 ± 3      H83A     126 ± 1      Y166A 0.40 ± 0.03      Y166F 2.29 ± 0.02      Y170F     325 ± 12  holoIsdAN- IsdCN       Wild-type 79 ± 7      H83A      138 ± 2      Y166A1  9.0 ± 0.5     65 ± 0.07      Y166F1  4.4 ± 0.3  2.41 ± 0.03      Y170F1      140 ± 3        28 ± 2 1Data fit optimally to a two-exponential. Fold-change related to slow phase.    71  Figure 4-5. Heme transfer rates between IsdBN2 and IsdAN variants. All curves (black) represent a single exponential fit of the change in absorbance at 375.9 nm over time for heme transfer reactions between 2 µM holo-IsdBN2 and 20 µM. ApoIsdAN was used in 10-fold excess to maintain pseudo-first order reaction conditions.  (A) IsdAN wild-type, (B) IsdAN H83A, (C) IsdAN Y166A, (D) IsdAN Y166F, (E) IsdAN Y170F. Points represent the mean   72 and standard error from five independent experiments. Residuals for the fit are shown beneath each curve.  4.2.5. IsdAN to IsdCN heme transfer rates  To investigate the roles of His83, Tyr166 and Tyr170 in heme transfer out of IsdA to IsdC, the in vitro transfer rates were determined by stopped-flow spectroscopy for variants at these positions. The largest spectral change in the holo-IsdAN (2 µM) to IsdCN (20 µM) transfer was at ~ 375.9 nm and its change over time could be fit by a single exponential to establish a kobs of ~ 79 s-1 (See Table	
  4-­‐3 for heme transfer rates). The kobs for heme transfer between full-length IsdA and IsdC (at 10-fold excess) was ~ 25 s-1 (72). As for the IsdB-IsdA transfers between full- length proteins, the rates are not directly comparable since they were performed in 20 mM Tris pH 8.0 at 22°C (72) and the NEAT domain transfers reaction were in 50 mM Tris, pH 8.0, 100 mM NaCl at 25°C. However, the rates for the NEAT domains are similar to those determined for full-length protein and the kobs is much greater than the koff for the IsdAN-heme complex (> 105- fold), suggesting transfer between IsdA and IsdC occurs directly between NEAT domains. The IsdAN H83A variant had a 2-fold higher heme transfer rate from IsdAN to IsdCN relative to wild- type (Table	
  4-­‐3). The transfer rates for the Y166A and Y166F IsdAN variants were better described by a two-exponential equation (Table	
  4-­‐3, Figure	
  4-­‐6); regardless, for both variants the rates were reduced by ~26 to 29 fold compared with wild-type. Strikingly, the decrease in the rate of heme transfer to IsdCN were observed despite increased off rates for these variants to apomyoglobin. 	
     73  Figure 4-6. Heme transfer rates between IsdAN variants and IsdCN. All curves represent a single exponential fit (black) or double exponential (red) of the change in absorbance at 375.9 nm over time for heme transfer reactions between 2 mM (A) holo-IsdAN wild-type, (B) holo-IsdAN H83A, (C) holo-IsdAN Y166A, (D) holo-IsdAN Y166F, (E) holo- IsdAN Y170F and 20 mM apo-IsdCN. ApoIsdCN was used in 10-fold excess to maintain pseudo- first order conditions.   74 4.3. Discussion     S. aureus uses the multicomponent Isd system to transport heme across the cell wall and cytoplasmic membrane to satisfy its iron requirement (63). The cell wall anchored Isd proteins relay heme from the cell surface to the membrane for import (73,74). To provide insight into the roles of specific residues in IsdA ligand binding and transfer, heme binding pocket point variants were analyzed by a combination of x-ray crystallography and stopped-flow spectroscopy. The sites probed were Tyr166 and Tyr170 on the proximal and Lys75 and His83 on the distal side of the heme pocket.  A loop on the distal side of NEAT domains makes several direct heme contacts. Recently, the NMR structure of IsdCN demonstrated that porphyrin binding stabilizes an otherwise flexible distal loop (176). In contrast, the apo-IsdAN structure closely resembles the holo-structure apart from rotation of the side chain of His83, and a shift of Met84 into the pocket (83). Nonetheless, the distal heme loop in the IsdAN binding pocket is sufficiently malleable to accommodate both CoPPIX and Fe(II)heme by exploiting the availability of His83 and Tyr166 for metal coordination (Figure	
  4-­‐3A and B). Crystal contacts in apo-IsdAN may have stabilized the distal loop in the same conformation as the holo form and the loop could undergo a similar disorder to ordered transition as seen in IsdCN. In the presence of heme, the Lys75 or His83 substitutions alone were not sufficient to disrupt the IsdAN distal loop, suggesting stability is a cumulative result of several interactions, including Lys75, Ser82, and His83 H-bonding, Met84 hydrophobic interactions and a stacked π-bonding interaction between Tyr87 and the most buried heme pyrrole ring. Since the main distal loop contacts are directly to heme (83), flexibility in this region is likely a general feature of apo NEAT domains.   75 Residues that form the distal loop are surprisingly variable in S. aureus heme-binding NEAT domains (177). Not a single position is absolutely conserved, including His83, which is replaced by Met, Ile and Val in IsdBN2, IsdCN and IsdHN3, respectively. The x-ray crystal structures of IsdAN Y166A, IsdAN-Co(III)PPIX and IsdAN-Fe(II)PPIX demonstrate that His83 can replace Tyr166 as the metal ligand. Furthermore, the ability of His83 to coordinate the ferrous iron in heme is supported by spectroscopic data on full length IsdA (162). Since the Isd system likely transports predominantly Fe(III)heme rather than Fe(II)heme, the physiological role of His83 in IsdA is unclear. Previously, His83 was proposed to occlude access to the distal side of heme-iron by small molecules (161). Additionally, since histidine is a common iron ligand, it is tempting to speculate His83 may have a role in heme transfer. Two different mechanisms of heme transfer between the Streptococcus pyogenes NEAT domain homolog, Shp and the IsdE homolog SpHtsA have been proposed (178). In the first mechanism for heme transfer between holoShp to apoHtsA, a Shp-SpHtsA complex is formed followed by heme release by from Shp coordinating Met residues. The heme is transferred by release from Shp before iron coordination by the His-Met pair of SpHtsA (178). The second mechanism, supported by spectroscopic data on Ala replacements of either Shp coordinating Met, involves a bridged six-coordinate heme-iron intermediate. In the intermediate, the remaining Met from Shp and one of the SpHtsA ligands coordinate heme iron, followed by replacement of the Met ligand of Shp by the second SpHtsA ligand (178). Heme transfer from full-length IsdA to IsdC was suggested to occur in a two step kinetic mechanism, with association of a holoIsdA-apoIsdC complex followed by transfer of heme to give apoIsdA-holoIsdC prior to dissociation (72). Within the NEAT domain, Tyr166 and His83 are both situated such that either residue could receive heme-iron from the donor or pass heme-   76 iron to the acceptor in a transfer complex similar to that proposed for Shp-SpHtsA. To determine if a specific residue plays a key role in receiving and donating heme, transfer rates to and from IsdAN variants were determined and compared to wild-type rates. Relative to wild-type, the IsdAN H83A variant decreases the off-rate and slightly increases transfer rates to and from IsdAN. Additionally, the IsdAN H83A variant crystal structure reveals that the distal iron coordination site remains unoccupied in the absence of the His83 imidazole ring. Therefore, the modest increase in transfer rate is reasoned to be a consequence of destabilizing the distal loop through the loss of the propionate H-bond and by increasing access to the distal side of the heme- iron. These findings suggest His83 does not play a direct role in heme transfer. Furthermore, the apparent non-essential role for His83 in heme binding and transfer is supported by the lack of His83 conservation among heme binding NEAT domains (179). The primary role of His83 in IsdA is likely to secure the more solvent exposed heme propionate and potentially gate access to the distal heme-iron position. In contrast to the His83 variant, Tyr166 variants were significantly impaired in heme transfer rates both to and from IsdAN despite an increase in the heme off-rate. The IsdAN Y166A crystal structure reveals His83 serves as the heme-iron ligand on the distal side in this variant (Figure	
  4-­‐2A). Since His168 can also form an iron ligand blocking the proximal coordination site (Figure	
  4-­‐2B), a sterically more subtle Y166F variant was made, but transfer rates were comparable to Y166A (Table	
  4-­‐3). The impaired heme transfer rates both from holoIsdBN2 to apoIsdAN Y166A/F, and from holoIsdAN Y166A/F to apoIsdCN, suggest one of two possibilities: Either tyrosinate-iron coordination is required for heme movement into or out of the pocket through an open distal site blocked by His83 coordination in these variants, or coordination by His83 alone is enough to impede efficient transfer. The two possibilities cannot be differentiated   77 with the current data and additional heme transfer assays under pseudo first-order conditions are being performed to investigate the order of the transfer reaction to better explain the transfer kinetics. Despite the shortcomings in the current data, based on the homology with the Shp- SpHtsA system, heme transfer in the Isd NEAT domains is likely driven by the former mechanism, through access to the distal side of the heme-iron, analogous to the model proposed for the single Met-Ala variants in Shp described above (178). The combination of structural and kinetic data support a model where heme is transferred from holo-IsdBN2 Tyr440 directly to IsdAN Tyr166 by accessing the distal side of the IsdBN2 heme- iron. From holo-IsdAN Tyr166 heme-iron is then directly passed to apo-IsdC Tyr132, which also accesses iron through the distal Fe(II)heme-iron face (Figure	
  4-­‐7). This model of transfer offers an explanation for the ability of IsdAN to bind heme in both orientations related by 180° about the heme α,γ-meso axis, which would allow heme to enter in either of the two orientations as required for direct access through the sixth-coordinate position (83). Whether heme is directly passed through a stable six-coordinate TyrDONOR-Fe3+-TyrACCEPTOR intermediate, or released by one Tyr and bound by the other while in complex, could not be determined in these experiments. No intermediate absorbance signatures were seen in full-spectrum stopped-flow experiments.      78  Figure 4-7. Model of NEAT domain heme transfer. (A) Model of the IsdBN2-IsdAN heme transfer complex. IsdBN2 (dark blue) and IsdAN (cyan) are shown as cartoons with heme and Tyr carbons shown as red and orange sticks, respectively. The model was generated manually using PyMol (151)with crystal packing and ClusPro (180,181) docking as a guide (B) Model of the IsdAN-IsdCN heme transfer complex. Shown as in part A with IsdAN shown in cyan and IsdCN in purple.  To date, S. aureus Isd protein-protein complexes have eluded detection, but it is clear that complexes form in solution because of the rapid rates of heme transfer (72,74). Rapid heme transfer by means of direct protein-protein interaction has been documented in the related Bacillus anthracis heme transport system between two NEAT domain containing proteins, BslK and IsdC (182). Crystal contacts can provide indications of the structure of complex formation in solution. Though the complexes in crystals are formed between the same protein, this may represent a biologically relevant transfer reaction as well because the relay occurs across the ~ 30   79 nm cell wall in S. aureus (58), and thus may require self exchange reactions. The crystal structures of IsdA-heme (83) and its variants from different crystal forms (this work), IsdC-heme (183), and IsdHN3-heme (184), all display strikingly similar crystal packing interactions. Two molecules come together across the heme binding pocket related by a ~180° rotation (Figure 4-8). Though two heme groups are present in the interface of the holo-proteins, the domains are oriented such that the coordinating Tyr of one molecule forms the sixth heme iron ligand of the neighboring molecule in all cases. If the same complex were to form with heme in only the donor molecule, the model proposed based on our kinetic data would support heme transfer from Tyr of the donor to Tyr of the recipient, possibly with a bis Tyr intermediate. To examine the potential to form these complexes in solution, the protein-protein complex modeling server, ClusPro (180,185), was used to predict unbiased optimal IsdBN2-IsdAN and IsdAN-IsdCN complexes. The heme ligand at the interface was not modeled in ClusPro, but again the top solutions were strikingly similar to the formations in IsdAN, IsdCN, IsdHN3 crystal packing (Figure 4-9) (183,184). The protein-protein complexes, though packed much more closely than in the models shown in Figure 4-7 because of the absence of heme, were in the same orientations. The proteins are related by a 180° rotation relative to one another such that the heme-iron coordinating Tyr residues could occupy opposite sides of a heme iron. With the crystal packing and ClusPro docking as a guide, a model of the proposed heme transfer pair was constructed using PyMol (Figure 4-7) (151).    80  Figure 4-8. Crystal contacts between NEAT domain heme interfaces. (A) Interface from the IsdAN Y166A structure. Different chains are shown in green or cyan cartoons with heme shown as sticks in the binding pocket (B) Interface from the IsdHN3 structure (C) Interface from the IsdC structure.            81   Figure 4-9. Isd NEAT domain protein-protein complexes predicted by ClusPro (180,185). (A) The top scoring prediction for the IsdBN2 (blue)-IsdAN (cyan) complex. (B) The top scoring prediction for the IsdAN (cyan)-IsdCN (purple) complex.   In the absence of direct experimental observation of complex formation, our structural, kinetic and modeling data support the idea that a heme transfer pair forms, which moves heme- iron from the Tyr of the donor to the Tyr of the acceptor. The absence of a detected transient spectroscopic signature for the intermediate may be explained by the high rates of transfer. Work is ongoing to stabilize transfer complexes for more detailed characterization and validation of the transfer model.   82 Chapter 5. Heme recognition by IsdE  5.1. Introduction  At the cell membrane heme import is driven through a specific ABC transporter encoded in the isd operon (63). The Isd system encodes a substrate binding protein (IsdE) and a permease (IsdF) with homology to known ABC transporters (63). Additionally, the S. aureus ferric hydroxamate uptake ATPase, FhuC, works with several iron transport systems, including the Isd system (98). The current model of the Isd system proposes that heme is relayed through the cell wall anchored receptors (IsdA, IsdB, IsdC and IsdH) to IsdE for ATP-driven import into the cytoplasm. At the time of publication of the material in this chapter, only a few homologous substrate binding protein structures were available with low amino acid sequence identity to IsdE (< 20 %) and no structures of the heme bound receptors were available. In this study, we show that inactivation of S. aureus isdE impairs growth on heme as a sole source of iron. To gain insight into the function of IsdE in heme binding and transport, the crystal structure of the IsdE-heme complex was determined. The structure revealed His-Met heme iron coordination unique to heme transport proteins. Corroborating the structure, alanine substitutions in binding pocket residues showed that mutation of Met78 and His229 resulted in significant loss of heme binding and that IsdE H229A was incapable of supporting IsdE- mediated growth on heme as a sole source of iron in growth promotion assays. This work added substantial mechanistic detail to the complex model of heme uptake by S. aureus and provides a framework for future studies into the mechanism of bacterial binding protein-dependent heme acquisition.      83 5.2. Results   5.2.1. Overall Protein Structure  The selenomethionine and native IsdE structures were solved to 1.95 and 2.15 Å resolution, respectively and data collection and refinement statistics are shown in Table 5-1. The structures reveal a protein with a bi-lobed conformation with two domains, each composed of a central- most parallel β-sheet surrounded by α-helices (Figure 5-1A). The N-terminal domain (Gly32- Arg138) β-sheet contains only two β-strands, whereas the C-terminal domain (Asn163-Lys289) contains a 5-stranded β-sheet. The two domains are connected by a single long α-helix (Lys139- Lys162) that spans the length of the molecule. A large interface is formed between the two domains and is contributed to by a mix of small hydrophobic and several hydrophilic residues. Although a Dali search (166) did not reveal any structurally similar heme-binding proteins, it did identify that the IsdE structure was distantly related to BtuF, a cobalamin transporter, and to the siderophore transporters CeuE and FhuD. For each structure, similarity is characterized by a Z- value >20, a root mean square deviation of 2.8-3.3 Å for 234-244 Cα atoms and a sequence alignment identity <18%. These proteins are all members of the helical backbone metal receptor superfamily.           84  Table 5-1. Data collection and refinement statistics for the IsdE-heme complex.   Native IsdE SeMet IsdE Data collection* Resolution range (Å) 50-2.15 (2.23-2.15) 50-1.95 (2.02-1.95) Space group P43212 P43212 Unit cell dimension (Å) a=64.22, b=64.22, c=144.99 a=63.53, b=63.53, c=144.25 Unique reflections 17319 22583 Completeness (%) 99.7 (99.6) 86.0 (79.0) Average I/σI 42.1 (5.9) 22.2 (5.3) Redundancy 6.3 (5.8) 12.1 (7.6) Rmerge 0.058 (0.363) 0.068 (0.359) Refinement Rwork (Rfree) 20.2 (25.3) 20.1 (26.3) B-factors (Å2)   Protein 28.1 31.7   Heme 31.1 32.6   Water 43.1 44.9 r.m.s.d bond length (Å) 0.012 0.013 * Values for the highest resolution shell are shown in parenthesis           85   Figure 5-1. The overall structure of the IsdE-heme complex. A, a schematic representation of IsdE illustrates the bi-lobed architecture of the protein. Secondary structural elements are represented by strands, loops, and helices colored in blue, green, and cyan, respectively. Propionate stabilizing helix-1 is represented in orange, and protein termini are labeled with N or C. Heme is shown as sticks within the binding pocket. Heme carbon and oxygen are shown as red, and nitrogen and iron are shown as blue and orange, respectively. B, view of the structure looking down into the binding pocket, rotated 90° about the horizontal axis. Atoms are colored as in A.    5.2.2. Heme Binding  Previous studies demonstrated that IsdE is a heme-binding protein (63,162). We demonstrate that a single heme molecule is bound to IsdE along the groove formed between the two lobes (Figure 5-1A and B). Heme is oriented within the pocket at approximately a 45° angle in relation to the longest axis of the protein such that the propionates interact primarily with the N-terminal   86 domain. Approximately 160 Å2 (19%) of the total heme surface area is solvent exposed (as determined with AREAIMOL (131)). Several hydrophobic residues line the interior of the pocket and interact with the largely hydrophobic porphyrin ring. The N-terminal domain contributes Pro77, Val96, and Ile99 to this hydrophobic environment, and the C-terminal domain contributes Val175, Pro176, Leu180, Tyr208, and Ile270 (Figure 5-2C). IsdE-bound heme iron is six-coordinate with axial coordination by the thioether of Met78 (2.3 Å) from the N-terminal domain and the imidazolate of His229 (2.0 Å) from the C-terminal domain (Figure 5-1A and Figure 5-2A). The angles formed between the tetrapyrrole nitrogen plane and the axial ligands are both ∼90°. The tetrapyrrole ring is close to planar, and the iron is displaced from the plane formed by the tetrapyrrole nitrogens by less than 0.04 Å (Figure 5-2A). His229 participates in a complex H-bond network that includes Glu265 from the C-terminal domain and residues Tyr61 and Lys62 from the N-terminal domain (Figure 5-2B). His229 Nδ1 forms an H-bond with HOH15 (2.9 Å), which, in turn, forms an H-bond to Glu265 Oϵ2 (2.9 Å). Glu265 also forms H-bonds directly to Lys62 Nζ (3.4 Å) and Tyr61 O H (2.7 Å) through carboxylate atoms Oϵ2 and Oϵ1, respectively.        87  Figure 5-2. Heme binding and surface structure of IsdE. (A) heme iron is six-coordinate with His229 and Met78 as axial ligands. The Fo - Fc omit difference map contoured at 2.5 σ is shown as a gray mesh. Heme is shown in sticks with heme carbon and oxygen shown in red and nitrogen and iron shown in blue and orange, respectively. Protein side chains are shown in yellow, with nitrogen, oxygen, and sulfur shown in blue, red, and orange, respectively. B, the H-bond network in the heme binding pocket is represented by dashed lines. Atoms are colored as in A. C, the molecular surface of the heme pocket is shown, colored according to atomic coloring scheme, revealing the large region of hydrophobic contacts. D, the conservation of residues in the alignment in Figure 5-3 is mapped onto the molecular surface of IsdE using the Consurf server (186,187). Red, white, and blue coloring indicates sequence conservation from 0 to 100% as indicated by the color bar.          88 The heme propionates are oriented approximately parallel to the binding groove and are essentially buried (Figure 5-2D). They are well ordered in the electron density map and form extensive interactions with IsdE (Figure 5-2B). Lys62 Nζ forms a H-bond to HOH13 (2.9 Å) that, in turn, forms H-bonds with both heme propionates (2.6 and 2.9 Å). One of the heme propionates forms additional direct H-bonds with the main chain amides of Val41 (2.7 Å) and Ala42 (3.0 Å). Val41 and Ala42 are located at the N terminus of α-helix 1 that is oriented such that the positive helix dipole is directed toward the propionate carboxylate group (Figure 5-1A). An additional HOH16 bridged (2.9 Å) interaction is formed with Thr40 OH (2.8 Å) and Thr271 OH (3.1 Å). The other propionate forms direct H-bonds to Ser60 Oγ (2.7 Å) and the main chain amide of Tyr61 (3.2 Å) (Figure 5-2B).  5.2.3. Multiple Sequence Alignments  A sequence search using BLAST (156) revealed several homologous proteins (E-value < 4x10-37) in other Gram-positive organisms, namely species of Bacillus, Listeria, Clostridia, Streptococcus, and Lactobacillus. Previously, the IsdE homologues from Listeria monocytogenes, Clostridium tetani, and Bacillus anthracis are shown to be associated with related Isd uptake systems (71). The sequences were aligned with ClustalX (154), and the alignments were manually edited in BioEdit (155). Each sequence shares greater than 28% identity over the 292 residues of S. aureus IsdE (Figure 5-3). In the identified sequences, the predicted secretion signal and ~15 N-terminal residues after the Cys lipidation site are poorly conserved. The alignments reveal several conserved residues within the heme pocket of the IsdE structure. Both of the iron axial ligands (i.e. Met78 and His229) are completely conserved in the homologous sequences (Figure 5-3). Residues forming the H-bonding network with His229 are   89 also generally conserved. Lys62 is conserved in the bacilli and listerial proteins, but it is replaced by Tyr in the streptococcal and clostridial species. Glu265 is generally conserved or replaced by Asn. Residues interacting with the propionates are also conserved as are the hydrophobic heme pocket residues; where different, they are substituted for residues with similar hydrophobic properties (Figure 5-3). The alignments reveal a conserved patch of residues located at the surface of the N- and C- terminal lobes traversing the heme pocket (Figure 5-2D). Several conserved, charged residues are evident within this conserved patch. Notably, Glu83 and Glu214 are completely conserved, whereas Glu242 differs only in the Clostridium perfringens sequence (Figure 5-3). Lys237 and Lys241 are also conserved as large charged residues at the lobe surface in all aligned sequences (Figure 5-3). Another striking feature revealed by mapping of amino acid conservation onto the structure is the maintenance of nine Pro residues between residues 32-105 in the N-terminal lobe of the homologues (Figure 5-4). Pro77 occurs in the heme iron coordinating Met78 loop, where it forces a tight turn necessary for orienting Met78 correctly in the heme binding pocket. However, Pro38, Pro58, Pro65, and Pro80 are present within loops traversing the N-terminal domain and are generally conserved.      90   Figure 5-3. Multiple sequence alignment of IsdE homologues. Homologous sequences with 28-60% identity to IsdE were identified and aligned. Absolutely conserved and highly conserved alignment positions are shaded black and gray, respectively. Residues are numbered according to the IsdE sequence. Residues forming the heme binding site are denoted by arrows. Heme iron ligating Met78 and His229 are indicated by Fe labels. Accession numbers are as follows: SauIsdE (BAB42229), SpyHtsA (NP_269807), SeHtsA (ABI79312), Lmonocy (CAD00262), Banthra (NP_846991), Bclausi (YP_176916), Bhalodu (NP_244163), Lbrevis (YP_794873), Cperfri (YP_697547), Ctetani (NP_781828), and Cnovyi (YP_877527).    91  Figure 5-4. Conservation of Pro residues within the N-terminal domain of IsdE. Several Pro residues are located in strands spanning the interior regions of the N-terminal domain. Secondary structural elements are shown as cartoons. Proline side chains are shown as sticks and coloured according to conservation from most conserved (blue) to least conserved (red) in the IsdE homologs identified in Figure 5-3. Heme is shown in sticks within the binding groove. Heme carbon, nitrogen and iron atoms are shown in red, blue and orange, respectively.    5.2.4. Contribution of Residues to Heme Binding and IsdE-mediated Heme Transport in vivo  As shown in (Figure 5-5A), spectroscopic analysis of GST-IsdE shows strong absorption in the Soret region as well as signals in the visible region around 650 nm; these signals are characteristic of heme binding. As expected, GST alone showed none of these signals. To validate the crystal structure of the IsdE-heme complex, Ala point mutations were constructed in several of the conserved heme binding pocket residues of IsdE. Mutation of the conserved heme iron-coordinating His229 and Met78, individually, resulted in a significant reduction in heme binding by IsdE. Moreover, mutation of both His229 and Met78 to Ala in the same protein completely abolished IsdE heme binding activity (Figure 5-5A). Notably, we also observed   92 altered IsdE heme binding properties upon mutation of other residues whose side chains coordinated directly or indirectly (via waters) to the heme structure (Figure 5-6).  Figure 5-5. Heme binding and transport by point mutants of IsdE. (A) electronic spectra of wild type and alanine substitution mutants of GST-IsdE fusion proteins expressed and isolated from E. coli. (B) IsdE contributes to growth of S. aureus on hemin as a sole source of iron, and His229 is absolutely required. Liquid culture growth assays were used to compare growth of S. aureus strains Newman containing pAW8 (empty vector) (circles), H834 (Newman isdE::Km) (down triangles), H834 containing pCLVEc (squares), H834 containing pCLVEc-Met78Ala (diamonds), and H834 containing pCLVEc-His229Ala (up triangles), in tris- minimal succinate media containing 5 µM ethylenediamine-di-(o-hydroxyphenyl)acetic acid with 50 µM FeSO4 (black shade), 5 µg/ml hemin (gray shade) or no further additions (no shade). Data points represent the mean of five replicates and error bars represent the standard deviation.     93  Figure 5-6. Heme binding by mutants of IsdE. Electronic spectra of wild-type and alanine substitution mutants of GST-IsdE fusion proteins expressed and isolated from E. coli.    We and others have shown previously that S. aureus is capable of growing on hemin as a sole source of iron (63,67,83,85). Also we demonstrated that IsdA, localized to the cell wall, contributes to this process (75). In the present study, we characterized Isd-mediated heme iron acquisition by defining the involvement of IsdE in this process at the cell membrane. In Figure 5-5B, we show that a S. aureus isdE knock-out mutant, H834, although still able to grow on hemin as a sole source of iron, grew slower than wild type. Notably, complementation of this mutant with pCLVEc (expresses isdE) restored growth on hemin to greater than wild type levels. These data confirm that IsdE contributes to heme iron acquisition in S. aureus and also suggest that other heme transport systems function in this bacterium. Ala substitutions were constructed in plasmid pCLVEc and used to show that although a M78A mutation in pCLVEc (pCLVEc- M78A) had no impact on the ability of IsdE to participate in S. aureus growth on hemin, a   94 H229A mutation in pCLVEc completely abolished the ability of the plasmid to complement the isdE knock-out phenotype. These results indicate that although Met78 is dispensable in vivo, His229 is required for the biological activity of IsdE.   5.3. Discussion  In this study, we demonstrate that S. aureus growth on heme as a sole iron source is impaired by inactivation of isdE. Moreover, IsdE-mediated heme transport in S. aureus is reliant on the key heme iron axial ligand, His229 (Figure 5-5). Our work implicates IsdE as a central component for heme iron uptake by the Isd system. The crystal structure of IsdE provides the first view of heme binding by a substrate-binding protein associated with an ABC transporter. Heme is bound within a deep groove of IsdE formed at the interface of the two domains. The heme is nearly completely buried with only a single edge of the porphyrin ring exposed (19% of the total heme surface). In contrast, heme protrudes from the surface of the NEAT domains of the cell wall anchored components IsdA (83) and IsdC (183). Heme bound to these NEAT domains is about 35% solvent exposed. The small amount of heme exposure in holo-IsdE is comparable with that of the transporter HemS (18%) from Yersinia enterocolitica (164). In addition to being more buried, the binding mode of heme to IsdE differs in many respects from that of the NEAT domains of IsdA and IsdC. Heme bound to the NEAT domains is five- coordinate with the single axial ligand provided by the phenolate of a Tyr residue (83,161,183). The phenolate oxygen in turn forms a H-bond to a second Tyr residue. In contrast, the structure of IsdE shows an extensive H-bond network attached to iron ligand His229, involving residues from both domains of IsdE (Figure 5-2B). The same H-bond network forms interactions with   95 both propionates through water molecules. This H-bond network, in combination with the dipole of α-helix-1, assists in neutralizing the negative charge on the buried propionates. The NEAT domains bind heme such that the propionates are largely solvent exposed (83,183) negating the need for charge neutralization. The more extensive interactions of IsdE with the heme iron and porphyrin group suggest greater specificity and tighter binding than by the NEAT domains of the cell wall anchored components of the Isd system. Indeed, previous spectroscopic studies demonstrated that IsdC is able to bind heme and protoporphyrin IX from the E. coli cytoplasm, whereas IsdE is associated predominantly with heme (162). The His-Met iron coordination observed in the IsdE-heme complex structure is unique to known heme transporters. This axial ligand combination is best known in several c-type cytochromes (188). His-Met coordination is in agreement with previous magnetic circular dichroism spectroscopic data for IsdE. Mack et al. (162) previously demonstrated that IsdE heme iron is six-coordinate, and though the axial ligand in addition to His could not be determined conclusively, His-Met was proposed as a potential configuration. In contrast, EPR studies of the close homologue, SpHtsA from S. pyogenes, suggested heme iron in this system was mediated through two axial N ligands because of similarities in the EPR spectrum from b-type cytochromes (189). However, molecular modeling of HtsA using the structure of IsdE as a template and the lack of a His in the N-terminal lobe of SpHtsA suggest Met, as in IsdE, is the second axial ligand in SpHtsA. Typically ABC transporter-binding proteins form a bi-lobed structure with the ligand bound in a groove formed between the lobes. Three classes of these proteins are described based on the structure of their interdomain connection. The majority of the proteins of known structure belong to either class I or II that contain three or two flexible domain-bridging β-strands, respectively.   96 The strands impart significant flexibility allowing for large interdomain conformational change upon ligand binding (36,190). However, IsdE belongs to the third class of bacterial-binding proteins that are characterized by a single domain spanning α-helix. The DALI search identified several class III members as structural homologues of IsdE with functions in metal (ZnuA), siderophore (FhuD, CeuE), and vitamin B12 (BtuF) transport (36,37,191,192). The general architecture of members of the class three binding proteins seems to be very conserved. The known members of this family all display a similar bi-lobed architecture with two lobes made up of central β-strands surrounded by several α-helices bridged by a single long α-helix. Because of the domain-spanning α-helix, members of class III are relatively inflexible and undergo minimal conformational change upon ligand binding and release (36,62,193). As is typical of class III- binding proteins, the interdomain interface of IsdE is much larger than those observed in class I and class II proteins. These large interaction surfaces are proposed to contribute to the inflexibility of these proteins (37). The IsdE interface is formed from several hydrophilic residues. However, in the structural homologue, FhuD, the interdomain interface is predominantly composed of hydrophobic residues, which Clarke et al. (37) suggest results in a greater restriction of domain movement during ligand binding. CeuE, lacking a discernable hydrophobic interface (192), is more similar to IsdE. Because of the extensive interaction of heme with IsdE for a transport protein, heme release is likely a function of the small structural changes induced by interaction with the transmembrane permease component of the ABC transporter. The transmembrane and ATPase components of the Isd system are not well characterized, however, the crystal structures of two bacterial substrate-binding protein-dependent ATPase transporters, BtuCD and HI1470/1, have been described (36,194). Analysis of sequence conservation between IsdE sequence homologues   97 revealed a highly conserved strip of residues spanning the width of the face involved in transporter interactions (Figure 5-2D). In particular, several charged residues are conserved that could form salt-bridged interactions with the permease. BtuF is the most similar structural homologue of IsdE as revealed by the DALI search. In BtuF, Glu72 and Glu202 are located on the surface of the N- and C-terminal lobes and are positioned to interact with complementary Arg residues when docked to the BtuC transmembrane component (36). In support of their importance to transport function, Sebulsky et al. (61) demonstrated that mutation to Ala of the corresponding Glu residues in S. aureus FhuD2 does not affect ligand (hydroxamate siderophore) binding but abrogated transport. In IsdE, Glu83 and Glu214 are highly conserved in IsdE sequence homologues (Figure 5-3) and may serve a similar role. In fact, Glu83 is in an equivalent position as Glu72 in BtuF. Glu202 (BtuF) and Glu214 (IsdE) are in topologically similar locations; however, this region is an α-helix in BtuF and a loop in IsdE (Figure 5-7). The electron density for Glu214 is weak, and this loop is involved in a crystal contact suggesting that an alternative conformation may exist in the presence of the permease. Nonetheless, the conservation and surface localization of these Glu residues suggest that the proteins dock via structurally conserved salt-bridge formation, similarly to BtuF (36). Thus, we would anticipate that mutation of the conserved Glu residues in IsdE would similarly disrupt heme transport whereas not affecting heme binding.   98  Figure 5-7. Superposition of IsdE and BtuF. S. aureus IsdE (green) and E. coli BtuF (1n2z) (orange) are superposed and represented as cartoons. The conserved negatively charged glutamate side-chains that are proposed to interact with the permease are shown in sticks with oxygen atoms represented in red. The BtuF and IsdE ligands, Vitamin B12 and heme, are shown in the binding pocket as blue and red sticks, respectively.  The IsdE structure is unique in this superfamily of proteins because of the lack of well defined β-strands in the N-terminal domain. This is because of the large prevalence of Pro residues that are unable to participate in β-sheet H-bonding. Many of these Pro residues are conserved in putative IsdE orthologues (Figure 5-3). Pro77 plays a necessary role in forming a tight turn that orients Met78; the conservation of additional Pro residues within domain suggests that they contribute rigidity to the backbone. Furthermore, in SpHtsA, position 58 (a Pro in IsdE) is a Cys, and a second Cys occurs at position 42, such that a disulfide bond could form between these two residues. Even though the Pro is not conserved in SpHtsA or SeHtsA (Figure 5-3), the disulfide would also add rigidity to the structure. Collectively, our data are consistent with a role for IsdE in relaying heme from the cell wall anchored surface receptors to the permease (63,71). A heme relay function for IsdE is supported by studies on the two IsdE orthologues, SpHtsA and SeHtsA, from Streptococcus equi subspecies   99 equi (each share ∼40% amino acid sequence identity with IsdE; (Figure 5-3). In streptococci, Shp is a cell surface protein that has been shown to relay heme to the HtsA lipoprotein (195,196). Those studies demonstrated that streptococcal apo-HtsA can bind and receive heme from a soluble domain of Shp. Transfer between streptococcal Shp and HtsA is believed to be driven by greater heme affinity of HtsA as compared with Shp (189,197). Based on the high sequence identity between IsdE and the two HtsA orthologues, including the conservation of residues in the heme binding pocket (Figure 5-3), both the structures and heme binding properties of these proteins are likely to be similar. Furthermore, parallels in the mechanism of heme transfer to IsdE from the cell wall anchored IsdC may exist to those of the Shp-HtsA system. As shown in Figure 5-5, mutation of isdE in S. aureus does not abrogate growth on heme as a sole source of iron, suggesting other means of heme iron acquisition in this bacterium. A second ABC transporter is recently identified and named HtsABC based on the evidence that a transposon mutation in the hts operon yields a strain demonstrating a decrease in the ratio of heme to transferrin iron uptake, relative to wild type (67), leading to the suggestion that this operon expresses an alternate heme uptake system in S. aureus. In this system, HtsA is believed to be the binding protein (lipoprotein) that would be analogous to IsdE. The S. aureus HtsA (SaHtsA), however, shows only a low degree of sequence similarity to either IsdE or S. pyogenes HtsA (<15% amino acid identity), and the protein lacks the key residues that are involved in IsdE-mediated heme and heme iron coordination (see above), indicating that if the SaHtsA binds heme, it does so using entirely different coordination. The results presented in this study add to the rapidly expanding body of knowledge about the Isd-mediated heme acquisition system in Gram-positive bacteria, providing important insight   100 into the mechanism of heme binding and transport via the IsdE component. These results provide a template for more mechanistic studies that will determine the details of heme transfer to the IsdE lipoprotein, presumably by cell wall anchored proteins.        101 Chapter 6. Staphyloferrin A recognition by HtsA  6.1. Introduction  Siderophore uptake systems are an important component of S. aureus iron acquisition strategies in an iron-restricted environment (60,102,107). To gain insight into the mechanism of siderophore uptake in S. aureus, the cell surface staphyloferrin A (SA) receptor was studied by x-ray crystallography and spectroscopy. SA is produced by most staphylococci and its has been shown to affect growth in iron minimal media and contribute to pathogenicity in infection models (102). The chemical structure of SA has been determined, but its iron-bound conformation is unknown (100,101,198). Recently, the SA biosynthetic locus was identified and an ABC transporter divergently expressed from the same genomic location (HtsABC) was shown to import ferric-bound SA (FeSA) (102,104). HtsABC comprises the substrate binding protein and heterodimeric permease components, respectively. In this study, we use recombinantly expressed HtsA to show it binds FeSA tightly and we present the first structure of a siderophore receptor from a Gram-positive bacterium. HtsA is a class III binding protein with separate N- and C-terminal domains bridged by a single α-helix. Three x-ray crystal structures were determined: open apo, open-FeSA-bound and closed-FeSA- bound. The structures provide insights into ligand recognition and the conformational change likely required for productive interaction with the permease (HtsBC).        102 6.2. Results  6.1.1. Affinity of HtsA for staphyloferrin A  Our previous studies showed that HtsABC was required for SA utilization in S. aureus (102). HtsA, a class III substrate binding protein, is tethered to the extracellular face of the cytoplasmic membrane via an N-terminal lipidation and functions as the receptor for staphyloferrin A. Changes in the intrinsic fluorescence of recombinant HtsA (lacking signal peptide) that occur upon ligand binding were examined to determine substrate affinity. Saturating concentrations of HPLC-purified ferric-SA (FeSA) resulted in an average 52.5% reduction in fluorescence emission. The dissociation constant (Kd) of HtsA and FeSA was in the low nM range but could not be accurately determined because the concentration of HtsA required to see fluorescence change (15 nM) was ~15-fold greater than the Kd (Figure 6-1). The specificity of HtsA for FeSA was demonstrated by the fact that ferric-staphyloferrin B (staphyloferrin B was synthesized as described (102) and HPLC-purified in a similar fashion to SA) failed to quench the intrinsic fluorescence of HtsA. These data are in agreement with previously published biological data (102,103).     103  Figure 6-1. Saturation curve of the binding of FeSA to HtsA. Recombinant HtsA was titrated with increasing concentrations (as determined by AAS) of FeSA and fluorescence knockdown is depicted here as percentage saturation. The experimental data is represented as the solid line with diamonds and the theoretical data (predicted by non-linear regression, one-site binding model) is the dashed line. Inset represents an emphasis in the region of FeSA concentrations used to determine the dissociation constant. Data is the result of three independent experiments and error bars represent standard deviation from the mean.  6.1.2. The crystal structure of apo-HtsA identifies a positively charged binding pocket  To provide insight into the function of HtsA in S. aureus, the X-ray crystal structure of HtsA was solved. Data collection and refinement statistics are shown in Table 6-1.The structure consists of residues Thr38–Lys327, which excludes 15 N-terminal residues following the Cys22 lipidation site that together likely form a flexible anchor. HtsA is comprised of mixed α/β N- terminal and C-terminal lobes bridged by a single α-helical backbone (Figure 6-2A). The ligand- binding groove is shallow and dominated by a large basic patch. The overall fold places HtsA among the class III periplasmic binding protein family.       104 Table 6-1. Data collection and refinement statistics for the HtsA structures.   Native HtsA Se-Met HtsA FeSA-HtsA open (form 1) FeSA-HtsA closed (form 2) Data collectiona    Resolution range (Å) 50 - 1.60 (1.66 - 1.60) 50 – 1.35 (1.40 - 1.35) 50 – 1.30 (1.35 - 1.30) 50 – 2.20 (2.32 – 2.20)    Space group P21 P21 P21 P21    Unit cell dimensions (Å) a = 44.70, b = 43.57, a = 44.95, b = 43.65, a = 44.56, b = 43.52, a = 52.28, b = 148.60,  c = 75.71, β = 100.59 c = 76.04, β = 100.57 c = 75.32, β = 100.6° c = 52.27, β = 117.1°    Unique reflections 38161 63781 70082 35958    Completeness (%) 96.8 (76.5) 97.9 (87.3) 99.2 (97.7) 99.2 (99.2)    Average I/σI 20.8 (6.0) 37.9 (5.9) 19.6 (3.1) 10.8 (4.1)    Redundancy 3.4 (2.6) 3.4 (2.5) 3.5 (3.1) 4.0 (4.0)    Rmerge 0.059 (0.166) 0.050 (0.166) 0.056 (0.355) 0.085 (0.309) Refinement    R-work (R-free) 16.6 (20.1) 13.0 (16.5) 15.3 (18.6) 16.5 (21.6)    No. of water molecules 382 494 313 220    Average B-value (Å2)         All atoms 13.9 13.9 16.6 37.1         Protein 12.5 11.7 15.0 37.1         Staphyloferrin A -- -- 24.3 25.5         Water 23.0 25.0 28.0 38.1    r.m.s.d. bond length (Å) 0.013 0.013 0.013 0.013 Ramachandran plot, % residues   In most-favourable region 92.5 91.4 92.5 86.7   In disallowed regions 0.0 0.0 0.0 0.0 a Values in parenthesis represent highest resolution shell     105  Figure 6-2. Apo-HtsA crystal structure. (A) HtsA belongs to the class three periplasmic binding protein family of folds with two domains bridged by a single α-helix. The structure is rendered as a cartoon with the N-terminal domain shown in cyan, the domain bridging α-helix in yellow and the C-terminal domain in green. (B) Residues within the binding pocket of HtsA. The structure of HtsA is rotated 90° out of the plane as indicated. Arg residues contributing to the large positive potential in the binding groove are shown in orange. Potential heme coordinating residues are shown in magenta. Oxygen and nitrogen atoms are represented in red and blue respectively. (C) Surface electrostatics of HtsA. HtsA is shown looking down into the binding groove between the opening of the N- and C- terminal lobes in the same orientation as Part B. The binding groove is dominated by a large positively charged region as well as several other charged regions. Blue, white and red indicate positive, neutral and negative potential respectively.      106 Recently, several protein structures from this class have been determined, all functioning as receptors for metal uptake systems. Of particular relevance, this family includes two siderophore-binding proteins, FhuD and CeuE (37,192), and three heme-binding proteins, IsdE, ShuT and PhuT (75,199). A search by the Dali server reveals the Campylobacter jejuni enterochelin receptor, CeuE (r.m.s.d. of 2.7 Å and 24% identity over 261 residues), the Bacillus subtilis bacillibactin receptor, FeuA (r.m.s.d. of 3.4 Å and 21% identity over 261 residues), and the E. coli ferrichrome uptake receptor, FhuD (r.m.s.d. of 2.9 Å and 23% identity over 254 residues), are the most similar structures in the database. To investigate ligand binding within the groove, the ligand-bound CeuE, FhuD, ShuT, PhuT and IsdE structures were superposed onto HtsA. When superposed, the ligands bound to these structures overlay in highly similar lateral locations within the binding groove. The corresponding region of HtsA contains a large patch of positive electrostatic potential contributed mainly by six Arg residues (Arg86, 104, 126, 299, 304 and 306) that are directed into the groove (Figure 6-2B and C). This arrangement of positively charged side-chains in the ligand-binding groove would favour an interaction with the anionic staphyloferrin A molecule.  6.1.3. Crystal structures of open and closed FeSA-HtsA  Crystal structures from two different FeSA-HtsA crystal forms were solved to examine FeSA binding (Figure 6-3A). The apoHtsA structure overlays with the FeSA-bound crystal form 1 (open) and crystal form 2 (closed) HtsA structures with an root mean square deviation over all Cα atoms of 0.5 and 1.6 Å, respectively (Figure 6-3B). Unlike the typical interdomain movement characteristic of many β-sheet bridged binding proteins, the N- and C-terminal domain cores overlay well with the apo structure. Relative to the apo structure, the closed holo structure does not undergo significant domain movement (hinged motion of less than 2°). Instead, the   107 predominant structural difference is centered at three loops at the surface of the C-terminal domain. These three loops are composed of residues 201–208 (Loop201–208), 228–258 (Loop228– 258), and 265–271 (Loop265–271) (Figure 6-3B). In the open structures, residues in the loops display elevated B-factors relative to core residues (Figure 6-3C), whereas in the closed structure, the B-factors are more similar (Figure 6-3D). Loop228–258 undergoes the largest structural change in the closed protein with Cα atoms moving as much as 12.1 Å (Tyr239) across the binding pocket relative to the apo or open structures. The large loop movement is accommodated by a slight unwinding of the α-helix230–239 preceding the loop. Tyr239 is located at the C-terminal end of the α-helix230–239 and is translated from the central portion of the C- terminal domain into the binding pocket to form an H-bond to the lateral side of FeSA. A second Tyr, Tyr244, is shifted across the pocket, forming a H-bond (2.5 Å) to the Phe146 main chain carbonyl of the N-terminal domain. The loop movement also creates several intradomain H- bonds. Lys238 forms a H-bond to Tyr212 (3.0 Å). Hydrophobic contacts are also created by the loop movement. Leu240 forms a hydrophobic contact with Phe146, again bridging the N- and C- terminal domains. Pro243 forms a stacked hydrophobic interaction with Tyr244 (3.8 Å), potentially stabilizing the interdomain contacts facilitated by Tyr244.       108  Figure 6-3. The overall structure of the HtsA-staphyloferrin A complex. (A) the open structure (crystal form 1) of HtsA is shown as a schematic colored in a gradual color change from the N terminus (blue) to the C terminus (red). Staphyloferrin A is shown in the binding pocket as sticks with carbon, nitrogen, oxygen, and iron shown in gray, blue, red, and orange, respectively. (B) shown is the overlay of open holoHtsA (blue, crystal form 1) and closed holoHtsA (red, crystal form 2) and apoHtsA (green, PDB entry 3EIW). The structure is rotated ∼90° relative to A to look into the binding pocket. Backbones are shown as tubes. Staphyloferrin A is shown in the binding pocket as in A. (C) shown is a B-factor tube diagram of open-HtsA. Regions of increasing B-factor are shown with larger diameter and coloring from blue (low) to red (high). (D) shown is a B-factor tube diagram of closed-HtsA. The structure is colored as in C.   109   In the closed structure, two Zn2+-mediated crystal contacts are formed between symmetry related C-terminal domains of each molecule. One site involves two metals bound by His266 and Lys270 from chain A and Glu250, His251, and Asp254 from a symmetry-related chain B. The other site involves the same five residues, this time with the Glu250, His251, and Asp254 from Chain A and His266 and Lys270 from a symmetry-related chain B. Zn2+ is present in the crystallization buffer and has been modeled into both sites. Because these crystal contacts involve residues from the loops with the largest structural changes, it is feasible that these crystal contacts induce a conformational change in holo-HtsA. Given the additional FeSA and interdomain HtsA contacts formed, a more likely explanation is that the closed conformer seen is biologically relevant and the crystal contacts simply form between two stable, closed structures.  6.1.4. Structure of Staphyloferrin A  SA is synthesized from two molecules of citrate forming amide bonds with the amino groups of a D-ornithine, forming N2,N5-di-(l-oxo-3-hydroxy-3,4-dicarboxylbutyl)-D-ornithine (Figure 6-4A) (100). Electron density for FeSA is present in the binding pocket formed between the N- and C-terminal domains in both the open and closed forms of HtsA. Electron density is present for the complete FeSA molecule in the closed structure, but weak density for the ornithine backbone and additional density around one terminal carboxylate group is apparent in the open structure (Figure 6-4B). Two waters have been modeled into the extra density but do not completely account for the extra positive peaks. The ligand is likely afforded additional flexibility in the absence of interaction by Loop228–258 that moves across the pocket in the closed structure.   110   Figure 6-4. The structure and chirality of staphyloferrin A. (A) a linear schematic of the staphyloferrin A molecule is shown. Stereochemistry at the three chiral centers is indicated. Atoms that directly interact with the iron are numbered according to Konetschny-Rapp et al. (100). (B) Shown is the conformation of staphyloferrin A from the open HtsA-SA structure. Extra density can be seen at the distal end of the terminal carboxylate group (group 3 from 2A). C, shown is the conformation of SA from the closed HtsA-SA structure. The omit Fo−Fc maps are contoured at 2.5 and 3.5 σ for B and C, respectively. Atoms are indicated by their element symbols.    The SA structure has the ornithine Cα atom in the R-configuration. The two chiral centers of the citrate components are modeled into the density for both structures with the more buried citrate in the S-configuration and the more solvent-exposed citrate in the R-configuration (Figure 6-4). The citrates on either side of the iron bind as mirror images of one another. The chirality at the citrate carbons are in line with predictions from an early characterization of SA, suggesting the chiral centers were likely R,S (100). However, subsequent findings by a different group based   111 on the CD spectra of model compounds suggested the chiral centers were S,S (198). Modeling of SA with the S,S configuration does not fit the observed electron density as well in either the open or closed structures. Modeling in the second S configuration clearly strains the siderophore providing poor fit to the density for O1, O2, and O3 groups (see Figure 6-5 for a comparison of S,S and S,R models). The S,R configurations at the chiral citrate carbon atoms suggest that the staphyloferrin A biosynthetic machinery is highly specific during the condensation of citrate to both the ornithine and the siderophore intermediate.   Figure 6-5. Staphyloferrin A models in omit difference (Fo-Fc) electron density. Density from the open structure (A-E, contoured at 2.5 σ) and the closed structure (F-G, contoured at 3.5 σ) are shown in grey mesh in several different orientations, with SA modeled in S,S (cyan) and S,R (green) configurations. Citrates are numbered as in Figure 6-4A with the more buried citrate denoted by “prime” and in the S configuration. The more solvent exposed citrate (“prime”) is a better match to the density in both structures modeled in the R configuration (green).      112 Ferric iron is coordinated by an oxygen atom from each of β-hydroxy,β-carboxyl-substituted carboxylates from the two citrate constituents. SA coordinates Fe3+ with distorted octahedral geometry and ligand bond lengths of 2.0–2.2 Å. The ligand bond angles are distorted from perfect octahedral geometry ranging from 75° to 101° (Figure 6-4B and C).  6.1.5. Siderophore bound in the open conformation of HtsA  The basic patch identified in the apo-HtsA structure contributes the majority of siderophore contacts. In the open structure, five Arg residues at the pocket surface form direct H-bonds to oxygen atoms of FeSA Figure 6-6A and Table 6-2. Arg104 and Arg126 form H-bonds to FeSA. Arg299 is modeled in two conformations, both within H-bonding distance of the FeSA ornithine hydroxyl group. Arg304 and Arg306 forms an additional H-bond to the terminal carboxylate group of the more buried terminus. His209 forms an additional H-bond to the hydroxyl of the ornithine component, and four ordered water molecules are modeled interacting with the ornithine carboxylate and the carbonyl as well as two carboxylates from one of the citrate moieties Figure 6-6A and Table 6-2. The B-factors of regions of the siderophore backbone are dependent on solvent accessibility. The more buried citrate group has the lowest B-factors (carbon atoms ranging from 14–18 Å2). The carbon atom B-factors of the ornithine increase from 20 to 36 Å2, moving from the inner citrate group to the fully solvent-exposed ornithine carboxylate. The outermost citrate group carbons have B-factors in the 24–30 Å2 range.      113   Figure 6-6. Staphyloferrin A in the HtsA binding pocket. (A) shown is the open HtsA binding pocket. Residues forming direct contacts with SA (cyan) are shown as sticks with carbon, nitrogen, oxygen, and iron shown as green, blue, red, and orange, respectively. Hydrogen bonds are indicated by dashed lines. Residues are numbered according to full-length HtsA. (B) the closed HtsA binding pocket shown colored as in A.  Table 6-2. FeSA-HtsA bond distances (Å). HtsA atom – FeSA atom1 Bond Distance (Å)      FeSA-HtsA open FeSA-HtsA closed Arg86 NΗ – O2 na2 3.1 Arg104 NΗ1, NΗ2 - O3’, O3’ 2.9, 3.0 2.8, 3.0 Arg126 NΗ – O3’  3.5 3.5 Arg126 NΗ1, NΗ2 – O3, O3  2.8, 3.1 2.7, 3.1 Lys203 O – Ornithine carboxylate na2 2.9 His209 Nε2 – Citrate’ carbonyl 2.8 2.7 Tyr239 OΗ – Ornithine N, O1 na2 2.8, 2.6 Arg299 Nε - O2’  3.0 or 2.73 na2 Arg299 NΗ - O2’, O2’ 2.8,3.6 or 3.1,3.33 2.7, 3.1 Arg299 NΗ - Citrate carbonyl 2.9 or na2,3 na2 Arg304 Nε - O2’ 3.4  3.4 Arg304 Nε - O2 3.3 3.1 Arg304 NH – O2 3.2 3.5 Arg306 NΗ - O3’ 2.8 2.4 Water – Ornithine carboxylate 2.9 na2 Water – Citrate carbonyl 3.0 na2 Water – O2 3.1 na2 Water – O3 3.3 3.0 1 FeSA atoms are numbered according to atom labels in Figure 6-4A. 2 Not applicable because residues contact not present in structure. 3 Refers to each of the two conformations modelled for Arg299 in open structure.    114 6.1.6. Siderophore bound in the closed conformation of HtsA  The major difference in HtsA-FeSA interactions between the open and closed conformations occur due to additional contacts from two of the loops undergoing large conformational changes. Relative to the open conformation, Tyr239 Cα translates 12.1 Å in Loop228–258 and forms a H- bond with the carbonyl group of the less buried citrate. Additionally, the translation of Loop201– 208 orients Lys203 and Arg83 to form H-bonds with the FeSA carboxylate groups (Figure 6-6B and Table 6-2). Despite these additional contacts, FeSA is bound within the binding pocket of the closed HtsA in a similar orientation to the open form; however, it is shifted slightly deeper into the pocket (Figure 6-6B). The result of the conformational changes is significant occlusion of solvent from the FeSA molecule, reducing the 33.0% solvent exposure in the open structure to 14.5% in the closed structure (as determined with AREAIMOL (131)). The distribution of siderophore backbone B-factors is similar to that of the open conformation. The lowest B-factors are associated with the more buried citrate group (15–20 Å2), a broad range is observed in the ornithine group (25–40 Å2), and the outer citrate group has elevated B-factors (27–30 Å2). In an overlay of the open and closed protein structures there is an ~1.5 Å average atom displacement over all 34 FeSA atoms. The half of FeSA located closest to the exterior of the HtsA binding pocket undergoes the largest displacement with an average shift of ∼2.1 Å into the pocket. Only slight intramolecular atomic displacements occur within FeSA with mean atom displacements relative to the protein core of ~0.5 Å (Figure 6-4B). Several of the additional key protein-FeSA contacts identified in the open structure are similar to those in the closed structure; however, H-bond-lengths are altered in many cases (Figure 6-6B and Table 6-2). In total, six Arg residues in the Arg-rich region form direct contacts with FeSA. Arg86, Arg104, Arg126, Arg299, Arg304, and Arg306 all form H-bonds to O atoms in the   115 citrate moieties of FeSA (Figure 6-6B and Table 6-2). Also similar to the open structure, His209 forms a H-bond to the carbonyl group on the ornithine component of FeSA.  6.1.7. Multiple sequence alignments  A BLAST (156) search of the NCBI non-redundant protein data base identified proteins with greater than 30% sequence identity. The list of sequences is of proteins from Gram-positive and Gram-negative bacteria. Because several staphylococcal species produce SA (100,101), the best matches correspond to homologous receptor proteins in Staphylococcus species aureus, epidermidis, warneri, hemeolyticus, capitis, saprophyticus, and hominus with amino acid sequence identities of >80%. Three proteins were identified from Bacillus sp.: (i) YfmC (~42% identity), annotated as a ferric citrate-binding protein (200), (ii) YhfQ (~37% identity), an unknown siderophore-binding protein (200,201), and (iii) YfiY (~30% identity), the binding protein for the siderophore schizokinen (202). Another interesting homolog identified was the ferric citrate-binding protein, FecB, found in many organisms but probably best studied in E. coli (E. coli FecB, ~35% identity). An alignment of 41 of the homologous sequences was made (for a subset of sequences, see Figure 6-7). The HtsA sequence contains an ~8-residue insertion relative to most homologous sequences in the alignment around Tyr239, in Loop228–258, which undergoes the largest structural change and forms a direct FeSA contact in the closed structure Figure 6-2B and Figure 6-7). This suggests the structural change will not be seen in many homologous structures and may be an adaptation for entrapment of specific substrates.     116   Figure 6-7. HtsA sequence alignments. A subset of the 34 non-redundant sequences (<80% sequence identity) is shown aligned. Residues are numbered as in full-length HtsA. Sequences are identified by the protein name or identifier followed by an underscore, and the first uppercase letter of the genus name followed by the first three letters of the species name. Arrows indicate HtsA residues that directly interact with SA.   The residues contacting FeSA are conserved to varying degrees (Arg86 (22/41), Arg104 (37/41), Arg126 (36/41), His209 (30/41), Tyr239 (9/41), Arg299 (21/41), Arg304 (20/34, 6 Lys), Arg306 (37/41)), although in many cases the amino acid substitutions are conservative. Furthermore, two conserved Glu residues, Glu110 (39/41, 2 Asp) and Glu250 (38/41, 2 Asp, 1 Ser), are located such that they could form salt bridges between the N- and C-terminal domains   117 of HtsA and conserved Arg residues on the ABC transporter permease components (HtsBC) to mediate protein docking. Interestingly, Glu250 is located on an α-helix that shifts ∼2.8 Å toward the domain interface in the closed structure. A model of the HtsBC permease based on the BtuF structure (PDB entry 2qi9 (39)) suggests the movement of Glu250 brings it toward a conserved Arg74 of HtsB or Arg56 of HtsC, which may mediate differentiation of ligand-bound or free HtsA. Several functionally interesting residues are highly conserved (Figure 6-7). Gly102 (41/41) and Pro107 (39/41) occur on either side of the conserved FeSA-interacting Arg104, orienting the arginine into the binding pocket. His127 (40/41) forms a H-bond to the main chain O of Arg126 and likely stabilizes the loop containing SA-interacting Arg126. Four sequential residues, Ile137—Thr140, are conserved in most sequences and occur on a tightly turning surface loop. Tyr150 (35/41) forms a H-bond between the N-terminal domain and the His177 (33/41) Nϵ from the domain-spanning α-helix, which would contribute to interdomain stability. Trp302 (41/41) is located close to two FeSA-ligands (Arg304 and Arg306) but is directed into the core of the protein where it forms several hydrophobic contacts in addition to a H-bond from its Nϵ1 group to Glu317. Trp302 burial likely imparts stability to the loop and anchors the ligand binding Arg residues. Similar to the N-terminal linkage, a domain linking interaction between the C-terminal domain and the bridging α-helix is mediated by a H-bond formed between Glu312 (32/41) and Arg173 in both the open and closed conformations. However, in the closed conformation, Arg173 models in two orientations, both bonding and non-bonding.  6.3. Discussion  The crystal structures of FeSA-bound HtsA in the open and closed forms have enabled identification of residues that interact with FeSA and demonstrated an unprecedented mode of   118 ligand entrapment not previously observed in this family of binding proteins. Several x-ray crystal structures of related class III binding proteins possessing similar structural folds have been determined in both apo and holo forms. BtuF (E. coli B12 uptake) (36,203), TroA (E. coli Zn2+ uptake) (36,203), FhuD (E. coli ferrichrome uptake) (37,38), and ShuT (Shigella dysenteriae heme uptake) (199) all display little to no conformational change between the apo and holo forms (< 4° hinge movement and small intradomain atom displacement). A few recent examples demonstrate that a larger interdomain movement is possible. The apo structure of E. coli FitE was recently presented with both open and closed conformations found within the four molecules of the asymmetric unit. Within each domain FitE undergoes little conformational change (0.4 - 0.75 Å root mean square deviation for Cα atoms). However, the domain-bridging α-helix undergoes a significant hinged motion (~18.5°) (204). The structure of the Bacillus subtilis bacillibactin receptor (FeuA) undergoes a hinged movement of ~20° between the apo (PDB entry 2phz) and holo structures (205). Consistent with these crystal structures, molecular dynamics simulations of FhuD predict a greater hinge motion (~6°) than what is observed between the apo and holo structures (2°) (38). HtsA undergoes significant conformational changes upon FeSA binding. However, the conformational changes do not mirror the rigid interdomain movement seen in the FitE or FeuA structures or FhuD simulations. Instead, the large conformational changes are isolated to specific regions within the C-terminal domain. These conformational changes allow HtsA to clamp around FeSA, providing additional contacts to the siderophore as well as interdomain contacts that may facilitate the slight hinge closing motion. Similar isolated conformational changes of this magnitude have not previously been observed upon ligand binding in class III binding proteins.   119 The ligand-dependent conformational changes in class III binding proteins likely affect docking to the permease component of the ABC transporter, thereby providing a means of discriminating between the ligand-bound versus ligand-free receptor. The BtuCD-F complex crystal structure has been determined, demonstrating that the binding of BtuF to BtuC is mediated by salt bridges between Glu72 and Glu202 from BtuF and Arg56 and Arg295 from BtuB (39). Alignments and subsequent site-directed mutagenesis of two similar class III binding proteins, E. coli FecB (206) and S. aureus FhuD2 (61), suggest that similar Glu-Arg salt bridges mediate docking as variants affect ligand transport but not ligand binding. Because docking is mediated by salt bridge formation, even minimal hinged motion would allow discrimination of open and closed conformations. The closed HtsA structure illustrates an alternative mechanism of discrimination of the ligand-bound form of the receptor. The movement of Loop228–258 to enclose FeSA alters the placement of the homologous Glu250, which likely mediates salt-bridge formation with the permease HtsBC. The HtsA-FeSA complex is the first complex of an α-hydroxycarboxylate-type siderophore bound to its cognate binding protein, and to our knowledge this study reports the first examination of affinity between an α-hydroxycarboxylate siderophore and its receptor. The Kd of HtsA for FeSA, in the low nm range, provides the explanation for how HtsA, when exposed to ferrated S. aureus culture supernatant, was able to complex and crystallize with FeSA. The combination of a large number of charge interactions and receptor closures around the siderophore afford specificity to the interaction of SA with HtsA. Many siderophore-binding proteins and transporters have broad specificity for siderophores of a similar type; however, the Hts system is highly specific. SB is also an α-hydroxycarboxylate-type siderophore (207), yet the Hts system cannot transport sufficient amounts for growth (102). The only common constituent   120 is a single citric acid component. Staphyloferrin B (SB) is also composed of a L-2,3- diaminopropionic acid, 1,2-diaminoethane, and a succinic semialdehyde (207). The specificity of the Hts system for one negatively charged α-hydroxycarboxylate siderophore over another is likely a reflection of the specific ionic contacts formed in the closed structure that would not properly accommodate SB. Although the advantage gained by this specificity is unclear, it could be tailored to the low concentrations the siderophores expected to be present in serum at the point of infection. The affinity of HtsA for FeSA is within the range of several outer membrane siderophore receptors in Gram-negative bacteria. Compared with other class III substrate binding proteins, the binding affinity of HtsA for SA is greater than the presented Bacillus sp. receptors and orders of magnitude stronger than E. coli FhuD (Table 6-3). These apparent differences in affinity may be a reflection of the extent and type of residues involved in siderophore binding or may result from differences in conformational changes or tryptophan masking that can affect fluorescence emission. The disparity between binding affinities for the E. coli hydroxamate-binding protein, FhuD, and HtsA could reflect the differences in specificity. FhuD is a receptor for a broad array of ferric hydroxamates, so the binding pocket sacrifices affinity for diversity, where HtsA is specialized for SA alone.               121 Table 6-3. Dissociation constants (Kd) for receptor-ferric-siderophore complexes.  Protein-siderophore (organism)a Kd (nM) Reference HtsA-staphyloferrin A (S. aureus) Low nM This Study FeuA-bacillibactin (Bacillus cereus) 19 (202) FeuA-enterobactin (B. cereus) 12 (202) FatB-3,4-DHB (B. cereus) 1.2 (202) FatB-petrobactin (B. cereus) 127 (202) FpuA-petrobactin (B. cereus) 175 (202) YfiY-schizokinen (B. cereus) 34 (202) YxeB-desferroximine (B. cereus) 18 (202) YclQ-petrobactin (B. subtilis) 113 (208) FhuD-ferric hydroxamates (E. coli) 300-7900 (209) FpvA*-pyoverdin (Pseudomonas aeruginosa) 0.37 (210) FhuA*-ferrichrome (P. aeruginosa) 0.65 (210) FptA*-pyochelin (P. aeruginosa) 0.54 (210) aProteins listed are class III ligand binding proteins, except for those with asterisks which are outer membrane receptor proteins in Gram-negative bacteria   The chemical characteristics of siderophore binding pockets of receptors varies with the class of the cognate siderophore. Ferric complexes of hydroxamate-type siderophores are generally hydrophobic. Crystal structures of the outer membrane receptor FhuA (211) and the binding protein FhuD (37,212) from the E. coli hydroxamate uptake system have been determined. Siderophore binding in both structures is primarily mediated through hydrophobic contacts. Similarly, in Pseudomonas aeruginosa, receptors for the largely hydrophobic siderophore ferric chelates, pyoverdin (FpvA) (213) and pyochelin (FptA), have binding sites that are primarily composed of hydrophobic and aromatic residues (214). Catecholate-type siderophore ferric chelates generally have a net negative charge, and the receptor binding residues often mirror the net charge. Ferric-enterobactin carries a −3 net charge, so not surprisingly, the binding pockets of the E. coli outer membrane receptor, FepA (215), and the Campylobacter jejuni-binding protein, CeuE (192), contain several positively charged residues. In CeuE, the net negative charge is balanced by three Arg residues, and although the   122 binding residues in FepA could not be definitively identified, an Arg-rich binding site was found (192,215). Interestingly, a hydrophobic patch was identified in FepA and shown by mutagenesis data to contribute to the affinity for ferric-enterobactin (216). The structures of the catecholate receptors FeuA (Bacillus cereus) and YclQ (B. subtilis), which bind ferric-bacillibactin and ferric-petrobactin, respectively, represent the only other Gram-positive bacterial siderophore receptor structures determined to date. Recently, a FeuA-ferric-bacillibactin crystal structure was determined (205). Ferric-bacillibactin carries a net negative charge that is neutralized by three basic residues, Lys84, Lys105, and Arg180, that interact with deprotonated catecholate oxygen atoms (205). Gln181 and Gln215 also form direct H-bonds to bacillibactin. Furthermore, the YclQ ligand binding cleft contains a series of conserved positively charged residues (Arg104, Arg192, Arg236, and Try275) that are predicted to interact with a negatively charged petrobactin ligand (208). Crystal structures of mammalian siderocalin bound to ferric complexes of catecholate-type siderophores have been determined (217,218). The crystal structures show that two Lys residues and a single Arg mediate binding to the negatively charged catecholates. Recently, it was established that electrostatic interactions between ferric-siderophores and siderocalin are the prime determinants of binding affinity (219). Analogous to these examples of charged siderophores, FeSA carries a net negative charge that is neutralized by six Arg residues in HtsA. This large number of positively charged residues is expected to favor tight binding to a compound that would be present in low concentrations in the environment. The abundance of positive electrostatic potential, in concert with the occlusion of the binding site upon closing, likely determines the specificity of the transporter for FeSA.   123 Furthermore, the extensive protein-siderophore contacts that enclose the small siderophore likely serve to discriminate between Fe-free and Fe-bound SA. In summary, we have demonstrated that the S. aureus ABC transporter-associated binding protein HtsA binds FeSA and undergoes conformational changes upon binding involving a very small scale hinge motion and relatively large movements at loops in the C-terminal domain to enclose the ligand. Furthermore, binding is mediated primarily by six Arg, a Tyr, and a His residue in the binding pocket. The coordinating residues are well conserved in several proteins, suggesting that a similar means of coordination may be utilized in both siderophore and exogenous ferric citrate uptake pathways from a broad range of bacteria.    124 Chapter 7. Overview and future directions  Free iron levels in the human host are too low to support bacterial growth, so uptake systems directed at host iron sources are essential to the success of most bacterial pathogens (18). The goal of this thesis was to characterize the heme and siderophore uptake systems in the devastating bacterial pathogen, S. aureus. Gram-negative iron uptake systems generally require TonB-dependent outer membrane receptors and potentially accessory outer membrane anchored proteins (for a comprehensive review of iron uptake in Gram negative bacteria see (220)). Gram- positive bacteria lack an outer membrane and therefore do not possess homologues of Gram- negative bacterial receptors. Distinct strategies are required for Gram-positive iron reception and import. S. aureus uses cell wall anchored surface receptors for ligands that do not readily traverse the cell wall, such as heme and heme proteins (63). These receptors relay heme to the ABC transport substrate binding protein. Alternatively, for ligands that readily diffuse through the cell wall, such as ferric-bound siderophores, S. aureus uses high affinity substrate binding proteins as the outermost receptors (59,107). The work detailed in this thesis is the result of long standing collaborations that facilitated a multidisciplinary approach spanning biological, biochemical and x-ray crystallographic techniques, with the goals to characterize in molecular detail: 1) heme-binding at the cell wall; 2) iron-complex binding at the membrane; 3) to develop models for iron-complex transport.  7.1. Heme recognition by Gram-positive cell wall anchored receptors  The cell wall is the initial site for heme reception in Gram-positive bacteria, where heme is likely encountered in the form of methemoglobin or other heme containing proteins (63). Chapter 3 focuses on heme binding in the surface receptors and demonstrates that deletion of the   125 cell wall anchored IsdA impairs S. aureus growth with heme as a sole source of iron. To study the mechanism of binding by the cell wall anchored receptors, soluble recombinant expression systems were developed. The expression systems were designed to exclude the high probability disordered regions (81,82) that likely act as flexible tethers to anchor binding domains to traverse the cell wall, or to bind non-heme ligands at the cell surface (68,88). Since the goal of this work was to characterize the surface heme reception and transport, the minimal functional binding domains of the proteins were targeted. Chapter 3 presents the first successful definition of the 125 amino acid NEAT domain as the functional heme binding region in IsdA. Unlike the full- length protein, the binding domain is stable in solution and amenable to crystallization. The IsdAN-heme complex structure and S. aureus sequence alignments described in Chapter 3 suggest that Tyr166, the heme iron ligand, is the primary indicator of heme binding in S. aureus NEAT domains. Subsequently, the heme-bound structure of IsdCN (1.5 Å) was also determined by x-ray crystallography (221) and its apo and Zn(II)-protoporphyrin IX (ZnPPIX)- bound structures were determined by NMR (176). Additionally, crystal structures of apo (2.2 Å) and heme-bound IsdHN3 (1.9 Å) have also been presented (184,222). All of these NEAT domain structures are readily superimposed and employ a homologous Tyr residue to coordinate the bound heme iron. For instance, the crystal structure of holo-IsdAN overlays with the holo crystal structures of IsdCN and IsdHN with r.m.s.d values of 1.76 and 1.47 Å	
  over all Cα atoms, respectively (Figure 7-1B). The main differences are in loops on the protein surface and, not surprisingly, at the chain termini. Notably, in IsdCN, the loop joining β6 and β7 contains a five- residue insertion immediately preceding the heme-iron coordinating residue (Figure 7-1). Since this insertion is present only in IsdCN, it could reflect the unique ability of IsdC to pass heme to IsdE (73,74). The NMR structure of apo-IsdHN3 was published within a year before the IsdAN-   126 heme structure, and although the sequences are ~ 23 % identical, the overall structures reveal a similar eight-stranded immunoglobulin-like β-sandwich fold (Figure 7-1A) (160). IsdHN1 does not bind heme, but instead interacts with hemoglobin across the same region that forms the heme binding pocket in IsdAN (223).  Figure 7-1. NEAT Domain fold. (A) The IsdA NEAT domain (PDB ID: 2itf) is shown to represent a typical NEAT domain fold. It is comprised of eight β-strands (β1-8) and one well defined α-helix (α1). The backbone is shown as a cartoon, coloured from the N-terminus (blue) to the C-terminus (red). Heme is shown as sticks with carbon (purple), nitrogen (blue), oxygen (red) and iron (orange) atoms coloured independently. (B) Overlay of the heme-bound NEAT domains shown as backbone ribbons. IsdAN (PDB ID: 2itf) (cyan), IsdCN (PDB ID: 2o6p) (magenta) and IsdHN3 (PDB ID: 2z6f) (orange) are shown in the same orientation as (A). Heme from the IsdA-N1 structure is shown in the binding pocket and coloured as in (A).   A lone α-helix seen in the holo-structures of the IsdA NEAT domain between β1b and β2 forms one side of the heme-binding pocket (Figure 7-1). NMR data of apo-IsdHN1 and apo-IsdCN reveal that the region defined as this helix in the holo structure is flexible in the absence of ligand (176,222), and in the case of IsdCN, this region becomes stabilized upon titration with ZnPPIX   127 (176). Similarly, the corresponding helix in the holo-IsdHN3 crystal structures has reduced B- factors as compared to the apo structure. The IsdAN crystal structure does not reveal the same decreased B-factor in holo relative to apo structures; however, loop stabilization in the apo structure may be an artifact of the binding of a CHES molecule from the crystallization buffer in the apo protein binding pocket, or crystal packing. This clasping of a flexible region is reminiscent, though the movement is not as dramatic, of the mechanism of heme binding described for the hemophore HasA from Serratia marcescens, which undergoes a large clamping motion to secure heme in the binding pocket (224). Heme is bound in the hydrophobic pockets of the S. aureus NEAT domains through several conserved contacts. Structural and spectroscopic data have demonstrated that ferric heme-iron is five-coordinate with a Tyr phenolate at distances of 2.1-2.2 Å (162,175,184,221,225-227) (Figure 7-2). Another Tyr is invariably located four residues later in the sequence, and forms a H-bond (2.5 Å) with the iron-coordinating Tyr phenolate, and a π-stacking interaction with a heme pyrrole ring (184,221,225). A conserved Ser and a second polar residue form H-bonds with a heme propionate in all known structures of heme binding NEAT domains. Several hydrophobic contacts to the tetrapyrrole structure of heme are maintained through the heme pocket. For instance, a stacked π-bonding interaction of a benzene ring of Tyr87 (IsdAN) or a phenylalanine with a pyrrole ring at the base of the binding pocket is absolutely conserved. Another obviously conserved residue is Trp113 (IsdAN), at the base of the pocket where it lies next to the vinyl end of the porphyrin ring (221).      128   Figure 7-2. Heme binding by IsdA, IsdC and IsdH. (A) The x-ray crystal structure of IsdA-N1 (PDB ID:2itf) is shown in cyan cartoon, looking into the binding pocket for orientation reference. (B) The IsdA-N1 (PDB ID:2itf) binding pocket residues and heme are shown as sticks. Oxygen (red), nitrogen (blue), iron (orange), heme carbon (red) and side chain carbon (green) atoms are coloured accordingly. Residues are numbered based on the full length protein sequence in the NCBI database (NCBI ID: YP_001332075). (C) The IsdC-N1 (PDB ID: 2o6p) binding pocket, oriented as in (A). Residues are numbered according to NCBI ID: YP_001332076. (D) The IsdH-N3 (PDB ID: 2z6f) binding pocket, oriented as in (A). Residues are numbered according to NCBI ID: YP_001332658.  The S. aureus NEAT domain structures provide a general model for heme uptake machinery in many organisms. Sequence alignments for all identified NEAT domains from other Gram- positive bacteria demonstrate that several of the key heme binding residues in S. aureus domains   129 are conserved (data not shown). Additionally, although not originally annotated as a NEAT domain, the crystal structure of the binding domain from S. pyogenes Shp has been determined in complex with heme. Shp forms a highly similar eight-stranded β-sandwich domain (r.m.s.d ~ 2.0 Å over all Cα relative to IsdCN), with heme bound in a similarly located binding pocket to S. aureus NEAT domains (Figure 7-3) (228). However, heme-iron in Shp is coordinated by two Met residues instead of a single Tyr as in the S. aureus NEAT domains. Despite the different coordination mode, the structural similarity suggests Shp should be grouped among the NEAT domain family of protein folds. The sequence and mechanistic diversity suggests the NEAT domain fold may be more widespread than originally suggested and the differing heme-iron coordination in Shp illustrates that caution should be exercised when generalizing new information learned to distantly related systems.   Figure 7-3. Shp binding domain shares the NEAT domain fold. Due to low sequence identity between NEAT domains, several putative heme biding domains have been incompletely annotated. (A) IsdA and (B) Shp share the same overall fold consisting of 8 β-stranded β-sandwich fold with heme bound at a pocket formed at one end of the domain.    130  7.2. A mechanism for heme transfer through the cell wall  Following heme binding at the surface receptors, heme needs to be rapidly passed to the ABC transporter for import. Using insight gained from the IsdA-heme ternary complex structure detailed in Chapter 3, site directed mutants were designed to gain insight into the function of individual residues in the IsdA pocket for heme binding and transfer functions. From this work described in Chapter 5, the proximal iron-coordinating Tyr166 is shown to be required for efficient heme transfer into and out of the binding pocket. In contrast, His83 located in the distal heme pocket loop is involved in heme binding but does not facilitate heme transfer. Since the rates of heme release from the NEAT domains into solution is much slower than the rates of heme transfer, active complexes must form between the transfer partners (72,74). Since transfer complexes have eluded direct detection, two other lines of evidence were used to predict complex formation: crystal packing and computational protein-protein docking. Through a combination of the experimental data and theoretical modelling, two potential models for the heme transfer mechanism were developed (Figure 7-4). In both models, heme is transferred from IsdB to IsdA by direct access of IsdA Tyr166 to the sixth coordinate position of the iron of heme bound to IsdB by Tyr440. Likewise, heme moves out of IsdA to IsdC through direct access by Tyr166to the sixth-coordinate position of heme bound in IsdA. The two models differ in terms of the transfer intermediate and whether activated complex formation causes heme dissociation from the donor Tyr before coordination by the receiving Tyr (Figure 7-4A), or transfer proceeds through a six-coordinate TyrDonor-iron-TyrAcceptor intermediate (Figure 7-4B). Further work is required to determine the structure of intermediates in the heme transfer pathway and the potential direct involvement of additional amino acid residues.   131  Figure 7-4. Proposed models for heme transfer between S. aureus NEAT domains. (A) Heme transfer occurs through an intermediate following heme release from Tyr of the donor NEAT domain prior to binding to Tyr of the heme acceptor. (B) Heme transfer occurs through a intermediate in which Tyr from the donor and acceptor NEAT domains form a six-coordinate complex that rapidly dissociates as heme moves to the acceptor.   Support for the Isd heme transfer model also comes from recent investigation and modeling of the Streptococcus pyogenes Shp/HtsA (here referred to as SpHtsA) transfer mechanism (178). Shp has a NEAT domain fold and SpHtsA shares ~ 40 % sequence identity to IsdE, but is not a close homologue of S. aureus HtsA which is a siderophore rather than heme receptor (an unfortunate misnomer). The transfer mechanism between Shp/SpHtsA is likely analogous to the Isd system. However, the models of inter-NEAT domain heme transfer and Shp-SpHtsA transfer are not directly comparable, because Shp coordinates heme iron through two Met residues and SpHtsA is the substrate binding protein. Models for wild-type and single coordinating Met-Ala point variants demonstrate important parallels. Ran et al. observed stable intermediates for heme transfer when one Met residue was replaced with Ala (178). In these variants, Shp formed a complex with SpHtsA followed by the formation of a stable intermediate with heme iron bridging the two proteins through the sulfur of one Shp coordinating Met residue and one SpHtsA coordinating residue. SpHtsA access to an open distal sixth-coordinate heme-iron site in Shp is permitted, and given the structural similarities between Shp and the S. aureus NEAT   132 domains, supports a similar transfer mechanism. A stable intermediate is not readily observed in wild-type Shp-SpHtsA transfer, and Ran et al. proposed that wild-type Shp transfers via an unstable intermediate, where heme is released by both Shp ligands and is then rapidly bound by both SpHtsA ligands (178). Like the wild-type Shp-SpHtsA complex, no obvious stable intermediate was detected in IsdB-IsdA or IsdA-IsdC transfers, and only a single rate was observed for most variants, indicating that no stable intermediate is formed. Note that the rates of transfer between the NEAT domains are 10 to 100-fold faster than Shp variants and SpHtsA, so the lack of an intermediate signature could just be a reflection of a rapid progression through the intermediate. The NEAT domain transfer model proposed here is a working hypothesis and needs to be validated by observing complex formation by spectroscopic or other means.  7.3. Ligand reception by the substrate binding protein  Iron-complexes reach the substrate binding proteins in S. aureus by one of two means, depending on whether the iron source readily crosses the cell wall. Heme is stripped from heme proteins at the cell wall surface and transferred from the cell wall anchored components of the Isd system through IsdC to IsdE (73,74). Alternatively, FeSA diffuses through the cell wall and is bound by HtsA. Data presented in Chapters 5 and 6 demonstrate that IsdE and HtsA have the same overall fold, with N- and C-terminal domains that consist of central parallel β-sheet and peripheral helices and loops. The two domains are bridged by a long α-helix, which appears to be an emerging characteristic of metal transport binding proteins (229,230). Furthermore, ligands are bound within a similar region of the binding pocket, such that central iron atoms overlay < 1 Å apart. The iron ligands form extensive contacts to both domain surfaces (Figure 7-5).    133  Figure 7-5. Overlay of IsdE-heme and HtsA-FeSA crystal structures. IsdE (green cartoon) and HtsA (blue cartoon) overlay with heme (red sticks) and staphyloferrin A (yellow sticks) located in the same region within the binding pocket with heme-iron and SA- iron atoms (orange spheres) located ~0.9 Å apart.    Aside from gross structural features, IsdE and HtsA differ substantially in molecular detail and have very different binding affinities. The KD for heme binding by IsdE is estimated to be low µM (74), whereas the KD for the HtsA-FeSA complex was determined to be low nM (Chapter 6). The large variation in affinity is likely the result of their locations within the respective uptake pathways. For instance, IsdE receives heme iron passed directly from IsdC after heme is relayed through the other cell wall anchored components (73,74), and thereby effectively increasing the local heme concentration and diminishing the need for high affinity binding. In contrast, HtsA is the outermost receptor for FeSA, and due to the low concentrations of FeSA in most environments, high affinity binding is necessitated. A similar trend is evident in other Gram-positive siderophore receptors, such as the Bacillus cereus receptors for bacillibactin and enterobactin that bind their respective ligands with low nM dissociation constants (202).   134 These findings suggest that the different routes to ligand binding result in vastly different affinities for the various iron-complex substrate binding proteins. A key feature that likely contributes to the low Kd for the HtsA-FeSA complex is the unique structural change upon ligand binding. The open and closed structures described in Chapter 6 demonstrate local structural changes in HtsA to enclose FeSA. Unfortunately, apo- IsdE has not yet yielded diffraction quality crystals, so little can be gleaned as to the conformational changes accompanying heme binding. Also, the topic of interdomain movement in the class III binding proteins remains somewhat controversial. The crystal structures of ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa were subsequently determined and both bind heme in a structurally similar binding pocket to IsdE, but are five-coordinate through a single Tyr (199). The ShuT and PhuT structures are in apo, partially occupied and holo forms, but strikingly, only minor sidechain rearrangement exists between the three structures with minimal backbone or interdomain conformational change. This similarity in relative interdomain rigidity is a general theme for several crystal structures of class III binding proteins (163). The apo and holo crystal structures of the well characterized E. coli vitamin B12 binding protein overlay with minimal structural change, excluding small scale conformational differences within the binding pocket (36,203). Small angle x-ray scattering measurements of S. aureus FhuD1 and FhuD2 suggest only slight domain movement occurs upon hydroxamate binding (61,62). Interestingly, molecular dynamics simulations suggest the crystal structures may be misleading with much larger “venus fly trap” opening and closing predicted in of BtuF (231,232), E. coli FhuD (38), whereas simulations with ShuT and PhuT predict interdomain twisting along the α- helical backbone in addition to “venus fly trap” opening and closing (233). Given the theoretical flexibility of the other class III binding proteins, flexible interdomain motions could explain the   135 inability of IsdE to crystallize in the apo form. It is clear that diverse structural changes occur in the class III binding proteins and, while many of the observed structural changes are controversial, given the conformational differences observed in the HtsA crystal structures and the inability to crystallize apo-IsdE, the theoretical structural changes are likely closer to reality than the assumptions drawn from the few crystal structures.  7.4. Substrate binding protein docking on the permease  Once ligand-bound, the substrate binding protein interacts with the permease to deliver ligand into the cytoplasm. A few crystal structures of binding protein docked ABC transporters are published, including the E. coli maltose transporter (234), the Archaeoglobus fulgidus molybdate transporter (235) and the E. coli vitamin B12 transporter (39). The binding proteins for the maltose and molybdate transporters are flexible β-strand bridged proteins, while the vitamin B12 substrate binding protein is a comparatively rigid class III binding protein that shares ~ 20 % sequence identity with IsdE and HtsA. BtuC forms a homodimeric permease and BtuD is a homodimeric ATPase. In the Isd system, IsdF is suggested to form a homodimeric integral membrane permease (63) while HtsBC forms a heterodimeric permease of the FeSA transporter (102). The final component needed to drive heme uptake is an ATP hydrolase. However, the operons for several iron-compound ABC transporters from S. aureus lack a gene coding for the corresponding ATPase, including the transporters HtsABC and IsdEF (111,236). FhuC from the ferric hydroxamate transporter in S. aureus provides the ATPase function for the Isd and Hts systems, among others (98,102). A BLAST search of sequences derived from entries in the RCSB Protein Data Bank with the IsdF sequence revealed promising modeling templates for IsdF and HtsBC. The permease from   136 the E. coli vitamin B12 transporter, BtuC, shares ~ 22% identity with IsdF over 273 residues. Also, FhuC shares ~29% sequence identity with the B12 transport ATPase, BtuD, over 226 of the 265 residues. A model of the IsdF-FhuC and HtsBC-FhuC transporters was generated with the program ESyPred3D (237), using the crystal structure of the BtuCD complex (PDB ID: 2qi9) (39) as a template. To dock IsdE or HtsA onto the permease, the transporter was superposed onto the substrate binding protein, BtuF, from the structure of BtuCD-F (39). IsdE and HtsA share ~ 20 % sequence identity with BtuF. The components of metal uptake ABC transporters typically display conserved gross structural features even in the absence of high sequence identities (39,194); therefore, when structural evidence is combined with the sequence alignments, the docking model is likely a useful prediction of interaction. The resulting models are of complete IsdF-FhuC-IsdE and HtsABC-FhuC complexes (Figure 7-6).              137  Figure 7-6. Models of ABC transporters in S. aureus. (A) The IsdEF-FhuC heme transporter. The crystal structure of IsdE (orange, PDB ID: 2q8q) is shown docked against a model of the IsdF (green) and FhuC (blue). The model complex was generated by superposition over the BtuCD-F crystal structure (PDB ID: 2qi9). The backbones are shown as cartoons with each homodimer chain shown in a different shade of the same colour. In the IsdE structure, the domain-spanning α-helix (light orange), the propionate-stabilizing α- helix (yellow) and the heme-iron coordinating side chains (cyan) are shown. (B) The HtsABC- FhuC transporter. The crystal structure of HtsA (green, PDB ID: 3li2) is shown docked against a model of the heterodimeric permease HtsB and HtsC (cyan and yellow) and FhuC (blue). The model complex was generated by superposition over the BtuCD-F crystal structure (PDB ID: 2qi9) as in part A. The backbones are shown as cartoons and staphyloferrin A is shown in the HtsA binding pocket in magenta.   Docking of the binding protein to the permease allows analysis of residues involved in protein-protein interaction and transport. Analogous to those observed in the crystal structure of the BtuF-BtuC complex, two Arg-rich patches that potentially interact with Glu83 (equivalent to Glu74 in BtuF) and Glu214 (equivalent to Glu202 in BtuF) from IsdE or Glu110 and Glu250 from HtsA are present on the extracellular side of the IsdF and HtsBC models. These   138 electrostatic interactions would likely aid in the recognition of the substrate binding protein by the permease and induce ligand transport. Interestingly, the open and closed structures of HtsA reveal a decrease in the spacing between the conserved Glu residues from 48 to 45.6 Å (Figure 7-7). The closed distance of 45.6 Å is in line with the inter-glutamate spacing in the ligand bound structures of BtuF (46.2 Å) (203), FhuD (45.5 Å) (37) and FeuA (44.4 Å) (205). The agreement to other closed structures suggests that the small shift in inter-glutamate distance between the open and closed HtsA forms may be sufficient for discrimination of apo and holo binding protein by the permease, although these predictions await experimental confirmation.   Figure 7-7. FeSA binding induces a decrease in inter-glutamate spacing in HtsA. The open (light grey, green) and closed (dark grey, blue) structures of HtsA are overlayed and shown as tubes with salt bridge-forming Glu110 and Glu 250 shown as sticks. Ferric- staphyloferrin A is shown as ball and stick in the binding pocket.           139 7.5. Future work  The work detailed in this thesis provides mechanistic insight into ligand recognition, and models of heme and siderophore transport are proposed. The work has generated several hypotheses to be tested by future experimental work. Heme binding by the S. aureus NEAT domains has been well defined and seems to be a conserved mechanism in several other Gram- positive Isd systems (83,183,184). However, the structurally homologous Shp binding domain illustrates that annotating domains with low sequence identity is challenging, and that among the same structural family, the heme binding environment can be variable. Additional x-ray crystal or NMR structures of heme receptors from other Gram-positive bacteria need to be determined to investigate whether similar mechanisms are used by related systems. Furthermore, the related Isd systems have not been robustly compared bioinformatically or experimentally to identify potential differences among the systems. For example, the S. aureus Isd system encodes an unknown membrane protein annotated as IsdD that is absent in the other Isd systems (63). In place of this membrane protein, B. anthracis encodes an ATPase that was unfortunately also annotated as IsdD (71). Given the diversity among the Gram-positive bacteria possessing NEAT domains, detailed structural and bioinformatic comparison could reveal interesting differences between the systems. Chapter 3 presents the apo structure of IsdA, and only minimal structural differences were observed in comparison to IsdAN-heme. A recent NMR structure of IsdCN revealed that titration of ZnPPIX resulted in a stabilization of the flexible loop enclosing the distal side of the metal- PPIX face (176). Given that similar distal loop motions are observed in the NMR structure of IsdHN1 (160), it is possible that a similar mechanism of entrapment occurs in IsdA, and that the stabilization of the loop in the crystal structure is an artifact of the crystal. An experiment   140 analogous to the IsdCN titrations followed by NMR  (176) could be used to determine whether distal loop flexibility is a general feature of NEAT domains or is unique to IsdC. Chapter 4 proposes a model for heme transfer between NEAT domains, but this proposal requires further testing. Stable complexes have not been observed by the several biophysical techniques tested, and spectroscopic signatures that would indicate intermediate formation are not apparent during heme transfer experiments by stopped-flow. Since heme transfer is rapid, slowing the transfer rate could allow individual processes to be observed by stopped-flow spectroscopy experiments as described in Chapter 4, or it could enable intermediate complexes to be stabilized. Site-directed mutagenesis of heme-iron ligands has been successfully used in the Shp-SpHtsA heme transfer pathway, and allowed observation of transient intermediates not otherwise observed in the wild-type heme transfer experiments (178). Furthermore, since the S. aureus NEAT have similar spectroscopic absorbance between 400 and 700 nm (226), mutational variants that substitute the heme-iron ligand could be used to validate the heme transfer model. In Chapter 4 it was also shown that mutating the heme-iron coordinating Tyr166 to a residue unable to coordinate heme-iron resulted in heme binding by the distally located His83 and that heme transfer was impaired. However, it is unclear whether heme transfer requires a Tyr in the proximal position or just an unoccupied distal heme-iron position for efficient transfer. Variants at heme-iron coordinating position 166 that alter the absorption spectra but maintain a proximal ligand, such as His or Met, could be used to discriminate between the possible alternatives. Additionally, variants at position 166 could be useful to study heme transfer because the altered heme-iron ligand would change the visible absorbance relative to the typical NEAT domain. The spectral shift could allow a more readily observable spectral change when combined with a wild- type transfer partner, and could potentially reveal unique spectral properties of a stable   141 intermediate. Ultimately, a major goal of the mutagenesis work and metal-substituted porphyrin binding experiments is to understand transfer and find conditions that stabilize the heme transfer complex so it can be characterized by x-ray crystallography. So far, attempts to crystallize a complex have been unsuccessful, but with information gained from the structures and transfer experiments, alterations that stabilize formation will be investigated. The crystal structure of IsdE has provided insight into heme recognition and potential docking interactions of IsdE with the permease for heme import. However, the process of heme transfer from IsdC into IsdE is unknown. Parallels can be drawn between the Shp-SpHtsA (178,196) and the IsdC-IsdE heme transfer mechanism. However, given the differences in IsdC and Shp heme-iron coordination discussed in Section 7.1, the mechanisms of transfer are likely distinct (183,228). With the crystal structures of IsdC and IsdE now available as a guide, site- directed mutations could be generated, and heme transfer between them analyzed by stopped- flow spectroscopy similarly to the inter-NEAT domain transfer experiments described in Chapter 4. The rate of heme transfer between IsdC and IsdE (74) is much slower than transfer among the NEAT domains (Chapter 4), and could be amenable to kinetic analysis and complex stabilization for crystallization of a heme transfer intermediate. Models for IsdE-heme and HtsA-FeSA docking on the permease offer insight into the mechanism of docking on the permease through conserved salt bridges (Section 7.4). Mutagenesis of the surface Glu residues in the E. coli ferric-citrate transporter FecB, and the S. aureus ferric-hydroxamate transporters FhuD1 and FhuD2, disrupted ligand import while maintaining ligand binding (61,62,206). With the in vivo reconstitution system for IsdE detailed in Chapter 5, the Isd and Hts systems could be reconstituted with similar Glu variants to assess the ability to grow on heme or FeSA, respectively. Given the inherent inaccuracies with   142 homology modeling using moderate sequence identity, the experiments are necessary to validate the roles of salt-bridge formation in the absence of a receptor-permease complex structure. Although insight into docking can be drawn from homology modeling, models are not necessarily accurate enough to provide insight into ligand release and transfer into the permease. The crystal structure of BtuCD-F reveals permease surface loops in the vicinity of the substrate binding protein binding pocket, which likely facilitate ligand transfer (39). Additionally, molecular dynamics simulations of the complex indicate that conformational changes between open and closed states in the permease contribute to ligand transfer (231,232). Ultimately, crystal structures of the IsdEF-heme-FhuD and HtsABC-FeSA-FhuD complexes would allow unambiguous definition of residues involved in docking, and provide insight into ligand transfer to the permease. Finally, the HtsA-FeSA structures provided insight into conformational change upon ligand binding. The localized structural changes identified in Chapter 6 present a new mechanism for class III substrate binding. However, these finding would benefit from validation by other experimental means. As described for IsdE in Chapter 5, S. aureus systems can be reconstituted in vivo with point mutational variants to alter individual amino acids. Since the largest structural change in HtsA is accompanied by a H-bond between the Tyr239 phenol and FeSA, mutation of Tyr239 should destabilize the conformational change, and be manifested as a growth defect of a Tyr239Ala variant of S. aureus growing on FeSA as an iron source. Similarly, other potentially important binding residues could be systematically targeted to define their roles in binding and transport. Alternatively, since the receptor is ~ 30 kDa, other biophysical methods such as NMR could be used to follow local structural changes upon binding.   143 Prior to this work, little structural-mechanistic information was available for Gram-positive uptake systems. 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