Heme Coordination by Staphylococcus aureus IsdE
2007; Elsevier BV; Volume: 282; Issue: 39 Linguagem: Inglês
10.1074/jbc.m704602200
ISSN1083-351X
AutoresJ.C. Grigg, Christie Vermeiren, David E. Heinrichs, M.E.P. Murphy,
Tópico(s)Antimicrobial Resistance in Staphylococcus
ResumoStaphylococcus aureus is a Gram-positive bacterial pathogen and a leading cause of hospital acquired infections. Because the free iron concentration in the human body is too low to support growth, S. aureus must acquire iron from host sources. Heme iron is the most prevalent iron reservoir in the human body and a predominant source of iron for S. aureus. The iron-regulated surface determinant (Isd) system removes heme from host heme proteins and transfers it to IsdE, the cognate substrate-binding lipoprotein of an ATP-binding cassette transporter, for import and subsequent degradation. Herein, we report the crystal structure of the soluble portion of the IsdE lipoprotein in complex with heme. The structure reveals a bi-lobed topology formed by an N- and C-terminal domain bridged by a single α-helix. The structure places IsdE as a member of the helical backbone metal receptor superfamily. A six-coordinate heme molecule is bound in the groove established at the domain interface, and the heme iron is coordinated in a novel fashion for heme transporters by Met78 and His229. Both heme propionate groups are secured by H-bonds to IsdE main chain and side chain groups. Of these residues, His229 is essential for IsdE-mediated heme uptake by S. aureus when growth on heme as a sole iron source is measured. Multiple sequence alignments of homologues from several other Gram-positive bacteria, including the human pathogens pyogenes, Bacillus anthracis, and Listeria monocytogenes, suggest that these other systems function equivalently to S. aureus IsdE with respect to heme binding and transport. Staphylococcus aureus is a Gram-positive bacterial pathogen and a leading cause of hospital acquired infections. Because the free iron concentration in the human body is too low to support growth, S. aureus must acquire iron from host sources. Heme iron is the most prevalent iron reservoir in the human body and a predominant source of iron for S. aureus. The iron-regulated surface determinant (Isd) system removes heme from host heme proteins and transfers it to IsdE, the cognate substrate-binding lipoprotein of an ATP-binding cassette transporter, for import and subsequent degradation. Herein, we report the crystal structure of the soluble portion of the IsdE lipoprotein in complex with heme. The structure reveals a bi-lobed topology formed by an N- and C-terminal domain bridged by a single α-helix. The structure places IsdE as a member of the helical backbone metal receptor superfamily. A six-coordinate heme molecule is bound in the groove established at the domain interface, and the heme iron is coordinated in a novel fashion for heme transporters by Met78 and His229. Both heme propionate groups are secured by H-bonds to IsdE main chain and side chain groups. Of these residues, His229 is essential for IsdE-mediated heme uptake by S. aureus when growth on heme as a sole iron source is measured. Multiple sequence alignments of homologues from several other Gram-positive bacteria, including the human pathogens pyogenes, Bacillus anthracis, and Listeria monocytogenes, suggest that these other systems function equivalently to S. aureus IsdE with respect to heme binding and transport. Staphylococcus aureus is a leading cause of hospital acquired bacterial infections (1Weems J.J. Postgrad. Med. J. 2001; 110: 24-26Crossref Scopus (48) Google Scholar). It is rapidly being recognized as an emerging pathogen because of relentless increases in drug resistance (2Tenover F.C. Pearson M.L. Emerg. Infect. Dis. 2004; 10: 2052-2053Crossref PubMed Scopus (8) Google Scholar). The establishment of two strains is problematic in the clinic, methicillin-resistant S. aureus and vancomycin-resistant S. aureus. Methicillin resistant S. aureus strains are increasing in prevalence both in hospitals and, more recently, within the community (3Deurenberg R.H. Vink C. Kalenic S. Friedrich A.W. Bruggeman C.A. Stobberingh E.E. Clin. Microbiol. Infect. 2007; 13: 222-235Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar, 4Deresinski S. Clin. Infect. Dis. 2005; 40: 562-573Crossref PubMed Scopus (370) Google Scholar). This drug resistance has high-lighted the need to better understand S. aureus pathogenesis and physiology. Iron uptake pathways have received significant attention because of the essential requirement of iron for the growth of most organisms (5Wandersman C. Delepelaire P. Annu. Rev. Microbiol. 2004; 58: 611-647Crossref PubMed Scopus (749) Google Scholar). In aerobic environments, free iron concentrations are generally low. For human pathogens, iron concentrations are further limited by host storage, transport, and innate immune mechanisms (6Radtke A.L. O'Riordan M.X. Cell. Microbiol. 2006; 8: 1720-1729Crossref PubMed Scopus (63) Google Scholar, 7Ratledge C. Dover L.G. Annu. Rev. Microbiol. 2000; 54: 881-941Crossref PubMed Scopus (1166) Google Scholar). Many bacterial pathogens have sophisticated systems to directly utilize host iron sources to satisfy their physiological requirements. Heme iron represents the most abundant iron source in the human body, accounting for ∼75% of the total iron (8Stojiljkovic I. Perkins-Balding D. DNA Cell Biol. 2002; 21: 281-295Crossref PubMed Scopus (103) Google Scholar). This heme iron can be found within hemoglobin in circulating red blood cells, myoglobin, and many other heme proteins. Because of its abundance, an ability to acquire heme iron from host sources represents a significant advantage for bacterial pathogens (9Ahn S.H. Han J.H. Lee J.H. Park K.J. Kong I.S. Infect. Immun. 2005; 73: 722-729Crossref PubMed Scopus (21) Google Scholar, 10Murphy E.R. Sacco R.E. Dickenson A. Metzger D.J. Hu Y. Orndorff P.E. 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More recently, a transposon mutant that preferentially acquired iron from transferrin versus heme implicated the heme transport system (Hts) in heme uptake as well (12Skaar E.P. Humayun M. Bae T. DeBord K.L. Schneewind O. Science. 2004; 305: 1626-1628Crossref PubMed Scopus (317) Google Scholar). iron-regulated surface determinant heme transport system near transporter ATP-binding cassette 4-morpholineethanesulfonic acid. The Isd system in S. aureus consists of nine members. IsdA, IsdB, IsdC, and IsdH/HarA are cell wall anchored surface proteins (13Mazmanian S.K. Skaar E.P. Gaspar A.H. Humayun M. Gornicki P. Jelenska J. Joachmiak A. Missiakas D.M. Schneewind O. Science. 2003; 299: 906-909Crossref PubMed Scopus (465) Google Scholar). These four proteins contain the conserved NEAT domain in one to three copies (14Andrade M.A. Ciccarelli F.D. Perez-Iratxeta C. Bork P. Genome Biol. 2002; 3 (research 0047.0041-0047.0045)Crossref PubMed Google Scholar). IsdB and IsdH have been shown to bind hemoglobin and hemoglobin-haptoglobin, respectively (13Mazmanian S.K. Skaar E.P. Gaspar A.H. Humayun M. Gornicki P. Jelenska J. Joachmiak A. Missiakas D.M. Schneewind O. Science. 2003; 299: 906-909Crossref PubMed Scopus (465) Google Scholar, 15Dryla A. Gelbmann D. von Gabain A. Nagy E. Mol. Microbiol. 2003; 49: 37-53Crossref PubMed Scopus (126) Google Scholar, 16Pilpa R.M. Fadeev E.A. Villareal V.A. Wong M.L. Phillips M. Clubb R.T. J. Mol. Biol. 2006; 360: 435-437Crossref PubMed Scopus (79) Google Scholar, 17Torres V.J. Pishchany G. Humayun M. Schneewind O. Skaar E.P. J. Bacteriol. 2006; 188: 8421-8429Crossref PubMed Scopus (219) Google Scholar). Recently, for IsdA and IsdC, the heme binding properties and crystal structures of the NEAT domains in complex with heme were determined, demonstrating similar mechanisms of heme coordination (18Grigg J.C. Vermeiren C.L. Heinrichs D.E. Murphy M.E. Mol. Microbiol. 2007; 63: 139-149Crossref PubMed Scopus (129) Google Scholar, 19Mack J. Vermeiren C. Heinrichs D.E. Stillman M.J. Biochem. Biophys. Res. Commun. 2004; 320: 781-788Crossref PubMed Scopus (45) Google Scholar, 20Sharp K.H. Schneider S. Cockayne A. Paoli M. J. Biol. Chem. 2007; 282: 10625-10631Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 21Vermeiren C.L. Pluym M. Mack J. Heinrichs D.E. Stillman M.J. Biochemistry. 2006; 45: 12867-12875Crossref PubMed Scopus (62) Google Scholar). Based on the predicted localization of these four proteins within the cell wall, Skaar and Schneewind (22Skaar E.P. Schneewind O. Microbes. Infect. 2004; 6: 390-397Crossref PubMed Scopus (166) Google Scholar) proposed a model for Isd heme transport. In this model, heme is removed from host heme proteins bound by IsdB and IsdH at the cell surface, transferred to IsdA and subsequently to IsdC. From there, heme moves to the ABC transporter-binding protein, IsdE (a lipoprotein), and subsequently through the transporter (13Mazmanian S.K. Skaar E.P. Gaspar A.H. Humayun M. Gornicki P. Jelenska J. Joachmiak A. Missiakas D.M. Schneewind O. Science. 2003; 299: 906-909Crossref PubMed Scopus (465) Google Scholar, 19Mack J. Vermeiren C. Heinrichs D.E. Stillman M.J. Biochem. Biophys. Res. Commun. 2004; 320: 781-788Crossref PubMed Scopus (45) Google Scholar). Once in the cytoplasm, IsdG and IsdI liberate iron by heme degradation (23Skaar E.P. Gaspar A.H. Schneewind O. J. Biol. Chem. 2004; 279: 436-443Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 24Wu R. Skaar E.P. Zhang R. Joachimiak G. Gornicki P. Schneewind O. Joachimiak A. J. Biol. Chem. 2005; 280: 2840-2846Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). 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 reveals His-Met heme iron coordination that is 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 adds 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. Cloning and Protein Expression for Structure Determination—The IsdE coding region (Gly32-Lys289), excluding the signal sequence and 11 N-terminal amino acids following the Cys lipidation site, and three C-terminal amino acids were amplified from S. aureus N315 chromosomal DNA and cloned into the expression vector, pET-28a(+) (Novagen). This construct was designed to optimize crystallization based on an analysis using the DISOPRED2 disorder prediction server (25Ward J.J. Sodhi J.S. McGuffin L.J. Buxton B.F. Jones D.T. J. Mol. Biol. 2004; 337: 635-645Crossref PubMed Scopus (1613) Google Scholar). Recombinant protein was expressed with an N-terminal His6 tag in Escherichia coli BL21. Cultures containing the expression vector were grown at 30 °C to an optical density at 600 nm of ∼0.8 followed by induction with 0.5 mm isopropyl β-d-thiogalactopyranoside 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). His6-IsdE was purified using a Ni-Sepharose high performance column (GE Healthcare) and dialyzed against 50 mm Tris (pH 8), 100 mm NaCl prior to thrombin digestion to remove the His6 purification tag. Cleaved protein was dialyzed 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 described previously (26Chan A.C. Lelj-Garolla B. Rosell F.I. Pedersen K.A. Mauk A.G. Murphy M.E. J. Mol. Biol. 2006; 362: 1108-1119Crossref PubMed Scopus (39) Google Scholar). Selenomethionine-labeled IsdE was produced as described previously (27Van Duyne G.D. Standaert R.F. Karplus P.A. Schreiber S.L. Clardy J. J. Mol. Biol. 1993; 229: 105-124Crossref PubMed Scopus (1087) Google Scholar) and purified similarly to native IsdE. Cloning and Protein Expression for Spectroscopy—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 (28Sebulsky M.T. Shilton B.H. Speziali C.D. Heinrichs D.E. J. Biol. Chem. 2003; 278: 49890-49900Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) 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 A600 of ∼0.8. Isopropyl β-d-thiogalactopyranoside (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, 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. UV/Visible Absorption Spectroscopy—All proteins (wild type and mutant) were purified as expressed from E. coli, and relative heme binding was assessed based on the ability of the proteins, all expressed in identical fashion, to scavenge and retain association with heme derived from the cytoplasm. Proteins were adjusted to an equivalent concentration, and electronic spectra were recorded using a Cary 500 spectrophotometer (Varian) with a 1-cm path length and 1-ml quartz cuvettes. All recordings were taken at room temperature. IsdE Structure Determination—Heme-bound IsdE crystals were grown by hanging drop vapor diffusion at room temperature. The well solution contained 50 mm MES (pH 5.5), 0.2 m ammonium acetate, and 28% polyethylene glycol 4000. Drops were made from 1 μl of 30 mg/ml protein solution and 1-μl 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 Stanford Synchrotron Radiation Laboratory at 100 K on beam lines 11-1 and 9-2 for the selenomethionine and native crystals, respectively. Single wavelength anomalous diffraction data were collected at a wavelength of 0.978894 Å. Native crystal data were collected at a wavelength of 1.0 Å. Data were processed using HKL2000 (29Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38570) Google Scholar). Crystals grew in the space group P43212 with one IsdE molecule in the asymmetric unit. The programs Solve (30Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar) and Resolve (31Terwilliger T.C. Acta Crystallogr. D Biol. Crystallogr. 2000; 56: 965-972Crossref PubMed Scopus (1634) Google Scholar, 32Terwilliger T.C. Acta Crystallogr. D Biol. Crystallogr. 2003; 59: 38-44Crossref PubMed Scopus (593) Google Scholar) were used to obtain phases from the nine identified selenium sites and to build a preliminary model (supplemental Fig. S1). 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 (33Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23370) Google Scholar) and refined using translation libration screw parameters (34Painter J. Merritt E.A. Acta Crystallogr. D Biol. Crystallogr. 2006; 62: 439-450Crossref PubMed Scopus (1104) Google Scholar, 35Painter J. Merritt E.A. J. Appl. Crystallogr. 2006; 39: 109-111Crossref Scopus (649) Google Scholar) with Refmac5 (36Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13868) Google Scholar) from the CCP4 program suite (37CCP4Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19765) Google Scholar). 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 favored conformation, and none are in the disallowed regions. Data collection and refinement statistics are shown in Table 1. Structure figures were generated in PYMOL (DeLano Scientific, San Carlos, CA).TABLE 1Data collection and refinement statistics for the IsdE-heme complexNative IsdESeMet IsdEData collectionaValues for the highest resolution shell are shown in parentheses.Resolution range (Å)50-2.15 (2.23-2.15)50-1.95 (2.02-1.95)Space groupP43212P43212Unit cell dimension (Å)a = 64.22, b = 64.22, c = 144.99a = 63.53, b = 63.53, c = 144.25Unique reflections17,31922,583Completeness (%)99.7 (99.6)86.0 (79.0)Average I/σI42.1 (5.9)22.2 (5.3)Redundancy6.3 (5.8)12.1 (7.6)Rmerge0.058 (0.363)0.068 (0.359)RefinementRwork (Rfree)20.2 (25.3)20.1 (26.3)B-factors (Å2)Protein28.131.7Heme31.132.6Water43.144.9r.m.s.d. bond length (Å)br.m.s.d., root mean square deviation.0.0120.013a Values for the highest resolution shell are shown in parentheses.b r.m.s.d., root mean square deviation. Open table in a new tab Construction of S. aureus isdE::Km—Allelic replacement, using methodologies described previously (38Speziali C.D. Dale S.E. Henderson J.A. Vines E.D. Heinrichs D.E. J. Bacteriol. 2006; 188: 2048-2055Crossref PubMed Scopus (71) Google Scholar), was used to generate a non-polar insertion mutation in the chromosomal copy of isdE; the kanamycin cassette, from plasmid pDG782 (39Guerout-Fleury A.M. Shazand K. Frandsen N. Stragier P. Gene. 1995; 167: 335-336Crossref PubMed Scopus (515) Google Scholar), 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 RN4220, by standard methodologies. Site-directed Mutagenesis of isdE—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 (40Kreiswirth B.N. Lofdahl S. Betley M.J. O'Reilly M. Schlievert P.M. Bergdoll M.S. Novick R.P. Nature. 1983; 305: 709-712Crossref PubMed Scopus (1009) Google Scholar) and subsequently transduced to H834 (isdE-) 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. Heme-dependent Bacterial Growth Promotion Studies—S. aureus strains were pre-grown, from single colony, overnight in tris-minimal succinate growth media (41Sebulsky M.T. Speziali C.D. Shilton B.H. Edgell D.R. Heinrichs D.E. J. Biol. Chem. 2004; 279: 53152-53159Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The cells were washed with saline, and 107 CFU of each strain were inoculated into tris-minimal succinate containing 5 μm ethylenediamine-di-(o-hydroxyphenyl)acetic acid with or without, either 50 μm FeCl3 or 5 μm hemin. Cultures (300 μl) were incubated at 37 °C with continuous shaking, and bacterial growth was monitored every 30 min over 20 h using a Bioscreen C (MTX Lab Systems, Inc.). Growth curves were plotted using Sigma Plot 2000. Overall Protein Structure—The selenomethionine and native IsdE structures were solved to 1.95 and 2.15 Å resolution, respectively. The structures reveal a protein with bi-lobed conformation with two domains, each composed of a central-most parallel β-sheet surrounded by α-helices (Fig. 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 (42Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3563) Google Scholar) 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. Heme Binding—Previous studies demonstrated that IsdE is a heme-binding protein (13Mazmanian S.K. Skaar E.P. Gaspar A.H. Humayun M. Gornicki P. Jelenska J. Joachmiak A. Missiakas D.M. Schneewind O. Science. 2003; 299: 906-909Crossref PubMed Scopus (465) Google Scholar, 19Mack J. Vermeiren C. Heinrichs D.E. Stillman M.J. Biochem. Biophys. Res. Commun. 2004; 320: 781-788Crossref PubMed Scopus (45) Google Scholar). We demonstrate that a single heme molecule is bound to IsdE along the groove formed between the two lobes (Fig. 1, A 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 domain. Approximately 160 Å2 (19%) of the total heme surface area is solvent exposed (as determined with AREAIMOL (37CCP4Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19765) Google Scholar)). 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 (Fig. 2C). IsdE-bound heme iron is six-coordinate with axial coordination by the thioether of Met78 (2.28 Å) from the N-terminal domain and the imidazolate of His229 (2.00 Å) from the C-terminal domain (Figs. 1A and 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 (Fig. 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 (Fig. 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. The heme propionates are oriented approximately parallel to the binding groove and are essentially buried (Fig. 2D). They are well ordered in the electron density map and form extensive interactions with IsdE (Fig. 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 (Fig. 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) (Fig. 2B). Multiple Sequence Alignments—A sequence search using BLAST (43Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70693) Google Scholar) revealed several homologous proteins (E-value < 4 × 10-37) 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 (22Skaar E.P. Schneewind O. Microbes. Infect. 2004; 6: 390-397Crossref PubMed Scopus (166) Google Scholar). The sequences were aligned with ClustalX (44Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35485) Google Scholar), and the alignments were manually edited in BioEdit (45Hall T. Nucleic Acids Symp. Ser. 1999; 41: 95-98Google Scholar). Each sequence shares greater than 28% identity over the 292 residues of S. aureus IsdE (Fig. 3 and supplemental Fig. S2). 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 (Fig. 3). Residues forming the H-bonding network with His229 are 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 (Fig. 3). The alignments reveal a conserved patch of residues located at the surface of the N- and C-terminal lobes traversing the heme pocket (Fig. 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 (Fig. 3). Lys237 and Lys241 are also conserved as large charged residues at the lobe surface in all aligned sequences (Fig. 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 (supplemental Fig. S3). 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. Contribution of Residues to Heme Binding and IsdE-mediated Heme Transport in Vivo—As shown in Fig. 4A, 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 (Fig. 4A). Notably, we also observed altered IsdE heme binding properties upon mutation of other residues whose side chains coordinated directly or indirectly (via waters) to the heme structure (supplemental Fig. S4). We and others have shown previously that S. aureus is capable of growing on hemin as a sole source of iron (12Skaar E.P. Humayun M. Bae T. DeBord K.L. Schneewind O. Science. 2004; 305: 1626-1628Crossref PubMed Scopus (317) Google Scholar, 13Mazmanian S.K. Skaar E.P. Gaspar A.H. Humayun M. Gornicki P. Jelenska J. Joachmiak A. Missiakas D.M. Schneewind O. Science. 2003; 299: 906-909Crossref PubMed Scopus (465) Google Scholar, 17Torres V.J. Pishchany G. Humayun M. Schneewind O. Skaar E.P. J. Bacteriol. 2006; 188: 8421-8429Crossref PubMed Scopus (219) Google Scholar, 18Grigg J.C. Vermeiren C.L. Heinrichs D.E. Murphy M.E. Mol. Microbiol. 2007; 63: 139-149Crossref PubMed Scopus (129) Google Scholar). Also we demonstrated that IsdA, localized to the cell wall, contributes to this process (18Grigg J.C. Vermeiren C.L. Heinrichs D.E. Murphy M.E. Mol. Microbiol. 2007; 63: 139-149Crossref PubMed Scopus (129) Google Scholar). In the present study, we characterized Isd-mediated heme iron acquisition
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