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Originally published In Press as doi:10.1074/jbc.M704602200 on July 31, 2007

J. Biol. Chem., Vol. 282, Issue 39, 28815-28822, September 28, 2007
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Heme Coordination by Staphylococcus aureus IsdE*Formula

Jason C. Grigg{ddagger}1, Christie L. Vermeiren§, David E. Heinrichs§, and Michael E. P. Murphy{ddagger}2

From the {ddagger}Department of Microbiology and Immunology, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3 and the §Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada N6A 5C1

Received for publication, June 5, 2007 , and in revised form, July 7, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha}-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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Staphylococcus aureus is a leading cause of hospital acquired bacterial infections (1). It is rapidly being recognized as an emerging pathogen because of relentless increases in drug resistance (2). 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 (3, 4). 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 (5). 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 (6, 7). 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 (8). 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 (9-11).

Several heme uptake pathways have been characterized in Gram-negative bacteria (5, 7). In contrast, uptake systems in Gram-positive bacteria are not as well understood. The Gram-positive pathogen S. aureus utilizes heme iron through the iron-regulated surface determinant (Isd)3 pathway (see below). More recently, a transposon mutant that preferentially acquired iron from transferrin versus heme implicated the heme transport system (Hts) in heme uptake as well (12).

The Isd system in S. aureus consists of nine members. IsdA, IsdB, IsdC, and IsdH/HarA are cell wall anchored surface proteins (13). These four proteins contain the conserved NEAT domain in one to three copies (14). IsdB and IsdH have been shown to bind hemoglobin and hemoglobin-haptoglobin, respectively (13, 15-17). 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 (18-21). Based on the predicted localization of these four proteins within the cell wall, Skaar and Schneewind (22) 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 (13, 19). Once in the cytoplasm, IsdG and IsdI liberate iron by heme degradation (23, 24).

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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 (25). 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 beta-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 (26). Selenomethionine-labeled IsdE was produced as described previously (27) 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 (28) 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 beta-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 x 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 (29). Crystals grew in the space group P43212 with one IsdE molecule in the asymmetric unit. The programs Solve (30) and Resolve (31, 32) 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 (33) and refined using translation libration screw parameters (34, 35) with Refmac5 (36) from the CCP4 program suite (37). 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).


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  		TABLE 1
Data collection and refinement statistics for the IsdE-heme complex

 
Construction of S. aureus isdE::Km—Allelic replacement, using methodologies described previously (38), was used to generate a non-polar insertion mutation in the chromosomal copy of isdE; the kanamycin cassette, from plasmid pDG782 (39), 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 (40) and subsequently transduced to H834 (isdE-) using phage 80{alpha}. The constructs were isolated from H834 and sequenced to ensure that mutations had not been introduced during the transformation and transduction procedures.


Figure 1
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  		FIGURE 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.

 
Heme-dependent Bacterial Growth Promotion Studies—S. aureus strains were pre-grown, from single colony, overnight in tris-minimal succinate growth media (41). 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 beta-sheet surrounded by {alpha}-helices (Fig. 1A). The N-terminal domain (Gly32-Arg138) beta-sheet contains only two beta-strands, whereas the C-terminal domain (Asn163-Lys289) contains a 5-stranded beta-sheet. The two domains are connected by a single long {alpha}-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 (42) 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 C{alpha} 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 (13, 19). 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 (37)). 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 N{delta}1 forms an H-bond with HOH15 (2.9 Å), which, in turn, forms an H-bond to Glu265 O{epsilon}2 (2.9 Å). Glu265 also forms H-bonds directly to Lys62 N{zeta} (3.4 Å) and Tyr61 O H (2.7 Å) through carboxylate atoms O{epsilon}2 and O{epsilon}1, respectively.


Figure 2
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  		FIGURE 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 {sigma} 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 Fig. 3 is mapped onto the molecular surface of IsdE using the Consurf server (59, 60). Red, white, and blue coloring indicates sequence conservation from 0 to 100% as indicated by the color bar.

 
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 N{zeta} 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 {alpha}-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 O{gamma} (2.7 Å) and the main chain amide of Tyr61 (3.2 Å) (Fig. 2B).

Multiple Sequence Alignments—A sequence search using BLAST (43) revealed several homologous proteins (E-value < 4 x 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 (22). The sequences were aligned with ClustalX (44), and the alignments were manually edited in BioEdit (45). 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.


Figure 3
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  		FIGURE 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).

 
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 (12, 13, 17, 18). Also we demonstrated that IsdA, localized to the cell wall, contributes to this process (18). 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 Fig. 4B, 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 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.


Figure 4
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  		FIGURE 4.
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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
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 (Fig. 4). 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 (18) and IsdC (20). 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 (46).

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 (18, 20, 21). 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 (Fig. 2B). The same H-bond network forms interactions with both propionates through water molecules. This H-bond network, in combination with the dipole of {alpha}-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 (18, 20) 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 (19).

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 (47). His-Met coordination is in agreement with previous magnetic circular dichroism spectroscopic data for IsdE. Mack et al. (19) 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 (48). 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 beta-strands, respectively. The strands impart significant flexibility allowing for large interdomain conformational change upon ligand binding (49, 50). However, IsdE belongs to the third class of bacterial-binding proteins that are characterized by a single domain spanning {alpha}-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 (49, 51-53). 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 beta-strands surrounded by several {alpha}-helices bridged by a single long {alpha}-helix. Because of the domain-spanning {alpha}-helix, members of class III are relatively inflexible and undergo minimal conformational change upon ligand binding and release (41, 49, 54). 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 (52). 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. (52) suggest results in a greater restriction of domain movement during ligand binding. CeuE, lacking a discernable hydrophobic interface (53), 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 (49, 55). Analysis of sequence conservation between IsdE sequence homologues revealed a highly conserved strip of residues spanning the width of the face involved in transporter interactions (Fig. 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 (49). In support of their importance to transport function, Sebulsky et al. (28) 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 (Fig. 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 {alpha}-helix in BtuF and a loop in IsdE (supplemental Fig. S5). 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 (49). Thus, we would anticipate that mutation of the conserved Glu residues in IsdE would similarly disrupt heme transport whereas not affecting heme binding.

The IsdE structure is unique in this superfamily of proteins because of the lack of well defined beta-strands in the N-terminal domain. This is because of the large prevalence of Pro residues that are unable to participate in beta-sheet H-bonding. Many of these Pro residues are conserved in putative IsdE orthologues (Fig. 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 (Fig. 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 (13, 22). A heme relay function for IsdE is supported by studies on the two IsdE orthologues, SpHtsA and SeHtsA, from Streptococcus equi subspecies equi (each share ~40% amino acid sequence identity with IsdE; Fig. 3). In streptococci, Shp is a cell surface protein that has been shown to relay heme to the HtsA lipoprotein (56, 57). 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 (48, 58). Based on the high sequence identity between IsdE and the two HtsA orthologues, including the conservation of residues in the heme binding pocket (Fig. 3 and supplemental Fig. S2), 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 Fig. 4, 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 (12), 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 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.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2Q8Q, 2Q8P) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by Canadian Institutes of Health Research operating Grants MOP-49597 and MOP-38002 (to M. E. P. M. and D. E. H., respectively). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-5. Back

1 Recipient of a Natural Sciences and Engineering Research Council Post Graduate Scholarship and a Michael Smith Foundation for Health Research Junior Graduate Trainee award. Back

2 To whom correspondence should be addressed: Tel.: 604-822-0254; Fax: 604-822-6041; E-mail: Michael.Murphy{at}ubc.ca.

3 The abbreviations used are: Isd, iron-regulated surface determinant; Hts, heme transport system; NEAT, near transporter; ABC, ATP-binding cassette; MES, 4-morpholineethanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank Anson Chan for his critical reading of this manuscript and Woo Cheol Lee, Iain MacPherson, Elitza Tocheva, Stacey Tom-Yew, Naomi Muryoi, and Chris Melo for their significant technical guidance. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL), a national user facility operated by Stanford University on behalf of the United States Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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