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Structural biology of heme binding in the Staphylococcus aureus Isd system
Journal of Inorganic Biochemistry 104 (2010) 341–348
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Review article
Structural biology of heme binding in the Staphylococcus aureus Isd system Jason C. Grigg, Georgia Ukpabi, Catherine F.M. Gaudin, Michael E.P. Murphy * Department of Microbiology and Immunology, The University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, Canada V6T1Z4
a r t i c l e
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Article history: Received 10 June 2009 Received in revised form 1 September 2009 Accepted 18 September 2009 Available online 26 September 2009 Keywords: Heme Protein structure Iron Staphylococcus aureus Iron regulated surface determinant Gram-positive bacteria
a b s t r a c t Iron is an absolute requirement for nearly all organisms, but most bacterial pathogens are faced with extreme iron-restriction within their host environments. To overcome iron limitation pathogens have evolved precise mechanisms to steal iron from host supplies. Staphylococcus aureus employs the ironresponsive surface determinant (Isd) system as its primary heme–iron uptake pathway. Hemoglobin or hemoglobin–haptoglobin complexes are bound by Near iron-Transport (NEAT) domains within cell surface anchored proteins IsdB or IsdH. Heme is stripped from the host proteins and transferred between NEAT domains through IsdA and IsdC to the membrane transporter IsdEF for internalization. Once internalized, heme can be degraded by IsdG or IsdI, thereby liberating iron for the organism. Most components of the Isd system have been structurally characterized to provide insight into the mechanisms of heme binding and transport. This review summarizes recent research on the Isd system with a focus on the structural biology of heme recognition. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Iron is an essential component of nearly every biological system [1]. As a cofactor in heme moieties, it functions in electron transport as well as enzymatic roles in diverse processes from general metabolism to avoiding oxidative damage [1,2]. Free iron is not readily available in most environments, so organisms generally need high affinity systems to acquire iron [3,4]. In humans and other mammalian host systems, restricting the access of iron is a common strategy to limit pathogenic growth. Despite possessing approximately 4 g of iron, 99.9% of iron in the human body is located intracellularly and is unavailable to most invading bacteria [5]. Approximately 75% of the intracellular iron pools are heme– iron, predominantly found in the oxygen transporter, hemoglobin (Hb) [5]. Due to its abundance, heme–iron represents a good source of growth-limiting iron provided a pathogen can gain access, import the heme and liberate its iron. Staphylococcus aureus is a Gram-positive bacterium and one of the most common organisms responsible for hospital-acquired bacterial infections [6]. S. aureus has an immense repertoire of virulence factors, which, when coupled with escalating antibiotic resistance, makes S. aureus a serious healthcare concern [7,8]. During infection, S. aureus secretes toxic hemolysins which cause the rupture of red blood cells, resulting in the release of the primary Hb store in the body. This represents a significant source of iron
* Corresponding author. Tel.: +1 604 822 8022; fax: +1 604 822 6041. E-mail address: [email protected] (M.E.P. Murphy). 0162-0134/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2009.09.012
for the organism and not surprisingly, S. aureus has been shown to utilize heme preferentially as an iron source [9]. The primary S. aureus heme-uptake system is termed the ironresponsive surface determinant (Isd) system [10,11]. Since identification of the system in 2002, a great amount of work has been done to characterize the Isd system. This focused review describes the current knowledge of the Isd system in S. aureus with an emphasis on structural biology. 2. Biology of the Isd system 2.1. Isd system components The S. aureus genome encodes nine proteins directly involved in heme uptake by the Isd system, and sortase B (SrtB) (Fig. 1). IsdA, IsdB, IsdC and IsdH (also referred to as HarA) are cell wall anchored surface receptors [11–14]. 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 [15]. 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 [16]. 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 [11]. The cell wall is typically 15–30 nm thick [17] and is a substantial barrier for ligands bound at the surface to move to the membrane. Thus, transporter proteins
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Fig. 1. 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 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. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
may be used by the cell if passive diffusion through the cell wall is not sufficient. The Isd system provides such 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 completely exposed to the environment, while IsdA is partially exposed and IsdC is buried within the cell wall [11] (Fig. 1). The localization of the proteins led to the proposed mechanism of transfer from the outer proteins, inward [9]. IsdE and IsdF comprise the binding protein and permease components of an ABC transporter, respectively [11,18]. 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. However, obvious homologues are absent in Isd systems from other bacteria. Finally, IsdG and IsdI are cytoplasmic heme-degrading enzymes that liberate the central iron atom for use by the organism [19].
recent study demonstrated high levels of IsdA expression in S. aureus isolated from the murine heart and liver, but high levels of IsdB in the heart but not the liver [24]. Furthermore, inactivating IsdA impairs S. aureus growth in the heart and liver, while inactivating IsdB impaired infection of heart tissue alone [21,24]. Whereas IsdG and IsdI both contribute to bacterial growth in the heart, only IsdG is central for infection in kidneys [21]. 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 different 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) [12,13,25,26]. In addition, IsdA promotes resistance to host innate defenses and survival on human skin by altering the hydrophobicity of the cell surface [27]. Furthermore, IsdA promotes binding to nasal epithelial cells, promoting rat nasal colonization [25,28,29]. The 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 Isd system is highly expressed and the human body mounts a considerable humoral immune response against Isd components [28,30]. IsdA and IsdB have both been successfully used alone or in multi-component vaccine trials, preventing infection in a murine abscess model [31,32] and a rat nasal carriage model [28]. Though there has been some question about the possibility of developing an efficacious S. aureus vaccine, several multicomponent vaccines which include Isd system components have demonstrated promise [33]. 3. The cell wall anchored surface receptors (IsdA, IsdB, IsdC, IsdH) 3.1. The conserved NEAT domain In S. aureus, all cell wall anchored Isd proteins, IsdA, IsdB, IsdC and IsdH, contain one to three copies of a conserved near irontransport (NEAT) domain [34]. 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 [34]. A schematic representation of the NEAT domains of IsdA, IsdB, IsdC and IsdH is presented in Fig. 2. 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
2.2. Importance to growth and pathogenesis The Isd system is required for maximal S. aureus growth on heme as a 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 [11]. Subsequent work demonstrated that inactivating IsdA, IsdG or IsdI impaired growth in iron-chelated minimal media with heme as an iron source [20,21]. Furthermore, inactivating IsdH and IsdB also led to decreased growth on Hb as an iron source [22]. 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 [21–23]. Interestingly, a
Fig. 2. 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.
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15–70 highly charged amino acids with low sequence complexity and a high likelihood of disorder as determined by the program, Disopred [35]. Recombinant expression of individual NEAT domains has enabled their structural and functional analysis. Currently, several structures of NEAT domains are described in the literature. 3.2. The structure of the NEAT domain and heme binding The published structures of NEAT domains all reveal a similar eight-stranded immunoglobulin-like b-sandwich fold (Fig. 3A). The structures of apo (1.6 Å) and heme-bound IsdA-N1 (1.9 Å) were determined by X-ray crystallography [20]. The heme-bound structure of IsdC-N1 (1.5 Å) was also determined by X-ray crystallography [36] and subsequent solution structures of apo and Zn2+protoporphyrin IX (PPIX) IsdC-N1 by NMR were described [37]. Two NMR structures of apo IsdH-N1 and an X-ray crystal structure of apo (2.2 Å) and heme-bound IsdH-N3 (1.9 Å) have also been presented [38,39]. All of these NEAT domain structures are readily superimposed. For instance, the crystal structure of holo-IsdA-N1 overlays with the holo crystal structures of IsdC-N1 and IsdH-N3 with r.m.s.d values of 1.76 and 1.47 Å over all Ca atoms, respectively (Fig. 3B). The main differences are in loops on the protein surface and, not surprisingly, at the chain termini. Notably, in IsdC-N1, the loop joining b6 and b7 contains a five-residue insertion immediately preceding the heme–iron coordinating residue (Fig. 3B). Since this insertion is only present in IsdC-N1, it could reflect the unique ability of IsdC to pass heme to IsdE [40,41]. A lone a-helix seen in the holo-structures of NEAT domains between b1 and b2 forms one side of the heme binding pocket (Fig. 3A). NMR data of apo-IsdH-N1 and apo-IsdC-N1 reveals that the region defined as the helix in the holo structure is flexible in the absence of ligand [37,38] and in the case of IsdC-N1 this region becomes stabilized upon titration with Zn2+-PPIX [37]. Similarly, the corresponding helix in the holo IsdH-N3 crystal structures has reduced B-factors as compared to the apo structure. The IsdA-N1 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 [20]. 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 [42].
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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 fivecoordinate with a Tyr phenolate at distances of 2.1–2.2 Å [20,36,39,43–46] (Fig. 4). Another Tyr is invariably located four residues later in the sequence and forms a H-bond (2.5 Å) with the iron-coordinating Tyr and a p-bonding interaction with a heme pyrrole ring [20,36,39] (Figs. 4 and 5). A conserved Ser and a hydrophilic residue form H-bonds with a heme propionate in all known structures of heme binding NEAT domains (Figs. 4 and 5). Several hydrophobic contacts to the tetrapyrrole structure of heme are maintained through the heme pocket. For instance, a stacked pbonding interaction of a benzene ring of Tyr87 (IsdA-N1) or a phenylalanine with a pyrrole ring at the base of the binding pocket is absolutely conserved (Figs. 4 and 5). The other obvious conserved residue is Trp113 (IsdA-N1) at the base of the pocket where it lies next to the vinyl end of the porphyrin ring [36]. A functionally interesting characteristic of the heme binding pockets occurs on the distal side of the heme face. In IsdA-N1, His83 is located directly adjacent to heme–iron in the proposed flexible a-helix (Fig. 4B). Spectroscopic studies have demonstrated that His83 coordinates heme–iron upon reduction of holo-protein solution with dithionite [44,46]. However, the other two S. aureus holo-NEAT domain structures, IsdC-N1 and IsdH-N3, contain Ile and Val (respectively) at the equivalent location to His83 of IsdAN1 (Fig. 4C and D). As a likely result of these substitutions, reduction of IsdC-N1 causes release of heme from the protein [44,45]. These residues are situated in the axial heme plane such that a favorable heme–iron coordinating side chain could coordinate heme–iron. Therefore, the amino acid variations could reflect the individual domains’ roles in the heme transfer chain or the differences in interacting partners or the variation could simply be due to little selective pressure at that position. 3.3. Interaction of NEAT domains with host heme proteins Some NEAT domains have roles other than, or in addition to, heme binding. Isd-host protein interaction has been observed by several methods, including enzyme-linked immunosorbent assays, surface plasmon resonance, NMR and targeted alanine mutagenesis. Three of the outermost proteins in the Isd system, IsdA, IsdB and IsdH, bind host heme proteins [12,14,22,38,40,47,48]. IsdH has two NEAT domains which can bind Hb or haptoglobin (Hp) with low nM dissociation constants but not heme (IsdH-N1, IsdH-N2) [47,48] and one that binds heme alone with a low lM
Fig. 3. 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 b-strands (b1-8) and one well defined a-helix (a1). 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. IsdA-N1 (PDB ID: 2itf) (cyan), IsdC-N1 (PDB ID: 2o6p) (magenta) and IsdH-N3 (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).
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Fig. 4. 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. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Sequence alignments of S. aureus NEAT Domains. Structures of IsdA-N1, IsdC-N1, IsdH-N1 and IsdH-N3 were superposed to generate a preliminary primary structure alignment using the CE algorithm [87] in the program STRAP [88]. All remaining NEAT domains were aligned against this subset. Alignments were visualized with Jalview [89]. Residues are numbered according to full-length IsdA (NCBI ID: NP_374247).
dissociation constant (IsdH-N3) [39,47,48]. Gel filtration and analytical ultracentrifugation were used to determine that metHbIsdH-N1 forms a 2:1 complex of IsdH-N1:metHb with an apparent molecular mass of 66 kDa [38]. Interestingly, alanine mutagenesis data suggests interaction with metHb occurs on the same face
of IsdH-N1 as heme binding in other NEAT domains, namely involving the b1–b2 loop and the b7–b8 loop [48]. IsdB has been shown to interact with Hb and Hp–Hb with low nM dissociation constants, but not with Hp [47]. The specific regions of IsdB responsible for protein or heme binding have not
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been reported, but homology to IsdH suggests IsdB-N1 (46% identity to IsdH-N1, 65% identity to IsdH-N2) binds Hb and IsdB-N2 (56% identity to IsdH-N3) binds heme. Despite these recent advances, the mechanism of heme extraction and transfer by IsdB and IsdH remains unknown. Rates of heme extraction from metHb by the combination of IsdH-N1 and IsdH-N3 (kobs 10.9 h 1) [48] are very close to the off rates of heme from metHb (7.8 h 1 for bsubunit and 0.6 h 1 for the a-subunit) [49] yet the rate of heme transfer from metHb to full-length IsdB (0.31 s 1) [40] is much more rapid (150-fold). Heme transfer must therefore be driven by an activated complex which likely requires either regions of the protein outside the NEAT domains, a combination of IsdH-N1, IsdH-N2 and IsdH-N3 not tested or that the tethering of the domains to one another is essential for efficient transfer. 3.4. Current model of heme transfer by the cell surface Isd system A model was put forth that suggested the locations of Isd proteins in the cell wall and membrane define the relay mechanism of heme import. Recent in vitro evidence demonstrated heme transfer rates between purified Isd surface proteins that support the model of heme flow based on localization [40,41,50]. Heme is transferred from metHb to IsdB (kobs 0.31 s 1) or IsdH. Muryoi et al. demonstrated that heme moves bidirectionally between IsdB-N2 and IsdH-N3 [41]. From IsdB (or IsdH), heme transfer is rapid to apo IsdA (114 s 1) or apo-IsdC (15 s 1). Heme bound to IsdA can then move to IsdC (53 s 1). Finally, Zhu et al. demonstrated only IsdC efficiently transfers heme to IsdE (0.0062 s 1) [40]. Alternatively, Muryoi et al. demonstrated that heme transfer to IsdE may occur directly from IsdB-N2 or IsdH-N3 [41]. Additional experiments are needed to determine the biological relevance of these transfer experiments, since protein localization on the cell wall likely affects transfer rates. A current model of heme transfer based on kinetic transfer experiments and cellular localization is illustrated by arrows in Fig. 1. Each proposed heme transfer step is rapid and occurs between full-length recombinant proteins (excluding the secretion signal and the residues following the sortase anchoring motif) [40,50] and between recombinant NEAT domains alone [41]. Since heme transfer rates between NEAT domain-containing Isd proteins are at least 10,000 times greater than the rate of heme dissociation, transfer is likely mediated by activated complex formation [40,41,50]. Despite an estimated KD based on heme transfer kinetics of 17 lM for the holo-IsdA apo-IsdC complex [40,50], Isd protein–protein complexes have not yet been physically observed or characterized on a molecular level. 4. The Isd membrane transporter Heme is transferred from the cell wall anchored components of the system through IsdC to IsdE [40,41]. Typical ABC transporters for the import of nutrients in bacteria are comprised of three main components: a substrate-binding protein (IsdE), a membrane permease (IsdF) and an ATP hydrolase [51]. Since Gram-positive bacteria lack an outer membrane, IsdE is anchored to the external side of the membrane through a lipid moiety covalently attached to a cysteine residue at the N-terminus of the mature protein [52]. IsdE assumes an overall fold comprising N- and C-terminal lobes which consist of central b-stands and peripheral helices and loops [18]. The two domains are bridged by a long a-helix, which appears to be an emerging characteristic of metal transport binding proteins (Fig. 6) [53,54]. Heme is bound to IsdE in a large groove formed between the N- and C-terminal domains with residues from both lobes providing several substrate contacts. Unlike the orientation of heme in the NEAT domains, the heme propionate
Fig. 6. Model of the heme ABC transporter of the Isd system composed of IsdEF and FhuC. The X-ray 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 a-helix (light orange), the propionate-stabilizing a-helix (yellow) and the heme–iron coordinating side chains (cyan) are shown.
groups are directed into the binding pocket and are essentially buried. Electrostatic interactions with the propionate groups stabilize the buried charge, including hydrogen bonds to the main chain amides of Val41 and Ala42 to one propionate, and the alignment of the N-terminus of a-helix 1 such that the positive helix dipole stabilizes the charge of the other propionate (Fig. 6). Heme–iron is six-coordinate with an axial ligand from each of the domains. In line with magnetic circular dichroism spectra that demonstrate His–Met coordination, the crystal structure of IsdE revealed that Met78 and His229 coordinate the heme–iron [18,43,55] (Fig. 6). IsdF is suggested to form the permease, a homodimeric integral membrane protein [11]. The final component needed to drive heme uptake is an ATP hydrolase. However, the S. aureus Isd system lacks an obvious ATPase encoded in the isd locus; in fact, several iron-compound ABC transporters from S. aureus lack a corresponding ATPase, including the siderophore transporters HtsABC and SirABC [56,57]. In the Hts and Sir systems, FhuC provides the ATPase function, and evidence suggests FhuC serves as the ATPase for the Isd system as well [56,58].
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A BLAST search of the RCSB Protein Data Bank with the IsdF sequence revealed promising modeling templates for IsdF. The permease from 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 transporter was generated with the program ESyPred3D [59] using the crystal structure of the BtuCD complex (PDB ID: 2qi9) [60] as a template. To dock IsdE onto the IsdF–FhuC model, the transporter was superposed onto the substrate-binding protein, BtuF, from the structure of BtuCD-F [60]. IsdE and BtuF share a sequence identity of 18% and overlay with an r.m.s.d of 2.6 Å over 232 Ca atoms. The components of metal uptake ABC transporters typically display conserved structure even in the absence of high sequence identities; therefore, when structural evidence is combined with the sequence alignments, the docking model is likely a useful prediction of interaction. The resulting model is a complete IsdF–FhuC–IsdE complex (Fig. 6). The IsdF model based on BtuC is predicted to have nine membrane spanning helices per monomer, missing the first helix. 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 are present on the extracellular side of the IsdF model that potentially interact with Glu83 (equivalent to Glu74 in BtuF) and Glu214 (equivalent to Glu202 in BtuF) from IsdE. These electrostatic interactions likely aid in the recognition of IsdE by IsdF and induce heme transport. The presence of similar interactions as seen in the BtuCF system supports the validity of the IsdEF model; however, these predictions await experimental confirmation. 5. Isd heme-degrading enzymes Following heme transport through the permease, heme enters the bacterial cytoplasm where it has two potential fates. It may be degraded to release iron as a nutrient for growth or it may be
sorted as an intact heme moiety to be used directly in bacterial hemoproteins [9]. IsdG and IsdI have been identified as the heme-degrading components of the Isd system in S. aureus [61– 63]. These enzymes are atypical heme-degrading enzymes, as their protein sequences and structural folds are distinct from the wellcharacterized heme oxygenases, the first family of enzymes found to break down heme and identified in numerous bacteria and eukaryotes. IsdG and IsdI have been shown to bind heme, producing a Soret peak at 413 nm [62]. Biochemical assays in aerobic conditions show either enzyme can release iron from heme in vitro in the presence of an electron donor such as ascorbate, or NADPH and cytochrome P450 reductase [62–64]. However, a detailed investigation into the catalytic mechanism of IsdG and IsdI is lacking, as it is not yet known whether the reaction proceeds by oxygen activation. For instance, in heme oxygenases the oxidative cleavage of the heme porphyrin ring yields biliverdin, thereby releasing iron [65]. IsdG and IsdI, along with their homologues from Bacillus anthracis [66] and Bradyrhizobium japonicum [67], have also been suggested to produce biliverdin from heme [62]. However, electronic spectra of the product are not consistent with biliverdin and product analysis by direct structural methods is not yet complete. Although the structures of native IsdG–heme and IsdI–heme are not yet available, the apo structures of IsdG and IsdI have been solved [63]. More recently, structures of IsdI–CoPPIX, a substrate analogue of heme, and of a mutational variant of IsdG–heme provided the first insights into IsdG and IsdI–heme binding; the use of a substrate analogue or a catalytically inactive variant hinders heme degradation by the enzyme and thus allows for structural analysis with heme in the binding pocket [64]. IsdG and IsdI adopt a ferredoxin-like a + b-sandwich fold and exist as homodimers which form a b-barrel at the dimer interface (Fig. 7A) [63]. This is in contrast to the monomeric and predominantly a-helical fold of canonical heme oxygenases [65]. IsdG and IsdI also lack the distal hydrogen bonding solvent network essential for heme oxygenase activity [68]. Instead, each IsdG and IsdI dimer contains two
Fig. 7. Structures of the holo-forms of the IsdG–N7A variant and native IsdI [64]. (A) IsdI–CoPPIX structure (PDB ID: 2zdp) showing the dimeric fold (green/cyan). (B) Surface representation of IsdI–CoPPIX revealing the deep binding clefts. (C) The substrate-binding pocket in the structure of IsdG–N7A–heme (PDB ID: 2zdo) (yellow) and (D) IsdI– CoPPIX (cyan). The conserved binding residues of IsdG and IsdI are shown for comparison, with the exception of Asn7 of IsdG, which has been mutated to Ala. Metalloporphyrins (gray) are distorted from planarity and take on a highly ruffled conformation.
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deep hydrophobic clefts where the heme moieties are bound, limiting access by water molecules (Fig. 7B). Interestingly, the residues that interact with the heme are conserved between the paralogous IsdG and IsdI proteins, which share 64% sequence identity [64]. Biochemical analysis identified multiple residues important for activity of IsdG, including Asn7 and Trp67.[63]. The structure of the IsdG–N7A variant bound to heme [64] revealed a five-coordinate heme iron that is liganded to an axial histidine residue (His77, Fig. 7C). IsdG and IsdI enzyme activity is also substantially hindered by reconstituting the enzymes with analogues of heme containing metals other than Fe3+ [64]. The metalloporphyrins that have been tested include Co, Ga, Mn and Zn-containing PPIX. The structure of IsdI–CoPPIX shows the Co3+ is six-coordinate with a distal Cl ligand in addition to the axial His76 ligand (Fig. 7D). The coordinating Cl is stabilized by a H-bond with Asn6, and the Cd1 atom of Ile53 is pointing away from the iron, in contrast to the equivalent residue (Ile54) in the IsdG–N7A structure. Insights into the natural distal ligand of IsdG and IsdI may be gained by determining the structure of the heme-bound form of the native enzymes. Perhaps the most interesting feature of these structures is the extreme conformational distortion of the porphyrin ring that occurs upon binding by IsdG and IsdI (Fig. 7C and D). The porphyrin ring is distorted from planarity by extensive steric interactions with hydrophobic residues in the binding cleft, particularly by Trp67, Val80, Asn7, and Phe23 in IsdG (equivalent residues in IsdI are Trp66, Val79, Asn6, and Phe22) [64]. This distortion is described as ruffled; ruffling to this extent has not been seen in the known structures of other hemoproteins. How the unusually high degree of ruffling contributes to IsdG and IsdI enzyme catalysis is unclear.
6. Related Isd systems Heme-uptake systems homologous to the Isd system in S. aureus have been identified and characterized to different extents in several Gram-positive organisms, including Bacillus anthracis, Listeria monocytogenes and Streptococcus pyogenes [69]. These organisms possess NEAT domain-containing proteins, heme ABC transporters and heme-degrading enzymes [69–79]. The systems have generally been shown to contribute to virulence [71,74– 76,80–82]. The many details of binding and pathogenesis in these organisms are beyond the scope of this review so the reader is referred to the primary literature referred to above. Notable instances of variability and similarity between the S. aureus Isd system and homologous heme-uptake systems are found. For instance, all other Isd systems encode an ATPase for the transporter within the operon. Furthermore, recent data revealed that IsdX1 (one NEAT domain; also known as IsdJ) and IsdX2 (five NEAT domains; also known as IsdK) from B. anthracis are secreted instead of being anchored to the cell wall [71,76]. Similarities to the S. aureus Isd system are also found, often encompassing intriguing dissimilarities as well. Recently, the structure of a cell surface receptor, Shp, from S. pyogenes was determined. Shp itself was not originally annotated as a NEAT domain-containing protein, but the 2.1 Å resolution crystal structure of the heme binding domain (or Shp180) revealed the immunoglobulin-like b-sandwich fold characteristic of NEAT domains; Shp180 overlays with S. aureus IsdC and IsdA over all Ca atoms with an r.m.s.d. of 2.0 and 2.2 Å, respectively [83]. However, unlike the other published NEAT domain structures, heme–iron is coordinated in Shp180 by two Met residues (Met153 and Met66) in the binding pocket [83]. Shp transfers its heme to HtsA, the substrate-binding lipoprotein component of the heme-specific ABC
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transporter, through a proposed activated complex which requires both Met153 and Met66 to be intact for full efficiency [84–86]. It is worth noting that HtsA bears 40% sequence identity to IsdE in S. aureus, including conservation of the heme–iron coordinating residues [18], and appears to share a similarity in function as well [84]. 7. Summary The Isd system of S. aureus describes a simple yet elegant way for the opportunistic human pathogen to fulfill its iron requirement. Numerous advances have recently been made in characterizing this pathway and have provided us with an excellent working model for understanding heme uptake during infection. Hemolysins are secreted, releasing intracellular Hb stores and producing Hb–Hp complexes, which can be exploited using IsdB and IsdH. Through an efficient relay system involving the formation of putative activated complexes, heme is extracted from the host hemoproteins and transferred through IsdA to IsdC, where it is delivered to the bi-lobed substrate-binding protein IsdE which docks onto the membrane permease IsdF. Internalization of the heme is likely driven by the ATPase FhuC. Inside the cell the porphyrin ring is degraded by IsdG and IsdI which have a ferredoxin-like a + b-sandwich fold and contort the ring into a highly ruffled conformation; iron is subsequently liberated. The NEAT domain has been demonstrated to be an essential, functional component of the surface proteins, and through its conserved eight-stranded b-sandwich fold, binds host hemoproteins or heme using a hydrophobic pocket containing a conserved Tyr to coordinate the heme–iron. However, despite the considerable work that has been accomplished on the system, several major puzzles have yet to be solved. The mechanism of heme extraction from host hemoproteins and heme transfer between Isd surface proteins is unclear. The product and mode of action of the Isd heme-degrading enzymes, IsdG and IsdI, has yet to be adequately described. Lacking structural homology to the canonical heme monooxygenases as well as the solvent network required for activity of these enzymes, the heme degradation mechanism by IsdG and IsdI may be novel. Lastly, homologues of IsdD have not been found. Its function remains a mystery. 8. Abbreviations PPIX CoPPIX Hp Isd NEAT
protoporphyrin IX cobalt protoporphyrin IX haptoglobin iron-regulated surface determinant near iron-transporter
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Review article
Structural biology of heme binding in the Staphylococcus aureus Isd system Jason C. Grigg, Georgia Ukpabi, Catherine F.M. Gaudin, Michael E.P. Murphy * Department of Microbiology and Immunology, The University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, Canada V6T1Z4
a r t i c l e
i n f o
Article history: Received 10 June 2009 Received in revised form 1 September 2009 Accepted 18 September 2009 Available online 26 September 2009 Keywords: Heme Protein structure Iron Staphylococcus aureus Iron regulated surface determinant Gram-positive bacteria
a b s t r a c t Iron is an absolute requirement for nearly all organisms, but most bacterial pathogens are faced with extreme iron-restriction within their host environments. To overcome iron limitation pathogens have evolved precise mechanisms to steal iron from host supplies. Staphylococcus aureus employs the ironresponsive surface determinant (Isd) system as its primary heme–iron uptake pathway. Hemoglobin or hemoglobin–haptoglobin complexes are bound by Near iron-Transport (NEAT) domains within cell surface anchored proteins IsdB or IsdH. Heme is stripped from the host proteins and transferred between NEAT domains through IsdA and IsdC to the membrane transporter IsdEF for internalization. Once internalized, heme can be degraded by IsdG or IsdI, thereby liberating iron for the organism. Most components of the Isd system have been structurally characterized to provide insight into the mechanisms of heme binding and transport. This review summarizes recent research on the Isd system with a focus on the structural biology of heme recognition. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Iron is an essential component of nearly every biological system [1]. As a cofactor in heme moieties, it functions in electron transport as well as enzymatic roles in diverse processes from general metabolism to avoiding oxidative damage [1,2]. Free iron is not readily available in most environments, so organisms generally need high affinity systems to acquire iron [3,4]. In humans and other mammalian host systems, restricting the access of iron is a common strategy to limit pathogenic growth. Despite possessing approximately 4 g of iron, 99.9% of iron in the human body is located intracellularly and is unavailable to most invading bacteria [5]. Approximately 75% of the intracellular iron pools are heme– iron, predominantly found in the oxygen transporter, hemoglobin (Hb) [5]. Due to its abundance, heme–iron represents a good source of growth-limiting iron provided a pathogen can gain access, import the heme and liberate its iron. Staphylococcus aureus is a Gram-positive bacterium and one of the most common organisms responsible for hospital-acquired bacterial infections [6]. S. aureus has an immense repertoire of virulence factors, which, when coupled with escalating antibiotic resistance, makes S. aureus a serious healthcare concern [7,8]. During infection, S. aureus secretes toxic hemolysins which cause the rupture of red blood cells, resulting in the release of the primary Hb store in the body. This represents a significant source of iron
* Corresponding author. Tel.: +1 604 822 8022; fax: +1 604 822 6041. E-mail address: [email protected] (M.E.P. Murphy). 0162-0134/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2009.09.012
for the organism and not surprisingly, S. aureus has been shown to utilize heme preferentially as an iron source [9]. The primary S. aureus heme-uptake system is termed the ironresponsive surface determinant (Isd) system [10,11]. Since identification of the system in 2002, a great amount of work has been done to characterize the Isd system. This focused review describes the current knowledge of the Isd system in S. aureus with an emphasis on structural biology. 2. Biology of the Isd system 2.1. Isd system components The S. aureus genome encodes nine proteins directly involved in heme uptake by the Isd system, and sortase B (SrtB) (Fig. 1). IsdA, IsdB, IsdC and IsdH (also referred to as HarA) are cell wall anchored surface receptors [11–14]. 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 [15]. 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 [16]. 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 [11]. The cell wall is typically 15–30 nm thick [17] and is a substantial barrier for ligands bound at the surface to move to the membrane. Thus, transporter proteins
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Fig. 1. 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 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. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
may be used by the cell if passive diffusion through the cell wall is not sufficient. The Isd system provides such 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 completely exposed to the environment, while IsdA is partially exposed and IsdC is buried within the cell wall [11] (Fig. 1). The localization of the proteins led to the proposed mechanism of transfer from the outer proteins, inward [9]. IsdE and IsdF comprise the binding protein and permease components of an ABC transporter, respectively [11,18]. 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. However, obvious homologues are absent in Isd systems from other bacteria. Finally, IsdG and IsdI are cytoplasmic heme-degrading enzymes that liberate the central iron atom for use by the organism [19].
recent study demonstrated high levels of IsdA expression in S. aureus isolated from the murine heart and liver, but high levels of IsdB in the heart but not the liver [24]. Furthermore, inactivating IsdA impairs S. aureus growth in the heart and liver, while inactivating IsdB impaired infection of heart tissue alone [21,24]. Whereas IsdG and IsdI both contribute to bacterial growth in the heart, only IsdG is central for infection in kidneys [21]. 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 different 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) [12,13,25,26]. In addition, IsdA promotes resistance to host innate defenses and survival on human skin by altering the hydrophobicity of the cell surface [27]. Furthermore, IsdA promotes binding to nasal epithelial cells, promoting rat nasal colonization [25,28,29]. The 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 Isd system is highly expressed and the human body mounts a considerable humoral immune response against Isd components [28,30]. IsdA and IsdB have both been successfully used alone or in multi-component vaccine trials, preventing infection in a murine abscess model [31,32] and a rat nasal carriage model [28]. Though there has been some question about the possibility of developing an efficacious S. aureus vaccine, several multicomponent vaccines which include Isd system components have demonstrated promise [33]. 3. The cell wall anchored surface receptors (IsdA, IsdB, IsdC, IsdH) 3.1. The conserved NEAT domain In S. aureus, all cell wall anchored Isd proteins, IsdA, IsdB, IsdC and IsdH, contain one to three copies of a conserved near irontransport (NEAT) domain [34]. 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 [34]. A schematic representation of the NEAT domains of IsdA, IsdB, IsdC and IsdH is presented in Fig. 2. 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
2.2. Importance to growth and pathogenesis The Isd system is required for maximal S. aureus growth on heme as a 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 [11]. Subsequent work demonstrated that inactivating IsdA, IsdG or IsdI impaired growth in iron-chelated minimal media with heme as an iron source [20,21]. Furthermore, inactivating IsdH and IsdB also led to decreased growth on Hb as an iron source [22]. 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 [21–23]. Interestingly, a
Fig. 2. 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.
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15–70 highly charged amino acids with low sequence complexity and a high likelihood of disorder as determined by the program, Disopred [35]. Recombinant expression of individual NEAT domains has enabled their structural and functional analysis. Currently, several structures of NEAT domains are described in the literature. 3.2. The structure of the NEAT domain and heme binding The published structures of NEAT domains all reveal a similar eight-stranded immunoglobulin-like b-sandwich fold (Fig. 3A). The structures of apo (1.6 Å) and heme-bound IsdA-N1 (1.9 Å) were determined by X-ray crystallography [20]. The heme-bound structure of IsdC-N1 (1.5 Å) was also determined by X-ray crystallography [36] and subsequent solution structures of apo and Zn2+protoporphyrin IX (PPIX) IsdC-N1 by NMR were described [37]. Two NMR structures of apo IsdH-N1 and an X-ray crystal structure of apo (2.2 Å) and heme-bound IsdH-N3 (1.9 Å) have also been presented [38,39]. All of these NEAT domain structures are readily superimposed. For instance, the crystal structure of holo-IsdA-N1 overlays with the holo crystal structures of IsdC-N1 and IsdH-N3 with r.m.s.d values of 1.76 and 1.47 Å over all Ca atoms, respectively (Fig. 3B). The main differences are in loops on the protein surface and, not surprisingly, at the chain termini. Notably, in IsdC-N1, the loop joining b6 and b7 contains a five-residue insertion immediately preceding the heme–iron coordinating residue (Fig. 3B). Since this insertion is only present in IsdC-N1, it could reflect the unique ability of IsdC to pass heme to IsdE [40,41]. A lone a-helix seen in the holo-structures of NEAT domains between b1 and b2 forms one side of the heme binding pocket (Fig. 3A). NMR data of apo-IsdH-N1 and apo-IsdC-N1 reveals that the region defined as the helix in the holo structure is flexible in the absence of ligand [37,38] and in the case of IsdC-N1 this region becomes stabilized upon titration with Zn2+-PPIX [37]. Similarly, the corresponding helix in the holo IsdH-N3 crystal structures has reduced B-factors as compared to the apo structure. The IsdA-N1 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 [20]. 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 [42].
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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 fivecoordinate with a Tyr phenolate at distances of 2.1–2.2 Å [20,36,39,43–46] (Fig. 4). Another Tyr is invariably located four residues later in the sequence and forms a H-bond (2.5 Å) with the iron-coordinating Tyr and a p-bonding interaction with a heme pyrrole ring [20,36,39] (Figs. 4 and 5). A conserved Ser and a hydrophilic residue form H-bonds with a heme propionate in all known structures of heme binding NEAT domains (Figs. 4 and 5). Several hydrophobic contacts to the tetrapyrrole structure of heme are maintained through the heme pocket. For instance, a stacked pbonding interaction of a benzene ring of Tyr87 (IsdA-N1) or a phenylalanine with a pyrrole ring at the base of the binding pocket is absolutely conserved (Figs. 4 and 5). The other obvious conserved residue is Trp113 (IsdA-N1) at the base of the pocket where it lies next to the vinyl end of the porphyrin ring [36]. A functionally interesting characteristic of the heme binding pockets occurs on the distal side of the heme face. In IsdA-N1, His83 is located directly adjacent to heme–iron in the proposed flexible a-helix (Fig. 4B). Spectroscopic studies have demonstrated that His83 coordinates heme–iron upon reduction of holo-protein solution with dithionite [44,46]. However, the other two S. aureus holo-NEAT domain structures, IsdC-N1 and IsdH-N3, contain Ile and Val (respectively) at the equivalent location to His83 of IsdAN1 (Fig. 4C and D). As a likely result of these substitutions, reduction of IsdC-N1 causes release of heme from the protein [44,45]. These residues are situated in the axial heme plane such that a favorable heme–iron coordinating side chain could coordinate heme–iron. Therefore, the amino acid variations could reflect the individual domains’ roles in the heme transfer chain or the differences in interacting partners or the variation could simply be due to little selective pressure at that position. 3.3. Interaction of NEAT domains with host heme proteins Some NEAT domains have roles other than, or in addition to, heme binding. Isd-host protein interaction has been observed by several methods, including enzyme-linked immunosorbent assays, surface plasmon resonance, NMR and targeted alanine mutagenesis. Three of the outermost proteins in the Isd system, IsdA, IsdB and IsdH, bind host heme proteins [12,14,22,38,40,47,48]. IsdH has two NEAT domains which can bind Hb or haptoglobin (Hp) with low nM dissociation constants but not heme (IsdH-N1, IsdH-N2) [47,48] and one that binds heme alone with a low lM
Fig. 3. 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 b-strands (b1-8) and one well defined a-helix (a1). 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. IsdA-N1 (PDB ID: 2itf) (cyan), IsdC-N1 (PDB ID: 2o6p) (magenta) and IsdH-N3 (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).
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Fig. 4. 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. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Sequence alignments of S. aureus NEAT Domains. Structures of IsdA-N1, IsdC-N1, IsdH-N1 and IsdH-N3 were superposed to generate a preliminary primary structure alignment using the CE algorithm [87] in the program STRAP [88]. All remaining NEAT domains were aligned against this subset. Alignments were visualized with Jalview [89]. Residues are numbered according to full-length IsdA (NCBI ID: NP_374247).
dissociation constant (IsdH-N3) [39,47,48]. Gel filtration and analytical ultracentrifugation were used to determine that metHbIsdH-N1 forms a 2:1 complex of IsdH-N1:metHb with an apparent molecular mass of 66 kDa [38]. Interestingly, alanine mutagenesis data suggests interaction with metHb occurs on the same face
of IsdH-N1 as heme binding in other NEAT domains, namely involving the b1–b2 loop and the b7–b8 loop [48]. IsdB has been shown to interact with Hb and Hp–Hb with low nM dissociation constants, but not with Hp [47]. The specific regions of IsdB responsible for protein or heme binding have not
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been reported, but homology to IsdH suggests IsdB-N1 (46% identity to IsdH-N1, 65% identity to IsdH-N2) binds Hb and IsdB-N2 (56% identity to IsdH-N3) binds heme. Despite these recent advances, the mechanism of heme extraction and transfer by IsdB and IsdH remains unknown. Rates of heme extraction from metHb by the combination of IsdH-N1 and IsdH-N3 (kobs 10.9 h 1) [48] are very close to the off rates of heme from metHb (7.8 h 1 for bsubunit and 0.6 h 1 for the a-subunit) [49] yet the rate of heme transfer from metHb to full-length IsdB (0.31 s 1) [40] is much more rapid (150-fold). Heme transfer must therefore be driven by an activated complex which likely requires either regions of the protein outside the NEAT domains, a combination of IsdH-N1, IsdH-N2 and IsdH-N3 not tested or that the tethering of the domains to one another is essential for efficient transfer. 3.4. Current model of heme transfer by the cell surface Isd system A model was put forth that suggested the locations of Isd proteins in the cell wall and membrane define the relay mechanism of heme import. Recent in vitro evidence demonstrated heme transfer rates between purified Isd surface proteins that support the model of heme flow based on localization [40,41,50]. Heme is transferred from metHb to IsdB (kobs 0.31 s 1) or IsdH. Muryoi et al. demonstrated that heme moves bidirectionally between IsdB-N2 and IsdH-N3 [41]. From IsdB (or IsdH), heme transfer is rapid to apo IsdA (114 s 1) or apo-IsdC (15 s 1). Heme bound to IsdA can then move to IsdC (53 s 1). Finally, Zhu et al. demonstrated only IsdC efficiently transfers heme to IsdE (0.0062 s 1) [40]. Alternatively, Muryoi et al. demonstrated that heme transfer to IsdE may occur directly from IsdB-N2 or IsdH-N3 [41]. Additional experiments are needed to determine the biological relevance of these transfer experiments, since protein localization on the cell wall likely affects transfer rates. A current model of heme transfer based on kinetic transfer experiments and cellular localization is illustrated by arrows in Fig. 1. Each proposed heme transfer step is rapid and occurs between full-length recombinant proteins (excluding the secretion signal and the residues following the sortase anchoring motif) [40,50] and between recombinant NEAT domains alone [41]. Since heme transfer rates between NEAT domain-containing Isd proteins are at least 10,000 times greater than the rate of heme dissociation, transfer is likely mediated by activated complex formation [40,41,50]. Despite an estimated KD based on heme transfer kinetics of 17 lM for the holo-IsdA apo-IsdC complex [40,50], Isd protein–protein complexes have not yet been physically observed or characterized on a molecular level. 4. The Isd membrane transporter Heme is transferred from the cell wall anchored components of the system through IsdC to IsdE [40,41]. Typical ABC transporters for the import of nutrients in bacteria are comprised of three main components: a substrate-binding protein (IsdE), a membrane permease (IsdF) and an ATP hydrolase [51]. Since Gram-positive bacteria lack an outer membrane, IsdE is anchored to the external side of the membrane through a lipid moiety covalently attached to a cysteine residue at the N-terminus of the mature protein [52]. IsdE assumes an overall fold comprising N- and C-terminal lobes which consist of central b-stands and peripheral helices and loops [18]. The two domains are bridged by a long a-helix, which appears to be an emerging characteristic of metal transport binding proteins (Fig. 6) [53,54]. Heme is bound to IsdE in a large groove formed between the N- and C-terminal domains with residues from both lobes providing several substrate contacts. Unlike the orientation of heme in the NEAT domains, the heme propionate
Fig. 6. Model of the heme ABC transporter of the Isd system composed of IsdEF and FhuC. The X-ray 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 a-helix (light orange), the propionate-stabilizing a-helix (yellow) and the heme–iron coordinating side chains (cyan) are shown.
groups are directed into the binding pocket and are essentially buried. Electrostatic interactions with the propionate groups stabilize the buried charge, including hydrogen bonds to the main chain amides of Val41 and Ala42 to one propionate, and the alignment of the N-terminus of a-helix 1 such that the positive helix dipole stabilizes the charge of the other propionate (Fig. 6). Heme–iron is six-coordinate with an axial ligand from each of the domains. In line with magnetic circular dichroism spectra that demonstrate His–Met coordination, the crystal structure of IsdE revealed that Met78 and His229 coordinate the heme–iron [18,43,55] (Fig. 6). IsdF is suggested to form the permease, a homodimeric integral membrane protein [11]. The final component needed to drive heme uptake is an ATP hydrolase. However, the S. aureus Isd system lacks an obvious ATPase encoded in the isd locus; in fact, several iron-compound ABC transporters from S. aureus lack a corresponding ATPase, including the siderophore transporters HtsABC and SirABC [56,57]. In the Hts and Sir systems, FhuC provides the ATPase function, and evidence suggests FhuC serves as the ATPase for the Isd system as well [56,58].
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A BLAST search of the RCSB Protein Data Bank with the IsdF sequence revealed promising modeling templates for IsdF. The permease from 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 transporter was generated with the program ESyPred3D [59] using the crystal structure of the BtuCD complex (PDB ID: 2qi9) [60] as a template. To dock IsdE onto the IsdF–FhuC model, the transporter was superposed onto the substrate-binding protein, BtuF, from the structure of BtuCD-F [60]. IsdE and BtuF share a sequence identity of 18% and overlay with an r.m.s.d of 2.6 Å over 232 Ca atoms. The components of metal uptake ABC transporters typically display conserved structure even in the absence of high sequence identities; therefore, when structural evidence is combined with the sequence alignments, the docking model is likely a useful prediction of interaction. The resulting model is a complete IsdF–FhuC–IsdE complex (Fig. 6). The IsdF model based on BtuC is predicted to have nine membrane spanning helices per monomer, missing the first helix. 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 are present on the extracellular side of the IsdF model that potentially interact with Glu83 (equivalent to Glu74 in BtuF) and Glu214 (equivalent to Glu202 in BtuF) from IsdE. These electrostatic interactions likely aid in the recognition of IsdE by IsdF and induce heme transport. The presence of similar interactions as seen in the BtuCF system supports the validity of the IsdEF model; however, these predictions await experimental confirmation. 5. Isd heme-degrading enzymes Following heme transport through the permease, heme enters the bacterial cytoplasm where it has two potential fates. It may be degraded to release iron as a nutrient for growth or it may be
sorted as an intact heme moiety to be used directly in bacterial hemoproteins [9]. IsdG and IsdI have been identified as the heme-degrading components of the Isd system in S. aureus [61– 63]. These enzymes are atypical heme-degrading enzymes, as their protein sequences and structural folds are distinct from the wellcharacterized heme oxygenases, the first family of enzymes found to break down heme and identified in numerous bacteria and eukaryotes. IsdG and IsdI have been shown to bind heme, producing a Soret peak at 413 nm [62]. Biochemical assays in aerobic conditions show either enzyme can release iron from heme in vitro in the presence of an electron donor such as ascorbate, or NADPH and cytochrome P450 reductase [62–64]. However, a detailed investigation into the catalytic mechanism of IsdG and IsdI is lacking, as it is not yet known whether the reaction proceeds by oxygen activation. For instance, in heme oxygenases the oxidative cleavage of the heme porphyrin ring yields biliverdin, thereby releasing iron [65]. IsdG and IsdI, along with their homologues from Bacillus anthracis [66] and Bradyrhizobium japonicum [67], have also been suggested to produce biliverdin from heme [62]. However, electronic spectra of the product are not consistent with biliverdin and product analysis by direct structural methods is not yet complete. Although the structures of native IsdG–heme and IsdI–heme are not yet available, the apo structures of IsdG and IsdI have been solved [63]. More recently, structures of IsdI–CoPPIX, a substrate analogue of heme, and of a mutational variant of IsdG–heme provided the first insights into IsdG and IsdI–heme binding; the use of a substrate analogue or a catalytically inactive variant hinders heme degradation by the enzyme and thus allows for structural analysis with heme in the binding pocket [64]. IsdG and IsdI adopt a ferredoxin-like a + b-sandwich fold and exist as homodimers which form a b-barrel at the dimer interface (Fig. 7A) [63]. This is in contrast to the monomeric and predominantly a-helical fold of canonical heme oxygenases [65]. IsdG and IsdI also lack the distal hydrogen bonding solvent network essential for heme oxygenase activity [68]. Instead, each IsdG and IsdI dimer contains two
Fig. 7. Structures of the holo-forms of the IsdG–N7A variant and native IsdI [64]. (A) IsdI–CoPPIX structure (PDB ID: 2zdp) showing the dimeric fold (green/cyan). (B) Surface representation of IsdI–CoPPIX revealing the deep binding clefts. (C) The substrate-binding pocket in the structure of IsdG–N7A–heme (PDB ID: 2zdo) (yellow) and (D) IsdI– CoPPIX (cyan). The conserved binding residues of IsdG and IsdI are shown for comparison, with the exception of Asn7 of IsdG, which has been mutated to Ala. Metalloporphyrins (gray) are distorted from planarity and take on a highly ruffled conformation.
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deep hydrophobic clefts where the heme moieties are bound, limiting access by water molecules (Fig. 7B). Interestingly, the residues that interact with the heme are conserved between the paralogous IsdG and IsdI proteins, which share 64% sequence identity [64]. Biochemical analysis identified multiple residues important for activity of IsdG, including Asn7 and Trp67.[63]. The structure of the IsdG–N7A variant bound to heme [64] revealed a five-coordinate heme iron that is liganded to an axial histidine residue (His77, Fig. 7C). IsdG and IsdI enzyme activity is also substantially hindered by reconstituting the enzymes with analogues of heme containing metals other than Fe3+ [64]. The metalloporphyrins that have been tested include Co, Ga, Mn and Zn-containing PPIX. The structure of IsdI–CoPPIX shows the Co3+ is six-coordinate with a distal Cl ligand in addition to the axial His76 ligand (Fig. 7D). The coordinating Cl is stabilized by a H-bond with Asn6, and the Cd1 atom of Ile53 is pointing away from the iron, in contrast to the equivalent residue (Ile54) in the IsdG–N7A structure. Insights into the natural distal ligand of IsdG and IsdI may be gained by determining the structure of the heme-bound form of the native enzymes. Perhaps the most interesting feature of these structures is the extreme conformational distortion of the porphyrin ring that occurs upon binding by IsdG and IsdI (Fig. 7C and D). The porphyrin ring is distorted from planarity by extensive steric interactions with hydrophobic residues in the binding cleft, particularly by Trp67, Val80, Asn7, and Phe23 in IsdG (equivalent residues in IsdI are Trp66, Val79, Asn6, and Phe22) [64]. This distortion is described as ruffled; ruffling to this extent has not been seen in the known structures of other hemoproteins. How the unusually high degree of ruffling contributes to IsdG and IsdI enzyme catalysis is unclear.
6. Related Isd systems Heme-uptake systems homologous to the Isd system in S. aureus have been identified and characterized to different extents in several Gram-positive organisms, including Bacillus anthracis, Listeria monocytogenes and Streptococcus pyogenes [69]. These organisms possess NEAT domain-containing proteins, heme ABC transporters and heme-degrading enzymes [69–79]. The systems have generally been shown to contribute to virulence [71,74– 76,80–82]. The many details of binding and pathogenesis in these organisms are beyond the scope of this review so the reader is referred to the primary literature referred to above. Notable instances of variability and similarity between the S. aureus Isd system and homologous heme-uptake systems are found. For instance, all other Isd systems encode an ATPase for the transporter within the operon. Furthermore, recent data revealed that IsdX1 (one NEAT domain; also known as IsdJ) and IsdX2 (five NEAT domains; also known as IsdK) from B. anthracis are secreted instead of being anchored to the cell wall [71,76]. Similarities to the S. aureus Isd system are also found, often encompassing intriguing dissimilarities as well. Recently, the structure of a cell surface receptor, Shp, from S. pyogenes was determined. Shp itself was not originally annotated as a NEAT domain-containing protein, but the 2.1 Å resolution crystal structure of the heme binding domain (or Shp180) revealed the immunoglobulin-like b-sandwich fold characteristic of NEAT domains; Shp180 overlays with S. aureus IsdC and IsdA over all Ca atoms with an r.m.s.d. of 2.0 and 2.2 Å, respectively [83]. However, unlike the other published NEAT domain structures, heme–iron is coordinated in Shp180 by two Met residues (Met153 and Met66) in the binding pocket [83]. Shp transfers its heme to HtsA, the substrate-binding lipoprotein component of the heme-specific ABC
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transporter, through a proposed activated complex which requires both Met153 and Met66 to be intact for full efficiency [84–86]. It is worth noting that HtsA bears 40% sequence identity to IsdE in S. aureus, including conservation of the heme–iron coordinating residues [18], and appears to share a similarity in function as well [84]. 7. Summary The Isd system of S. aureus describes a simple yet elegant way for the opportunistic human pathogen to fulfill its iron requirement. Numerous advances have recently been made in characterizing this pathway and have provided us with an excellent working model for understanding heme uptake during infection. Hemolysins are secreted, releasing intracellular Hb stores and producing Hb–Hp complexes, which can be exploited using IsdB and IsdH. Through an efficient relay system involving the formation of putative activated complexes, heme is extracted from the host hemoproteins and transferred through IsdA to IsdC, where it is delivered to the bi-lobed substrate-binding protein IsdE which docks onto the membrane permease IsdF. Internalization of the heme is likely driven by the ATPase FhuC. Inside the cell the porphyrin ring is degraded by IsdG and IsdI which have a ferredoxin-like a + b-sandwich fold and contort the ring into a highly ruffled conformation; iron is subsequently liberated. The NEAT domain has been demonstrated to be an essential, functional component of the surface proteins, and through its conserved eight-stranded b-sandwich fold, binds host hemoproteins or heme using a hydrophobic pocket containing a conserved Tyr to coordinate the heme–iron. However, despite the considerable work that has been accomplished on the system, several major puzzles have yet to be solved. The mechanism of heme extraction from host hemoproteins and heme transfer between Isd surface proteins is unclear. The product and mode of action of the Isd heme-degrading enzymes, IsdG and IsdI, has yet to be adequately described. Lacking structural homology to the canonical heme monooxygenases as well as the solvent network required for activity of these enzymes, the heme degradation mechanism by IsdG and IsdI may be novel. Lastly, homologues of IsdD have not been found. Its function remains a mystery. 8. Abbreviations PPIX CoPPIX Hp Isd NEAT
protoporphyrin IX cobalt protoporphyrin IX haptoglobin iron-regulated surface determinant near iron-transporter
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