NEAr Transporter Research Articles

The Shr receptor from Streptococcus pyogenes uses a cap and release mechanism to acquire heme–iron from human hemoglobin

Streptococcus pyogenes (group A Streptococcus) is a clinically important microbial pathogen that requires iron in order to proliferate. During infections, S. pyogenes uses the surface displayed Shr receptor to capture human hemoglobin (Hb) and acquires its iron-laden heme molecules. Through a poorly understood mechanism, Shr engages Hb via two structurally unique N-terminal Hb-interacting domains (HID1 and HID2) which facilitate heme transfer to proximal NEAr Transporter (NEAT) domains. Based on the results of X-ray crystallography, small angle X-ray scattering, NMR spectroscopy, native mass spectrometry, and heme transfer experiments, we propose that Shr utilizes a "cap and release" mechanism to gather heme from Hb. In the mechanism, Shr uses the HID1 and HID2 modules to preferentially recognize only heme-loaded forms of Hb by contacting the edges of its protoporphyrin rings. Heme transfer is enabled by significant receptor dynamics within the Shr-Hb complex which function to transiently uncap HID1 from the heme bound to Hb's β subunit, enabling the gated release of its relatively weakly bound heme molecule and subsequent capture by Shr's NEAT domains. These dynamics may maximize the efficiency of heme scavenging by S. pyogenes, enabling it to preferentially recognize and remove heme from only heme-loaded forms of Hb that contain iron.

NEAr Transporter (NEAT) Domains: Unique Surface Displayed Heme Chaperones That Enable Gram-Positive Bacteria to Capture Heme-Iron From Hemoglobin.

Iron is an important micronutrient that is required by bacteria to proliferate and to cause disease. Many bacterial pathogens forage iron from human hemoglobin (Hb) during infections, which contains this metal within heme (iron–protoporphyrin IX). Several clinically important pathogenic species within the Firmicutes phylum scavenge heme using surface-displayed or secreted NEAr Transporter (NEAT) domains. In this review, we discuss how these versatile proteins function in the Staphylococcus aureus Iron-regulated surface determinant system that scavenges heme-iron from Hb. S. aureus NEAT domains function as either Hb receptors or as heme-binding chaperones. In vitro studies have shown that heme-binding NEAT domains can rapidly exchange heme amongst one another via transiently forming transfer complexes, leading to the interesting hypothesis that they may form a protein-wire within the peptidoglycan layer through which heme flows from the microbial surface to the membrane. In Hb receptors, recent studies have revealed how dedicated heme- and Hb-binding NEAT domains function synergistically to extract Hb’s heme molecules, and how receptor binding to the Hb-haptoglobin complex may block its clearance by macrophages, prolonging microbial access to Hb’s iron. The functions of NEAT domains in other Gram-positive bacteria are also reviewed.

Nucleotide Polymorphism in the Iron Utilization System Gene isdB NEAT Domain Affects Heme-Binding Ability of IsdB Protein in Various Human Strains of Staphylococcus aureus

Genetic heterogeneity of the isdB gene fragment encoding one of two heme-binding NEAr Transporter (NEAT) domains pfam05031 in different isolates of Staphylococcus aureus from the healthy carriers and the risk group patients (chronic inflammatory infections of the upper respiratory tract and middle ear) was investigated. All S. aureus strains included in the current study differed phenotypically from one another by degree of affinity to polystyrene with immobilized human hemoglobin. The isolates that displayed low affinity to hemoglobin were found to have more mutations within the isdB gene fragment which encodes one of the IsdB protein domains containing heme-binding sites.

Insight into a Rapid Heme Transfer Reaction between Near Transporter Domains of Staphylococcus Aureus: A Theoretical Study using QM/MM and MD Simulations

Iron is versatile in biological systems and required cofactor in the growth and activity of all living organisms. Some gram-positive pathogens such as Staphylococcus aureus have evolved an iron-regulated surface determinant (Isd) system across its thick cell wall to steal iron from human hemoglobin and heme. Recent studies have demonstrated that heme extracted from hemoglobin is rapidly transferred into cytoplasm through NEAr Transporter (NEAT) domain of the Isd system, despite the high affinity for each domain. These observations suggest the presence of an intermediate NEAT•heme•NEAT complex. In this study, we performed restrained molecular dynamics (MD) simulations to dock the acceptor NEAT domains to the donor NEAT•heme complexes and obtained models where the two NEAT domains were arranged with two-fold pseudo symmetry around the heme molecule. The overall structures of the complex models were stably maintained during subsequent more than 900-ns unrestrained simulations. Although the hydrogen bond between a propionate group of heme and the acceptor domain was completely maintained, the hydrogen bond between the other propionate group of heme and the donor domain was broken, which may facilitate the transition of heme from the donor to the acceptor. Moreover, subsequent structural optimization using the QM/MM method showed that the two tyrosine residues, one from each NEAT domain, were simultaneously coordinated to the ferric heme iron in the intermediate complex, only if they are deprotonated. Therefore, the heme-transfer reaction proceeds by protonation on the coordinating tyrosine residue. Thus, these theoretical studies provide an atomistic insight into the intermediate structure allowing rapid heme transfer between the NEAT domains.

Rapid Heme Transfer Reactions between NEAr Transporter Domains of Staphylococcus aureus: A Theoretical Study Using QM/MM and MD Simulations.

In vertebrates, most iron is present as heme or is chelated by proteins. Thus, Gram-positive pathogens such as Staphylococcus aureus have evolved an iron-regulated surface determinant (Isd) system that transports heme across thick cell walls into the cytoplasm. Recent studies have demonstrated that heme is rapidly transferred between the NEAr Transporter (NEAT) domains of the Isd system, despite its high affinity toward each domain, suggesting the presence of an intermediate NEAT•heme•NEAT complex. In the present study, we performed short restrained molecular dynamics (MD) simulations to dock the acceptor NEAT domain to the donor NEAT•heme complex and obtained models where the two NEAT domains were arranged with two-fold pseudo symmetry around the heme molecule. After turning off the restraints, complex structures were stably maintained during subsequent unrestrained MD simulations, except for the hydrogen bond between the propionate group of the heme molecule and the donor NEAT domain, potentially facilitating the transition of heme from the donor to the acceptor. Subsequent structural optimization using the quantum mechanics/molecular mechanics (QM/MM) method showed that two tyrosine residues, one from each NEAT domain, were simultaneously coordinated to the ferric heme iron in the intermediate complex only if they were deprotonated. Based on these results, we propose a reaction scheme for heme transfer between NEAT domains.

Novel Mechanism of Hemin Capture by Hbp2, the Hemoglobin-binding Hemophore from Listeria monocytogenes

Iron is an essential nutrient that is required for the growth of the bacterial pathogen Listeria monocytogenes. In cell cultures, this microbe secretes hemin/hemoglobin-binding protein 2 (Hbp2; Lmo2185) protein, which has been proposed to function as a hemophore that scavenges heme from the environment. Based on its primary sequence, Hbp2 contains three NEAr transporter (NEAT) domains of unknown function. Here we show that each of these domains mediates high affinity binding to ferric heme (hemin) and that its N- and C-terminal domains interact with hemoglobin (Hb). The results of hemin transfer experiments are consistent with Hbp2 functioning as an Hb-binding hemophore that delivers hemin to other Hbp2 proteins that are attached to the cell wall. Surprisingly, our work reveals that the central NEAT domain in Hbp2 binds hemin even though its primary sequence lacks a highly conserved YXXXY motif that is used by all other previously characterized NEAT domains to coordinate iron in the hemin molecule. To elucidate the mechanism of hemin binding by Hbp2, we determined crystal structures of its central NEAT domain (Hbp2(N2); residues 183-303) in its free and hemin-bound states. The structures reveal an unprecedented mechanism of hemin binding in which Hbp2(N2) undergoes a major conformational rearrangement that facilitates metal coordination by a non-canonical tyrosine residue. These studies highlight previously unrecognized plasticity in the hemin binding mechanism of NEAT domains and provide insight into how L. monocytogenes captures heme iron.

Solution structure and molecular determinants of hemoglobin binding of the first NEAT domain of IsdB in Staphylococcus aureus.

The human pathogen Staphylococcus aureus acquires heme iron from hemoglobin (Hb) via the action of a series of iron-regulated surface determinant (Isd) proteins. The cell wall anchored IsdB protein is recognized as the predominant Hb receptor, and is comprised of two NEAr transporter (NEAT) domains that act in concert to bind, extract, and transfer heme from Hb to downstream Isd proteins. Structural details of the NEAT 2 domain of IsdB have been investigated, but the molecular coordination between NEAT 2 and NEAT 1 to extract heme from hemoglobin has yet to be characterized. To obtain a more complete understanding of IsdB structure and function, we have solved the 3D solution structure of the NEAT 1 domain of IsdB (IsdBN1) spanning residues 125–272 of the full-length protein by NMR. The structure reveals a canonical NEAT domain fold and has particular structural similarity to the NEAT 1 and NEAT 2 domains of IsdH, which also interact with Hb. IsdBN1 is also comprised of a short N-terminal helix, which has not been previously observed in other NEAT domain structures. Interestingly, the Hb binding region (loop 2 of IsdBN1) is disordered in solution. Analysis of Hb binding demonstrates that IsdBN1 can bind metHb weakly and the affinity of this interaction is further increased by the presence of IsdB linker domain. IsdBN1 loop 2 variants reveal that phenylalanine 164 (F164) of IsdB is necessary for Hb binding and rapid heme transfer from metHb to IsdB. Together, these findings provide a structural role for IsdBN1 in enhancing the rate of extraction of metHb heme by the IsdB NEAT 2 domain.

Heme-Transfer Mechanism of Structurally Similar Isd NEAT Domains of Staphylococcus Aureus Exhibiting Different Affinities for Heme

Near Transporter (NEAT) domains of the iron-regulated surface determinant (Isd) proteins play an important role in importing heme from host animals to Staphylococcus aureus. IsdH, IsdA, and IsdC are anchored to the cell wall and display one or more copies of NEAT domain. The order of transfer of heme between the NEAT domains is as follows: IsdH-NEAT3 → IsdA-NEAT → IsdC-NEAT, though the three-dimensional structures are quite similar. In this study, we measured the difference of the affinities for heme between these NEAT domains and found that the affinity gradually increased in the downstream receptors. To gain insight into the atomistic mechanism for the difference, we performed in silico MD simulation and in vitro site-directed mutagenesis. The simulations showed that a cluster of negatively charged residues in the first loop between strand β1b and the 310 helix (loop 1 region) adversely influences the interaction with the propionate group of heme. Meanwhile, the higher affinity of IsdC was partly attributed to an internal salt-bridge formed between Glu88/Arg100 on binding to heme. On the other hand, we also observed that Phe130 of IsdC makes the β7-β8 hairpin region less flexible in the ligand-free form, which leads to reducing the entropy loss on binding to heme. Based on these calculations, we confirmed that substitution of these key residues decreased its affinity for heme. Importantly, these affinity-decreased IsdC mutants did transfer heme back to IsdA. Moreover, substitution of the loop 1 region in IsdH-NEAT3 for IsdC-type residues reversibly increased its affinity for heme and did not transfer to IsdA. Thus, NEAT domains have evolved the characteristic residues on the common structural scaffold so that they exhibit different affinities for heme and promote the efficient transfer of heme.

Heme Binding Mechanism of Structurally Similar Iron-Regulated Surface Determinant Near Transporter Domains ofStaphylococcus aureusExhibiting Different Affinities for Heme

Near transporter (NEAT) domains of the iron-regulated surface determinant (Isd) proteins are essential for the import of nutritional heme from host animals to Gram-positive pathogens such as Staphylococcus aureus. The order of transfer of heme between NEAT domains occurs from IsdH to IsdA to IsdC, without any energy input despite the similarity of their three-dimensional structures. We measured the free energy of binding of heme and various metalloporphyrins to each NEAT domain and found that the affinity of heme and non-iron porphyrins for NEAT domains increased gradually in the same order as that for heme transfer. To gain insight into the atomistic mechanism for the differential affinities, we performed in silico molecular dynamics simulation and in vitro site-directed mutagenesis. The simulations revealed that the negatively charged residues that are abundant in the loop between strand β1b and the 310 helix of IsdH-NEAT3 destabilize the interaction with the propionate group of heme. The higher affinity of IsdC was in part attributed to the formation of a salt bridge between its unique residue, Glu88, and the conserved Arg100 upon binding to heme. In addition, we found that Phe130 of IsdC makes the β7-β8 hairpin less flexible in the ligand-free form, which serves to reduce the magnitude of the entropy loss on binding to heme. We confirmed that substitution of these key residues of IsdC decreased its affinity for heme. Furthermore, IsdC mutants, whose affinities for heme were lower than those of IsdA, transferred heme back to IsdA. Thus, NEAT domains have evolved the characteristic residues on the common structural scaffold such that they exhibit different affinities for heme, thus promoting the efficient transfer of heme.

1H, 13C, 15N backbone and side chain NMR resonance assignments of the N-terminal NEAr iron transporter domain 1 (NEAT 1) of the hemoglobin receptor IsdB of Staphylococcus aureus

Staphylococcus aureus is an opportunistic pathogen that causes skin and severe infections in mammals. Critical to S. aureus growth is its ability to scavenge iron from host cells. To this effect, S. aureus has evolved a sophisticated pathway to acquire heme from hemoglobin (Hb) as a preferred iron source. The pathway is comprised of nine iron-regulated surface determinant (Isd) proteins involved in heme capture, transport, and degradation. A key protein of the heme acquisition pathway is the surface-anchored hemoglobin receptor protein IsdB, which is comprised of two NEAr transporter (NEAT) domains that act in concert to bind Hb and extract heme for subsequent transfer to downstream acquisition pathway proteins. Despite significant advances in the structural knowledge of other Isd proteins, the structural mechanisms and molecular basis of the IsdB-mediated heme acquisition process are not well understood. In order to provide more insights into the mode of function of IsdB, we have initiated NMR structural studies of the first NEAT domain of IsdB (IsdB(N1)). Herein, we report the near complete (1)H, (13)C and (15)N resonance assignments of backbone and side chain atoms, and the secondary structural topology of the 148-residue IsdB NEAT 1 domain. The NMR results are consistent with the presence of eight β-strands and one α-helix characteristic of an immunoglobulin-like fold observed in other NEAT domain family proteins. This work provides a solid framework to obtain atomic-level insights toward understanding how IsdB mediates IsdB-Hb protein-protein interactions critical for heme capture and transfer.

Heme binding to the IsdE(M78A; H229A) double mutant: challenging unidirectional heme transfer in the iron-regulated surface determinant protein heme transfer pathway of Staphylococcus aureus

The pathogenic bacterium Staphylococcus aureus has adopted specialized mechanisms for scavenging iron from its host. The cell-wall- and cell-membrane-associated iron-regulated surface determinant (Isd) proteins (IsdH, IsdB, IsdA, IsdC, IsdDEF, IsdG, and IsdI) allow S. aureus to scavenge iron from the heme in hemoglobin and haptoglobin-hemoglobin. Of these, IsdE chaperones heme to the ATP-binding-cassette-type transmembrane transporter (IsdF). IsdH, IsdB, IsdA, and IsdC contain at least one heme-binding near transporter (NEAT) domain. Previous studies have shown that ferric heme is transferred unidirectionally in the sequence IsdA-NEAT (Tyr-proximal amino acid)→IsdC-NEAT (Tyr)→IsdE (His). IsdA-NEAT does not transfer heme directly to IsdE. To challenge and probe this unusual unidirectional mechanism, the double mutant IsdE(M78A; H229A)-IsdE(MH)-was constructed and used in studies of heme transfer between IsdA-NEAT, IsdC-NEAT, and IsdE. This study probed the specific requirements in the heme binding site that enforce the unidirectional property of the system. Significantly, heme transfer from holo-IsdE(MH) to apo-IsdA-NEAT now occurs, breaking the established mechanism. The unique unidirectional heme-transfer properties now function under an affinity-driven mechanism. Overall, the heme proximal and distal ligands must play a crucial role controlling a gate that stops heme transfer between the native IsdE and IsdA-NEAT. We propose that these amino acids are the key control elements in the specific unidirectional protein-protein-gated release mechanism exhibited by the Isd system.

Mapping Ultra-weak Protein-Protein Interactions between Heme Transporters of Staphylococcus aureus

Iron is an essential nutrient for the proliferation of Staphylococcus aureus during bacterial infections. The iron-regulated surface determinant (Isd) system of S. aureus transports and metabolizes iron porphyrin (heme) captured from the host organism. Transportation of heme across the thick cell wall of this bacterium requires multiple relay points. The mechanism by which heme is physically transferred between Isd transporters is largely unknown because of the transient nature of the interactions involved. Herein, we show that the IsdC transporter not only passes heme ligand to another class of Isd transporter, as previously known, but can also perform self-transfer reactions. IsdA shows a similar ability. A genetically encoded photoreactive probe was used to survey the regions of IsdC involved in self-dimerization. We propose an updated model that explicitly considers self-transfer reactions to explain heme delivery across the cell wall. An analogous photo-cross-linking strategy was employed to map transient interactions between IsdC and IsdE transporters. These experiments identified a key structural element involved in the rapid and specific transfer of heme from IsdC to IsdE. The resulting structural model was validated with a chimeric version of the homologous transporter IsdA. Overall, our results show that the ultra-weak interactions between Isd transporters are governed by bona fide protein structural motifs.

Insight into blocking heme transfer by exploiting molecular interactions in the core Isd heme transporters IsdA-NEAT, IsdC-NEAT, and IsdE of Staphylococcus aureus

The pathogenic bacterium Staphylococcus aureus has adopted specialized mechanisms for scavenging iron from its host. The nine cell wall and membrane-associated iron regulated surface determinant (Isd) proteins (IsdH, IsdB, IsdA, IsdC, IsdDEF, IsdG and IsdI) allow Staphylococcus aureus to scavenge iron from the heme in hemoglobin and haptoglobin-hemoglobin. Of these, it is IsdE that chaperones the heme to the ATP binding cassette-type transmembrane transporter (IsdF). IsdH, IsdB, IsdA and IsdC contain at least one heme binding Near Transporter (NEAT) domain. Previous studies have shown that ferric heme is transferred unidirectionally in the sequence IsdA-NEAT (Tyr - proximal amino acid) → IsdC-NEAT (Tyr) → IsdE (His). IsdA-NEAT does not transfer heme directly to IsdE. In this paper we investigated PPIX transfer through the core cell wall proteins of the Isd system (IsdA-NEAT, IsdC-NEAT and IsdE) with FePPIX-dimethylester, and the metal substituted CoPPIX and MnPPIX using ESI-MS, UV-visible absorption and MCD spectroscopy. IsdA binds each of the rings but the subsequent transfer properties to IsdC-N or IsdE are not the same as found with heme. FePPIX-DME transfers from IsdA-N to IsdC-N but neither protein transfers the ring to IsdE. IsdA-N does not transfer CoPPIX to IsdC-N or IsdE. IsdA-N does transfer MnPPIX to both IsdC-N and IsdE. Significantly, it is possible that since CoPPIX and FePPIX-DME bind to IsdA-N, the lack of transfer to IsdC-N and subsequently to IsdE for CoPPIX could prove to be used as a potential disruption agent to the S. aureus heme transfer system and may identify a possible anti-microbial.

Transient Weak Protein–Protein Complexes Transfer Heme Across the Cell Wall of Staphylococcus aureus

Iron is an essential nutrient for the bacterial pathogen Staphylococcus aureus . Heme in hemoglobin (Hb) is the most abundant source of iron in the human body and during infections is captured by S. aureus using iron-regulated surface determinant (Isd) proteins. A central step in this process is the transfer of heme between the cell wall associated IsdA and IsdC hemoproteins. Biochemical evidence indicates that heme is transferred via an activated IsdA:heme:IsdC heme complex. Transfer is rapid and occurs up to 70,000 times faster than indirect mechanisms in which heme is released into the solvent. To gain insight into the mechanism of transfer, we modeled the structure of the complex using NMR paramagnetic relaxation enhancement (PRE) methods. Our results indicate that IsdA and IsdC transfer heme via an ultraweak affinity "handclasp" complex that juxtaposes their respective 3(10) helices and β7/β8 loops. Interestingly, PRE also identified a set of transient complexes that could represent high-energy pre-equilibrium encounter species that form prior to the stereospecific handclasp complex. Targeted amino acid mutagenesis and stopped-flow measurements substantiate the functional relevance of a PRE-derived model, as mutation of interfacial side chains significantly slows the rate of transfer. IsdA and IsdC bind heme using NEAr Transporter (NEAT) domains that are conserved in many species of pathogenic Gram-positive bacteria. Heme transfer in these microbes may also occur through structurally similar transient stereospecific complexes.

Characterization of the haem-uptake system of the equine pathogen Streptococcus equi subsp. equi

Streptococcus equi possesses a haem-uptake system homologous to that of Streptococcus pyogenes and Streptococcus zooepidemicus. The system consists of two ligand-binding proteins (Shr and Shp) and proteins (HtsA-C) with homology to an ABC transporter. The haem-uptake system of S. equi differs from that of S. pyogenes and S. zooepidemicus in that Shr is truncated by two-thirds. This study focused on the SeShr, SeShp and SeHtsA proteins of S. equi. Analysis of shr, shp and shphtsA knockout mutants showed that all three proteins were expressed in vitro and that expression was upregulated under conditions of iron limitation. SeShr possesses no membrane-/cell wall-spanning sequences and was shown to be secreted. Both SeShp and SeHtsA were confirmed to be envelope-associated. Recombinant SeShp and SeHtsA proteins have been previously shown to bind haem and SeHtsA could capture haem from SeShp. This report extends these studies and shows that both SeShp and SeHtsA can sequester haem from haemoglobin but not from haemoglobin-haptoglobin complexes. Like full-length Shr, SeShr possesses haemoglobin and haemoglobin-haptoglobin binding ability but unlike full-length Shr, it lacks haem- or fibronectin-binding capabilities. Analysis of SeShr truncates showed that residues within and upstream of the near transporter (NEAT) domain are required for this ligand binding. Structural predictions suggest that truncation of NEAT1 in SeShr accounts for its impaired ability to bind haem. Haem and haemoglobin restored to almost normal the impaired growth rates of wild-type S. equi cultured under iron-limiting conditions. However, no difference in the growth rates of wild-type and mutants could be detected under the in vitro growth conditions tested.

Functionally Distinct NEAT (NEAr Transporter) Domains within the Staphylococcus aureus IsdH/HarA Protein Extract Heme from Methemoglobin

The pathogen Staphylococcus aureus uses iron-regulated surface determinant (Isd) proteins to scavenge the essential nutrient iron from host hemoproteins. The IsdH protein (also known as HarA) is a receptor for hemoglobin (Hb), haptoglobin (Hp), and the Hb-Hp complex. It contains three NEAT (NEAr Transporter) domains: IsdH(N1), IsdH(N2), and IsdH(N3). Here we show that they have different functions; IsdH(N1) binds Hb and Hp, whereas IsdH(N3) captures heme that is released from Hb. The staphylococcal IsdB protein also functions as an Hb receptor. Primary sequence homology to IsdH indicates that it will also employ functionally distinct NEAT domains to bind heme and Hb. We have used site-directed mutagenesis and surface plasmon resonance methods to localize the Hp and Hb binding surface on IsdH(N1). High affinity binding to these structurally unrelated proteins requires residues located within a conserved aromatic motif that is positioned at the end of the beta-barrel structure. Interestingly, this site is quite malleable, as other NEAT domains use it to bind heme. We also demonstrate that the IsdC NEAT domain can capture heme directly from Hb, suggesting that there are multiple pathways for heme transfer across the cell wall.

The IsdC Protein from Staphylococcus aureus Uses a Flexible Binding Pocket to Capture Heme

Staphylococcus aureus scavenges heme-iron from host hemoproteins using iron-regulated surface determinant (Isd) proteins. IsdC is the central conduit through which heme is passed across the cell wall and binds this molecule using a NEAr Transporter (NEAT) domain. NMR spectroscopy was used to determine the structure of IsdC in complex with a heme analog, zinc-substituted protoporphyrin IX (ZnPPIX). The backbone coordinates of the ensemble of conformers representing the structure exhibit a root mean square deviation to the mean structure of 0.53 +/- 0.11 angstroms. IsdC partially buries protoporphyrin within a large hydrophobic pocket that is located at the end of its beta-barrel structure. The central metal ion of the analog adopts a pentacoordinate geometry in which a highly conserved tyrosine residue serves as a proximal ligand. Consistent with the structure and its role in heme transfer across the cell wall, we show that IsdC weakly binds heme (K(D) = 0.34 +/- 0.12 microm) and that ZnPPIX rapidly dissociates from the protein at a rate of 126 +/- 30 s(-1). NMR studies of the apo-form of IsdC reveal that a 3(10) helix within the binding pocket undergoes a flexible to rigid transition as heme is captured. This structural plasticity may increase the efficiency of heme transfer across the cell wall by facilitating protein-protein interactions between apoIsdC and upstream hemoproteins.

Characterization of Bacillus anthracis iron‐regulated surface determinant (Isd) proteins containing NEAT domains

Three iron-regulated surface determinant (Isd) proteins, containing NEAr Transporter (NEAT) domains (GBAA4789-7), constitute part of an eight-member Bacillus anthracis operon. GBAA4789 (IsdC), previously characterized by others as a haem-binding protein, and two novel Isd proteins characterized in this study, GBAA4788 (IsdJ) and GBAA4787 (IsdK) proteins, can be translated from two alternative overlapping transcriptional units. The three NEAT-containing Isd proteins are shown to be expressed in vivo during B. anthracis infection. Expression in vitro is regulated by iron ions independent of the virulence plasmids pXO1 and pXO2, yet their presence affects the range of response to iron ion concentration. The expression of IsdC, J and K is strongly repressed under high CO(2) tension, conditions that are optimal for B. anthracis toxin and capsule expression, suggesting that these Isd proteins are elements of a B. anthracis'air-regulon'. Deletion mutants of isdC, isdK or the entire isdCJK locus are as virulent and pathogenic to guinea pigs as the fully virulent wild-type Vollum strain. The isdC-deleted mutant is defective in sequestration of haemin, consistent with previous biochemical observations, while the DeltaisdK mutant is defective in haemoglobin uptake. Studies with recombinant IsdK demonstrate specific binding to haemoglobin.

Direct Hemin Transfer from IsdA to IsdC in the Iron-regulated Surface Determinant (Isd) Heme Acquisition System of Staphylococcus aureus

The iron-regulated surface determinants (Isd) of Staphylococcus aureus, including surface proteins IsdA, IsdB, IsdC, and IsdH and ATP-binding cassette transporter IsdDEF, constitute the machinery for acquiring heme as a preferred iron source. Here we report hemin transfer from hemin-containing IsdA (holo-IsdA) to hemin-free IsdC (apo-IsdC). The reaction has an equilibrium constant of 10 +/- 5 at 22 degrees C in favor of holo-IsdC formation. During the reaction, holo-IsdA binds to apo-IsdC and then transfers the cofactor to apo-IsdC with a rate constant of 54.3 +/- 1.8 s(-1) at 25 degrees C. The transfer rate is >70,000 times greater than the rate of simple hemin dissociation from holo-IsdA into solvent (k transfer = 54.3 s(-1) versus k -hemin = 0.00076 s(-1)). The standard free energy change, Delta G 0, is -27 kJ/mol for the formation of the holo-IsdA-apo-IsdC complex. IsdC has a higher affinity for hemin than IsdA. These results indicate that the IsdA-to-IsdC hemin transfer is through the activated holo-IsdA-apo-IsdC complex and is driven by the higher affinity of apo-IsdC for the cofactor. These findings demonstrate for the first time in the Isd system that heme transfer is rapid, direct, and affinity-driven from IsdA to IsdC. These results also provide the first example of heme transfer from one surface protein to another surface protein in Gram-positive bacteria and, perhaps most importantly, indicate that the mechanism of activated heme transfer, which we previously demonstrated between the streptococcal proteins Shp and HtsA, may apply in general to all bacterial heme transport systems.

Crystal Structure of the Heme-IsdC Complex, the Central Conduit of the Isd Iron/Heme Uptake System in Staphylococcus aureus

Pathogens such as Staphylococcus aureus require iron to survive and have evolved specialized proteins to steal heme from their host. IsdC is the central conduit of the Isd (iron-regulated surface determinant) multicomponent heme uptake machinery; staphylococcal cell-surface proteins such as IsdA, IsdB, and IsdH are thought to funnel their molecular cargo to IsdC, which then mediates the transfer of the iron-containing nutrient to the membrane translocation system IsdDEF. The structure of the heme-IsdC complex reveals a novel heme site within an immunoglobulin-like domain and sheds light on its binding mechanism. The folding topology is reminiscent of the architecture of cytochrome f, cellobiose dehydrogenase, and ethylbenzene dehydrogenase; in these three proteins, the heme is bound in an equivalent position, but interestingly, IsdC features a distinct binding pocket with the ligand located next to the hydrophobic core of the beta-sandwich. The iron is coordinated with a tyrosine surrounded by several non-polar side chains that cluster into a tightly packed proximal side. On the other hand, the distal side is relatively exposed with a short helical peptide segment that acts as a lip clasping onto almost half of the porphyrin plane. This structural feature is argued to play a role in the mechanism of binding and release by switching to an open conformation and thus loosening the interactions holding the heme. The structure of the heme-IsdC complex provides a template for the understanding of other proteins, such as IsdA, IsdB, and IsdH, that contain the same heme-binding module as IsdC, known as the NEAT (near transporter) domain.