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.     

Iron-coordinating tyrosine is a key determinant of NEAT domain heme transfer

In humans, heme iron is the most abundant iron source, and bacterial pathogens such as Staphylococcus aureus acquire it for growth. IsdB of S. aureus acquires Fe(III)-protoporphyrin IX (heme) from hemoglobin for transfer to IsdC via IsdA. These three cell-wall-anchored Isd (iron-regulated surface determinant) proteins contain conserved NEAT (near iron transport) domains. The purpose of this work was to delineate the mechanism of heme binding and transfer between the NEAT domains of IsdA, IsdB, and IsdC using a combination of structural and spectroscopic studies. X-ray crystal structures of IsdA NEAT domain (IsdA-N1) variants reveal that removing the native heme-iron ligand Tyr166 is compensated for by iron coordination by His83 on the distal side and that no single mutation of distal loop residues is sufficient to perturb the IsdA-heme complex. Also, alternate heme-iron coordination was observed in structures of IsdA-N1 bound to reduced Fe(II)-protoporphyrin IX and Co(III)-protoporphyrin IX. The IsdA-N1 structural data were correlated with heme transfer kinetics from the NEAT domains of IsdB and IsdC. We demonstrated that the NEAT domains transfer heme at rates comparable to full-length proteins. The second-order rate constant for heme transfer from IsdA-N1 was modestly affected (< 2-fold) by the IsdA variants, excluding those at Tyr166. Substituting Tyr166 with Ala or Phe changed the reaction mechanism to one with two observable steps and decreased observed rates > 15-fold (to 100-fold excess IsdC). We propose a heme transfer model wherein NEAT domain complexes pass heme iron directly from an iron-coordinating Tyr of the donor protein to the homologous Tyr residues of the acceptor protein.     

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.     

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.     

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.     

Structural biology of heme binding in the Staphylococcus aureus Isd system

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 iron-responsive 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.     

Demonstration of the iron-regulated surface determinant (Isd) heme transfer pathway in Staphylococcus aureus

In this study, we report experimental results that provide the first complete challenge of a proposed model for heme acquisition by Staphylococcus aureus via the Isd pathway first put forth by Mazmanian, S. K., Skaar, E. P., Gaspar, A. H., Humayun, M., Gornicki, P., Jelenska, J., Joachmiak, A., Missiakas, D. M., and Schneewind, O. (2003) Science 299, 906-909. The heme-binding NEAT domains of Isd proteins IsdA, IsdB (domain 2), IsdC, and HarA/IsdH (domain 3), and the heme-binding IsdE protein, were overexpressed and purified in apo (heme-free) form. Absorption and magnetic circular dichroism spectral data, together with electrospray ionization mass spectrometry were used to unambiguously identify that heme transfers from NEAT-A through NEAT-C to IsdE. Heme transfer was demonstrated to occur in a unidirectional fashion in the sequence NEAT-B2 --> NEAT-A --> NEAT-C --> IsdE or, alternatively, initiating from NEAT-H3 instead of NEAT-B2: NEAT-H3 --> NEAT-A --> NEAT-C --> IsdE. Under the conditions of our experiments, only NEAT-H3 and NEAT-B2 could transfer bidirectionally, which is in the reverse direction as well, and only with each other. Whereas apo-IsdE readily accepted heme from holo-NEAT-C, it would not accept heme from holo-NEAT-A. Heme transfer to IsdE requires the presence of holo-NEAT-C, in agreement with the proposal that IsdC serves as the central conduit of the heme transfer pathway. These experimental findings corroborate the heme transfer model first proposed by the Schneewind group. Our data show that heme transport from the wall-anchored IsdH/IsdB proteins proceeds directly to IsdE at the membrane and, for this to occur, we propose that specific protein-protein interactions must take place.     

Pathway for heme uptake from human methemoglobin by the iron-regulated surface determinants system of Staphylococcus aureus

The iron-regulated surface proteins IsdA, IsdB, and IsdC and transporter IsdDEF of Staphylococcus aureus are involved in heme acquisition. To establish an experimental model of heme acquisition by this system, we have investigated hemin transfer between the various couples of human methemoglobin (metHb), IsdA, IsdB, IsdC, and IsdE by spectroscopic and kinetic analyses. The efficiencies of hemin transfer from hemin-containing donors (holo-protein) to different hemin-free acceptors (apo-protein) were examined, and the rates of the transfer reactions were compared with that of indirect loss of hemin from the relevant donor to H64Y/V68F apomyoglobin. The efficiencies, spectral changes, and kinetics of the transfer reactions demonstrate that: 1) metHb directly transfers hemin to apo-IsdB, but not to apo-IsdA, apo-IsdC, and apo-IsdE; 2) holo-IsdB directly transfers hemin to apo-IsdA and apo-IsdC, but not to apo-IsdE; 3) apo-IsdE directly acquires hemin from holo-IsdC, but not from holo-IsdB and holo-IsdA; and 4) IsdB and IsdC enhance hemin transfer from metHb to apo-IsdC and from holo-IsdB to apo-IsdE, respectively. Taken together with our recent finding that holo-IsdA directly transfers its hemin to apo-IsdC, these results provide direct experimental evidence for a model in which IsdB acquires hemin from metHb and transfers it directly or through IsdA to IsdC. Hemin is then relayed to IsdE, the lipoprotein component of the IsdDEF transporter.     

Iron-Regulated Surface Determinant (Isd) Proteins of Staphylococcus lugdunensis

Staphylococcus lugdunensis is the only coagulase-negative Staphylococcus species with a locus encoding iron-regulated surface determinant (Isd) proteins. In Staphylococcus aureus, the Isd proteins capture heme from hemoglobin and transfer it across the wall to a membrane-bound transporter, which delivers it into the cytoplasm, where heme oxygenases release iron. The Isd proteins of S. lugdunensis are expressed under iron-restricted conditions. We propose that S. lugdunensis IsdB and IsdC proteins perform the same functions as those of S. aureus. S. lugdunensis IsdB is the only hemoglobin receptor within the isd locus. It specifically binds human hemoglobin with a dissociation constant (K_d) of 23 nM and transfers heme on IsdC. IsdB expression promotes bacterial growth in an iron-limited medium containing human hemoglobin but not mouse hemoglobin. This correlates with weak binding of IsdB to mouse hemoglobin in vitro. Unlike IsdB and IsdC, the proteins IsdJ and IsdK are not sorted to the cell wall in S. lugdunensis. In contrast, IsdJ expressed in S. aureus and Lactococcus lactis is anchored to peptidoglycan, suggesting that S. lugdunensis sortases may differ in signal recognition or could be defective. IsdJ and IsdK are present in the culture supernatant, suggesting that they could acquire heme from the external milieu. The IsdA protein of S. aureus protects bacteria from bactericidal lipids due to its hydrophilic C-terminal domain. IsdJ has a similar region and protected S. aureus and L. lactis as efficiently as IsdA but, possibly due to its location, was less effective in its natural host.     

Heme binding in the NEAT domains of IsdA and IsdC of Staphylococcus aureus

Absorption, magnetic circular dichroism (MCD), and electrospray mass spectral (ESI-MS) data are reported for the heme binding NEAr iron Transporter (NEAT) domains of IsdA and IsdC, two proteins involved in heme scavenging by Staphylococcus aureus. The mass spectrometry data show that the NEAT domains are globular in structure and efficiently bind a single heme molecule. In this work, the IsdA NEAT domain is referred to as NEAT-A, the IsdC NEAT domain is referred to as NEAT-C, heme-free NEAT-C is NEAT-A and NEAT-C are inaccessible to small anionic ligands. Reduction of the high-spin Fe(III) heme iron to 5-coordinate high-spin Fe(II) in NEAT-A results in coordination by histidine and opens access, allowing for CO axial ligation, yielding 6-coordinate low-spin Fe(II) heme. In contrast, reduction of the high-spin Fe(III) heme iron to 5-coordinate high-spin Fe(II) in NEAT-C results in loss of the heme from the binding site of the protein due to the absence of a proximal histidine. The absorption and MCD data for NEAT-A closely match those previously reported for the whole IsdA protein, providing evidence that heme binding is primarily a property of the NEAT domain.     

The iron-regulated surface proteins IsdA, IsdB, and IsdH are not required for heme iron utilization in Staphylococcus aureus

The iron-regulated surface determinant proteins (Isd) of Staphylococcus aureus are expressed during iron limitation and have been proposed to be involved in the scavenging of iron from heme. In this study, the genes encoding the surface proteins IsdA, IsdB, and IsdH were inactivated in order to determine their combined role. The triple mutant was found to have no defect in growth under any conditions of iron limitation tested. Also using a mouse septic arthritis model of S.?aureus systemic disease, no significant difference in bacterial load was observed for the triple mutant, compared with its otherwise isogenic parent.     

Unique heme-iron coordination by the hemoglobin receptor IsdB of Staphylococcus aureus

Iron is an essential requirement for life for nearly all organisms. The human pathogen Staphylococcus aureus is able to acquire iron from the heme cofactor of hemoglobin (Hb) released from lysed erythrocytes. IsdB, the predominant Hb receptor of S. aureus, is a cell wall-anchored protein that is composed of two NEAT domains. The N-terminal NEAT domain (IsdB-N1) binds Hb, and the C-terminal NEAT domain (IsdB-N2) relays heme to IsdA for transport into the cell. Here we present the 1.45 ? resolution X-ray crystal structure of the IsdB-N2-heme complex. While the structure largely conforms to the eight-strand β-sandwich fold seen in other NEAT domains such as IsdA-N and uses a conserved Tyr residue to coordinate heme-iron, a Met residue is also involved in iron coordination, resulting in a novel Tyr-Met hexacoordinate heme-iron state. The kinetics of the transfer of heme from IsdB-N2 to IsdA-N can be modeled as a two-step process. The rate of transfer of heme between the isolated NEAT domains (82 s(-1)) was found to be similar to that measured for the full-length proteins. Replacing the iron coordinating Met with Leu did not abrogate high-affinity heme binding but did reduce the heme transfer rate constant by more than half. This unusual Met-Tyr heme coordination may also bestow properties on IsdB that help it to bind heme in different oxidation states or extract heme from hemoglobin.     

Staphylococcus aureus IsdB is a hemoglobin receptor required for heme iron utilization

The pathogenesis of human infections caused by the gram-positive microbe Staphylococcus aureus has been previously shown to be reliant on the acquisition of iron from host hemoproteins. The iron-regulated surface determinant system (Isd) encodes a heme transport apparatus containing three cell wall-anchored proteins (IsdA, IsdB, and IsdH) that are exposed on the staphylococcal surface and hence have the potential to interact with human hemoproteins. Here we report that S. aureus can utilize the host hemoproteins hemoglobin and myoglobin, but not hemopexin, as iron sources for bacterial growth. We demonstrate that staphylococci capture hemoglobin on the bacterial surface via IsdB and that inactivation of isdB, but not isdA or isdH, significantly decreases hemoglobin binding to the staphylococcal cell wall and impairs the ability of S. aureus to utilize hemoglobin as an iron source. Stable-isotope-tracking experiments revealed removal of heme iron from hemoglobin and transport of this compound into staphylococci. Importantly, mutants lacking isdB, but not isdH, display a reduction in virulence in a murine model of abscess formation. Thus, IsdB-mediated scavenging of iron from hemoglobin represents an important virulence strategy for S. aureus replication in host tissues and for the establishment of persistent staphylococcal infections.     

Solution structure of the NEAT (NEAr Transporter) domain from IsdH/HarA: the human hemoglobin receptor in Staphylococcus aureus

During infections the pathogen Staphylococcus aureus procures the essential nutrient iron from its host using iron-regulated surface determinant (Isd) proteins, which scavenge heme bound iron from host hemoproteins. Four Isd proteins are displayed in the cell wall, where they function as receptors for host proteins and heme. Each of the receptors contains one or more copies of a recently discovered domain called NEAT (NEAr Transporter) that has been shown to mediate protein binding. Here we report the three-dimensional solution structure of the NEAT domain from the IsdH/HarA protein, which is the hemoglobin receptor in the Isd system. This is the first structure of a NEAT domain and reveals that they adopt a beta sandwich fold that consists of two five-stranded antiparallel beta sheets. Although unrelated at the primary sequence level, our results indicate that NEAT domains belong to the immunoglobulin superfamily. Binding studies indicate that two IsdH/HarA NEAT domains bind a single molecule of methemoglobin, while the distantly related NEAT domain from the S. aureus IsdC protein binds only heme. A comparison of their primary sequences in light of the new structure is used to predict the hemoglobin and heme binding surfaces on NEAT domains.     

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.     

Structure of the Hemoglobin-IsdH Complex Reveals the Molecular Basis of Iron Capture by Staphylococcus aureus

Staphylococcus aureus causes life-threatening disease in humans. The S. aureus surface protein iron-regulated surface determinant H (IsdH) binds to mammalian hemoglobin (Hb) and extracts heme as a source of iron, which is an essential nutrient for the bacteria. However, the process of heme transfer from Hb is poorly understood. We have determined the structure of IsdH bound to human Hb by x-ray crystallography at 4.2 Å resolution, revealing the structural basis for heme transfer. One IsdH molecule is bound to each α and β Hb subunit, suggesting that the receptor acquires iron from both chains by a similar mechanism. Remarkably, two near iron transporter (NEAT) domains in IsdH perform very different functions. An N-terminal NEAT domain binds α/β globin through a site distant from the globin heme pocket and, via an intervening structural domain, positions the C-terminal heme-binding NEAT domain perfectly for heme transfer. These data, together with a 2.3 Å resolution crystal structure of the isolated N-terminal domain bound to Hb and small-angle x-ray scattering of free IsdH, reveal how multiple domains of IsdH cooperate to strip heme from Hb. Many bacterial pathogens obtain iron from human hemoglobin using proteins that contain multiple NEAT domains and other domains whose functions are poorly understood. Our results suggest that, rather than acting as isolated units, NEAT domains may be integrated into higher order architectures that employ multiple interaction interfaces to efficiently extract heme from host proteins.     

Staphylococcus aureus Uses a Novel Multidomain Receptor to Break Apart Human Hemoglobin and Steal Its Heme

Staphylococcus aureus is a leading cause of life-threatening infections in the United States. It requires iron to grow, which must be actively procured from its host to successfully mount an infection. Heme-iron within hemoglobin (Hb) is the most abundant source of iron in the human body and is captured by S. aureus using two closely related receptors, IsdH and IsdB. Here we demonstrate that each receptor captures heme using two conserved near iron transporter (NEAT) domains that function synergistically. NMR studies of the 39-kDa conserved unit from IsdH (IsdH^N2N3, Ala³²⁶–Asp⁶⁶⁰) reveals that it adopts an elongated dumbbell-shaped structure in which its NEAT domains are properly positioned by a helical linker domain, whose three-dimensional structure is determined here in detail. Electrospray ionization mass spectrometry and heme transfer measurements indicate that IsdH^N2N3 extracts heme from Hb via an ordered process in which the receptor promotes heme release by inducing steric strain that dissociates the Hb tetramer. Other clinically significant Gram-positive pathogens capture Hb using receptors that contain multiple NEAT domains, suggesting that they use a conserved mechanism.     

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 ¹H, ¹³C and ¹⁵N 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.     

Backbone 1H, 13C and 15N resonance assignments of the 39?kDa staphylococcal hemoglobin receptor IsdH

During infections Stahpylococcus aureus preferentially uses heme as an iron source, which it captures from human hemoglobin using the Iron regulated surface determinant (Isd) system. On the cell surface two related staphylococcal surface receptors called IsdH and IsdB bind to hemoglobin and extract its heme. Both receptors contain multiple NEAr iron Transporter (NEAT) domains that either bind to hemoglobin, or to heme. All previous structural studies have investigated individual NEAT domains and have not explored how the domains might interact with one another to synergistically extract heme from hemoglobin. Here, we report the near complete (1)H, (13)C and (15)N backbone resonance assignments of a bi-domain unit from IsdH that contains the N2 and N3 NEAT domains, which bind to hemoglobin and heme, respectively (IsdH(N2N3), residues 326-660, 39 kDa). The assigned backbone resonances lay the foundation for future NMR studies that will explore the molecular basis of IsdH function.     

Structure and role of the linker domain of the iron surface-determinant protein IsdH in heme transportation in Staphylococcus aureus

Staphylococcus aureus is a major cause of deadly nosocomial infections, a severe problem fueled by the steady increase of resistant bacteria. The iron surface determinant (Isd) system is a family of proteins that acquire nutritional iron from the host organism, helping the bacterium to proliferate during infection, and therefore represents a promising antibacterial target. In particular, the surface protein IsdH captures hemoglobin (Hb) and acquires the heme moiety containing the iron atom. Structurally, IsdH comprises three distinctive NEAr-iron Transporter (NEAT) domains connected by linker domains. The objective of this study was to characterize the linker region between NEAT2 and NEAT3 from various biophysical viewpoints and thereby advance our understanding of its role in the molecular mechanism of heme extraction. We demonstrate the linker region contributes to the stability of the bound protein, likely influencing the flexibility and orientation of the NEAT3 domain in its interaction with Hb, but only exerts a modest contribution to the affinity of IsdH for heme. Based on these data, we suggest that the flexible nature of the linker facilitates the precise positioning of NEAT3 to acquire heme. In addition, we also found that residues His45 and His89 of Hb located in the heme transfer route toward IsdH do not play a critical role in the transfer rate-determining step. In conclusion, this study clarifies key elements of the mechanism of heme extraction of human Hb by IsdH, providing key insights into the Isd system and other protein systems containing NEAT domains.