Direct heme transfer reactions in the Group A Streptococcus heme acquisition pathway

The heme acquisition machinery in Group A Streptococcus (GAS) consists of the surface proteins Shr and Shp and ATP-binding cassette transporter HtsABC. Shp cannot directly acquire heme from methemoglobin (metHb) but directly transfers its heme to HtsA. It has not been previously determined whether Shr directly relays heme from metHb to Shp. Thus, the complete pathway for heme acquisition from metHb by the GAS heme acquisition machinery has remained unclear. In this study, the metHb-to-Shr and Shr-to-Shp heme transfer reactions were characterized by spectroscopy, kinetics and protein-protein interaction analyses. Heme is efficiently transferred from the β and α subunits of metHb to Shr with rates that are 7 and 60 times greater than those of the passive heme release from metHb, indicating that Shr directly acquires heme from metHb. The rapid heme transfer from Shr to Shp involves an initial heme donor/acceptor complex and a spectrally and kinetically detectable transfer intermediate, implying that heme is directly channeled from Shr to Shp. The present results show that Shr speeds up heme transfer from metHb to Shp, whereas Shp speeds up heme transfer from Shr to HtsA. Furthermore, the findings demonstrate that Shr can interact with metHb and Shp but not HtsA. Taken together with our published results on the Shp/HtsA reaction, these findings establish a model of the heme acquisition pathway in GAS in which Shr directly extracts heme from metHb and Shp relays it from Shr to HtsA.     

Shr of group A streptococcus is a new type of composite NEAT protein involved in sequestering haem from methaemoglobin

A growing body of evidence suggests that surface or secreted proteins with NEAr Transporter (NEAT) domains play a central role in haem acquisition and trafficking across the cell envelope of Gram‐positive bacteria. Group A streptococcus (GAS), a β‐haemolytic human pathogen, expresses a NEAT protein, Shr, which binds several haemoproteins and extracellular matrix (ECM) components. Shr is a complex, membrane‐anchored protein, with a unique N‐terminal domain (NTD) and two NEAT domains separated by a central leucine‐rich repeat region. In this study we have carried out an analysis of the functional domains in Shr. We show that Shr obtains haem in solution and furthermore reduces the haem iron; this is the first report of haem reduction by a NEAT protein. More specifically, we demonstrate that both of the constituent NEAT domains of Shr are responsible for binding haem, although they are missing a critical tyrosine residue found in the ligand‐binding pocket of other haem‐binding NEAT domains. Further investigations show that a previously undescribed region within the Shr NTD interacts with methaemoglobin. Shr NEAT domains, however, do not contribute significantly to the binding of methaemoglobin but mediate binding to the ECM components fibronectin and laminin. A protein fragment containing the NTD plus the first NEAT domain was found to be sufficient to sequester haem directly from methaemoglobin. Correlating these in vitro findings to in vivo biological function, mutants analysis establishes the role of Shr in GAS growth with methaemoglobin as a sole source of iron, and indicates that at least one NEAT domain is necessary for the utilization of methaemoglobin. We suggest that Shr is the prototype of a new group of NEAT composite proteins involved in haem uptake found in pyogenic streptococci and Clostridium novyi.     

The surface protein Shr of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> binds heme and transfers it to the streptococcal heme-binding protein Shp

Background  

The heme acquisition machinery in Streptococcus pyogenes is believed to consist of the surface proteins, Shr and Shp, and heme-specific ATP-binding cassette transporter HtsABC. Shp has been shown to rapidly transfer its heme to the lipoprotein component, HtsA, of HtsABC. The function of Shr and the heme source of Shp have not been established.     

Bis-methionyl coordination in the crystal structure of the heme-binding domain of the streptococcal cell surface protein Shp

Surface proteins Shr, Shp, and the ATP-binding cassette (ABC) transporter HtsABC are believed to make up the machinery for heme uptake in Streptococcus pyogenes. Shp transfers its heme to HtsA, the lipoprotein component of HtsABC, providing the only experimentally demonstrated example of direct heme transfer from a surface protein to an ABC transporter in Gram-positive bacteria. To understand the structural basis of heme transfer in this system, the heme-binding domain of Shp (Shp¹⁸⁰) was crystallized, and its structure determined to a resolution of 2.1 Å. Shp¹⁸⁰ exhibits an immunoglobulin-like β-sandwich fold that has been recently found in other pathogenic bacterial cell surface heme-binding proteins, suggesting that the mechanisms of heme acquisition are conserved. Shp shows minimal amino acid sequence identity to these heme-binding proteins and the structure of Shp¹⁸⁰ reveals a unique heme-iron coordination with the axial ligands being two methionine residues from the same Shp molecule. A negative electrostatic surface of protein structure surrounding the heme pocket may serve as a docking interface for heme transfer from the more basic outer cell wall heme receptor protein Shr. The crystal structure of Shp¹⁸⁰ reveals two exogenous, weakly bound hemins, which form a large interface between the two Shp¹⁸⁰ molecules in the asymmetric unit. These “extra” hemins form a stacked pair with a structure similar to that observed previously for free hemin dimers in aqueous solution. The propionates of the protein-bound heme coordinate to the iron atoms of the exogenous hemin dimer, contributing to the stability of the protein interface. Gel filtration and analytical ultracentrifugation studies indicate that both full-length Shp and Shp¹⁸⁰ are monomeric in dilute aqueous solution.     

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.     

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.     

The five near-iron transporter (NEAT) domain anthrax hemophore, IsdX2, scavenges heme from hemoglobin and transfers heme to the surface protein IsdC

Pathogenic bacteria require iron to replicate inside mammalian hosts. Recent studies indicate that heme acquisition in Gram-positive bacteria is mediated by proteins containing one or more near-iron transporter (NEAT) domains. Bacillus anthracis is a spore-forming, Gram-positive pathogen and the causative agent of anthrax disease. The rapid, extensive, and efficient replication of B. anthracis in host tissues makes this pathogen an excellent model organism for the study of bacterial heme acquisition. B. anthracis secretes two NEAT hemophores, IsdX1 and IsdX2. IsdX1 contains a single NEAT domain, whereas IsdX2 has five, a novel property among hemophores. To understand the functional significance of harboring multiple, non-identical NEAT domains, we purified each individual NEAT domain of IsdX2 as a GST fusion and analyzed the specific function of each domain as it relates to heme acquisition and transport. NEAT domains 1, 3, 4, and 5 all bind heme, with domain 5 having the highest affinity. All NEATs associate with hemoglobin, but only NEAT1 and -5 can extract heme from hemoglobin, seemingly by a specific and active process. NEAT1, -3, and -4 transfer heme to IsdC, a cell wall-anchored anthrax NEAT protein. These results indicate that IsdX2 has all the features required to acquire heme from the host and transport heme to the bacterial cell wall. Additionally, these results suggest that IsdX2 may accelerate iron import rates by acting as a "heme sponge" that enhances B. anthracis replication in iron-starved environments.     

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.     

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.     

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.     

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.     

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.     

The mechanism of direct heme transfer from the streptococcal cell surface protein Shp to HtsA of the HtsABC transporter

The heme-binding proteins Shp and HtsA are part of the heme acquisition machinery found in Streptococcus pyogenes. The hexacoordinate heme (Fe(II)-protoporphyrin IX) or hemochrome form of holoShp (hemoShp) is stable in air in Tris-HCl buffer, pH 8.0, binds to apoHtsA with a K(d) of 120 +/- 18 microm, and transfers its heme to apoHtsA with a rate constant of 28 +/- 6s(-1) at 25 degrees C, pH 8.0. The hemoHtsA product then autoxidizes to the hexacoordinate hemin (Fe(III)-protoporphyrin IX) or hemichrome form (hemiHtsA) with an apparent rate constant of 0.017 +/- 0.002 s(-1). HemiShp also rapidly transfers hemin to apoHtsA through a hemiShp.apoHtsA complex (K(d) = 48 +/- 7 microM) at a rate approximately 40,000 times greater than the rate of simple hemin dissociation from hemiShp into solvent (k(transfer) = 43 +/- 3s(-1) versus k(-hemin) = 0.0003 +/- 0.00006 s(-1)). The rate constants for hemin binding to and dissociation from HtsA (k'(hemin) approximately 80 microm(-1) s(-1), k(-hemin) = 0.0026 +/- 0.0002 s(-1)) are 50- and 10-fold greater than the corresponding rate constants for Shp (k(hemin) approximately 1.6 microM(-1) s(-1), k(-hemin) = 0.0003 s(-1)), which implies that HtsA has a more accessible active site. However, the affinity of apoHtsA for hemin (k(hemin) approximately 31,000 microm(-1)) is roughly 5-fold greater than that of apoShp (k(hemin) approximately 5,300 microM(-1)), accounting for the net transfer from Shp to HstA. These results support a direct, rapid, and affinity-driven mechanism of heme and hemin transfer from the cell surface receptor Shp to the ATP-binding cassette transporter 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.     

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.     

Heme Transfer to the Bacterial Cell Envelope Occurs via a Secreted Hemophore in the Gram-positive Pathogen Bacillus anthracis

To initiate and sustain an infection in mammals, bacterial pathogens must acquire host iron. However, the host''s compartmentalization of large amounts of iron in heme, which is bound primarily by hemoglobin in red blood cells, acts as a barrier to bacterial iron assimilation. Bacillus anthracis, the causative agent of the disease anthrax, secretes two NEAT (near iron transporter) proteins, IsdX1 and IsdX2, which scavenge heme from host hemoglobin and promote growth under low iron conditions. The mechanism of heme transfer from these hemophores to the bacterial cell is not known. We present evidence that the heme-bound form of IsdX1 rapidly and directionally transfers heme to IsdC, a NEAT protein covalently attached to the cell wall, as well as to IsdX2. In both cases, the transfer of heme is mediated by a physical association between the donor and recipient. Unlike Staphylococcus aureus, whose NEAT proteins acquire heme from hemoglobin directly at the bacterial surface, B. anthracis secretes IsdX1 to capture heme in the extracellular milieu and relies on NEAT-NEAT interactions to deliver the bound heme to the envelope via IsdC. Understanding the mechanism of NEAT-mediated iron transport into pathogenic Gram-positive bacteria may provide an avenue for the development of therapeutics to combat infection.     

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.     

Benfang Lei's research on heme acquisition in Gram-positive pathogens and bacterial pathogenesis

Benfang Lei's laboratory conducts research on pathogenesis of human pathogen Group A Streptococcus (GAS) and horse pathogen Streptococcus equi (S. equi). His current research focuses on heme acquisition in Gram-positive pathogens and molecular mechanism of GAS and S. equi pathogenesis. Heme is an important source of essential iron for bacterial pathogens. Benfang Lei and colleagues identified the first cell surface heme-binding protein in Gram-positive pathogens and the heme acquisition system in GAS, demonstrated direct heme transfer from one protein to another, demonstrated an experimental pathway of heme acquisition by the Staphylococcus aureus Isd system, elucidated the activated heme transfer mechanism, and obtained evidence for a chemical mechanism of direct axial ligand displacement during the Shp-to-HtsA heme transfer reaction. These findings have considerably contributed to the progress that has been made over recent years in understanding the heme acquisition process in Gram-positive pathogens. Pathogenesis of GAS is mediated by an abundance of extracellular proteins, and pathogenic role and functional mechanism are not known for many of these virulence factors. Lei laboratory identified a secreted protein of GAS as a CovRS-regulated virulence factor that is a protective antigen and is critical for GAS spreading in the skin and systemic dissemination. These studies may lead to development of novel strategies to prevent and treat GAS infections.     

Differential Function of Lip Residues in the Mechanism and Biology of an Anthrax Hemophore

To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin. Bacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positive hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 3₁₀-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain of B. anthracis on hemoglobin as the sole iron source. These data indicate that not only is the 3₁₀-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands.     

The Near-iron Transporter (NEAT) Domains of the Anthrax Hemophore IsdX2 Require a Critical Glutamine to Extract Heme from Methemoglobin

Several Gram-positive pathogenic bacteria employ near-iron transporter (NEAT) domains to acquire heme from hemoglobin during infection. However, the structural requirements and mechanism of action for NEAT-mediated heme extraction remains unknown. Bacillus anthracis exhibits a rapid growth rate during systemic infection, suggesting that the bacterium expresses efficient iron acquisition systems. To understand how B. anthracis acquires iron from heme sources, which account for 80% of mammalian iron stores, we investigated the properties of the five-NEAT domain hemophore IsdX2. Using a combination of bioinformatics and site-directed mutagenesis, we determined that the heme extraction properties of IsdX2 are dependent on an amino acid with an amide side chain within the 3₁₀-helix of the NEAT domain. Additionally, we used a spectroscopic analysis to show that IsdX2 NEAT domains only scavenge heme from methemoglobin (metHb) and that autoxidation of oxyhemoglobin to metHb must occur prior to extraction. We also report the crystal structures of NEAT5 wild type and a Q29T mutant and present surface plasmon resonance data that indicate that the loss of this amide side chain reduces the affinity of the NEAT domain for metHb. We propose a model whereby the amide side chain is first required to drive an interaction with metHb that destabilizes heme, which is subsequently extracted and coordinated in the aliphatic heme-binding environment of the NEAT domain. Because an amino acid with an amide side chain in this position is observed in NEAT domains of several genera of Gram-positive pathogenic bacteria, these results suggest that specific targeting of this or nearby residues may be an entry point for inhibitor development aimed at blocking bacterial iron acquisition during infection.