Molecular basis of recognition of antibacterial porphyrins by heme-transporter IsdH-NEAT3 of Staphylococcus aureus

Antibiotic resistance is increasingly seen as a serious problem that threatens public health and erodes our capacity to effectively combat disease. So-called non-iron metalloporhyrins have shown promising antibacterial properties against a number of pathogenic bacteria including Staphylococcus aureus. However, little is known about the molecular mechanism(s) of action of these compounds and in particular how they reach the interior of the bacterial cells. A popular hypothesis indicates that non-iron metalloporphyrins infiltrate into bacterial cells like a "Trojan horse" using heme transport systems. Iron-regulated surface determinant (Isd) is the best characterized heme transport system of S. aureus. Herein we studied the molecular mechanism by which the extracellular heme-receptor IsdH-NEAT3 of Isd recognizes antimicrobial metalloporphyrins. We found that potent antibacterial porphyrins Ga(III)-protoporphyrin IX (PPIX) and Mn(III)-PPIX closely mimicked the properties of the natural ligand heme, namely (i) stable binding to IsdH-NEAT3 with comparable affinities for the receptor, (ii) nearly undistinghuishable three-dimensional structure when complexed with IsdH-NEAT3, and (iii) similar transfer properties to a second receptor IsdA. On the contrary, weaker antibacterial porphyrins Mg(II)-PPIX, Zn(II)-PPIX, and Cu(II)-PPIX were not captured effectively by IsdH-NEAT3 under our experimental conditions and displayed lower affinities. Moreover, reduction of Fe(III)-PPIX to Fe(II)-PPIX with dithionite abrogated stable binding to receptor. These data revealed a clear connection between oxidation state of metal and effective attachment to IsdH-NEAT3. Also, the strong correlation between binding affinity and reported antimicrobial potency suggested that the Isd system may be used by these antibacterial compounds to gain access to the interior of the cells. We hope these results will increase our understanding of Isd system of S. aureus and highlight its biomedical potential to deliver new and more efficient antibacterial treatments.     

革兰氏阴性菌血红素载体蛋白Hemophore的结构及作用机制

血红素作为宿主体内最丰富的铁离子来源,是致病菌营养竞争的主要目标,尤其对于血红素自身合成途径部分丧失的细菌。革兰氏阴性菌血红素转运系统由血红素载体蛋白(Hemophore)、外膜血红素受体、TonB-ExbB-ExbD复合物、ABC转运体等组成。Hemophore是存在于细菌细胞膜上或分泌到胞外环境中的一种蛋白。它能从宿主血红素结合蛋白中捕获血红素并将其传递给外膜受体。目前,在不同革兰氏阴性菌中已发现3种类型的Hemophore,分别是HasA、HxuA和HmuY型。本文将详细描述这3种Hemophore捕获血红素及与外膜受体相互作用的机制,以期为进一步研究其他细菌血红素载体蛋白的功能及作用机制奠定基础。     

Iron-containing lipoprotein SiaA in SiaABC, the primary heme transporter of Streptococcus pyogenes

The cell-surface lipoprotein SiaA, a component of the SiaABC transporter, acts as the primary receptor for heme in the infamous human pathogen Streptococcus pyogenes. However, little is known about the molecular mechanism of heme binding and release as well as the role of heme-binding ligands that contribute to the uptake of heme into the pathogenic bacteria. The present report aims to clarify the coordination properties of heme iron in SiaA. By substitution of either Met79 or His229 with alanine, the mutant M79A and H229A proteins display significantly decreased heme-binding affinity and substantially increased heme-release rates, as compared with wild-type SiaA protein. Both fluorescence and circular dichroism spectra indicated that heme binding results in alterations in the secondary structure of the protein. Heme release from SiaA is a stepwise process in which heme disassociates firstly from Met79 and then from His229 with distinct conformational changes. His229 may serve as an anchor for heme binding in SiaA and thus may play a major role in the stability of the coordination between heme and the protein.     

Evolutionarily consistent families in SCOP: sequence, structure and function

Background

HutZ is the sole heme storage protein identified in the pathogenic bacterium Vibrio cholerae and is required for optimal heme utilization. However, no heme oxygenase activity has been observed with this protein. Thus far, HutZ??s structure and heme-binding mechanism are unknown.

Results

We report the first crystal structure of HutZ in a homodimer determined at 2.0 ? resolution. The HutZ structure adopted a typical split-barrel fold. Through a docking study and site-directed mutagenesis, a heme-binding model for the HutZ dimer is proposed. Very interestingly, structural superimposition of HutZ and its homologous protein HugZ, a heme oxygenase from Helicobacter pylori, exhibited a structural mismatch of one amino acid residue in ??6 of HutZ, although residues involved in this region are highly conserved in both proteins. Derived homologous models of different single point variants with model evaluations suggested that Pro¹⁴⁰ of HutZ, corresponding to Phe²¹⁵ of HugZ, might have been the main contributor to the structural mismatch. This mismatch initiates more divergent structural characteristics towards their C-terminal regions, which are essential features for the heme-binding of HugZ as a heme oxygenase.

Conclusions

HutZ??s deficiency in heme oxygenase activity might derive from its residue shift relative to the heme oxygenase HugZ. This residue shift also emphasized a limitation of the traditional template selection criterion for homology modeling.     

Insights into mechanism and functional consequences of heme binding to hemolysin-activating lysine acyltransferase HlyC from Escherichia coli

Background

Tight regulation of heme homeostasis is a critical mechanism in pathogenic bacteria since heme functions as iron source and prosthetic group, but is also toxic at elevated concentrations. Hemolysin-activating lysine-acyltransferase (HlyC) from Escherichia coli is crucial for maturation of hemolysin A, which lyses several mammalian cells including erythrocytes liberating large amounts of heme for bacterial uptake. A possible impact and functional consequences of the released heme on events employing bacterial HlyC have remained unexplored.

Heme-binding properties of heme detoxification protein from Plasmodium falciparum

The heme detoxification protein of the malaria parasite Plasmodium falciparum is involved in the formation of hemozoin, an insoluble crystalline form of heme. Although the disruption of hemozoin formation is the most widely used strategy for controlling the malaria parasite, the heme-binding properties of heme detoxification protein are poorly characterized. In this study, we established a method for the expression and purification of the non-tagged protein and characterized heme-binding properties. The spectroscopic features of non-tagged protein differ from those of the His-tagged protein, suggesting that the artificial tag interferes with the properties of the recombinant protein. The purified recombinant non-tagged heme detoxification protein had two heme-binding sites and exhibited a spectrum typical of heme proteins. A mechanism for hemozoin formation is proposed.     

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.     

Novel heme-binding component in the serum of the channel catfish (Ictalurus punctatus)

The serum of the channel catfish (Ictalurus punctatus) was examined for heme- and hemoglobin-binding proteins. Electrophoretic mobility retardation assays failed to detect a hemoglobin-binding material similar to mammalian haptoglobin; however, a heme-binding component (not previously described) was identified in catfish seru. The heme-binding component was purified by gel filtration chromatography; electrophoretic analyses suggested it to be composed of two polypeptide subunits of molecular masses about 115 and 98 kDa. This composition is inconsistent with hemopexin, the known heme-binding serum protein of mammals. Although it was not fully saturated with heme, the catfish component contained detectable heme in normal sera. When complexed by the binding material, heme was used as an iron source by isolates of the bacterial Gram-negative genusAeromonas; the capacity of other bacteria to use the complex was not tested. The physiological function of the catfish heme-binding serum protein is presently not clear.     

Glutamate-induced metabolic changes influence the cytoplasmic redox state of hippocampal neurons

The heme oxygenase ChuZ is part of the iron acquisition mechanism of Campylobacter jejuni, a major pathogen causing enteritis in humans. ChuZ is required for C. jejuni to use heme as the sole iron source. The crystal structure of ChuZ was resolved at 2.5 Å, and it was revealed to be a homodimer with a split-barrel fold. One heme-binding site was at the dimer interface and another novel heme-binding site was found on the protein surface. Heme was bound in this site by four histidine side-chains through hydrophobic interactions. Based on stoichiometry studies and comparisons with other proteins, the possibility that similar heme-binding site exists in homologous proteins and its possible functions are discussed. The structural and mutagenesis analyses reported here establish ChuZ and ChuZ homologs as a new bacterial heme oxygenase family apart from the canonical and IsdG/I families. Our studies provide insight into the enzymatic mechanisms and structure–function relationship of ChuZ.     

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.     

Characterization of the periplasmic heme-binding protein shut from the heme uptake system of Shigella dysenteriae

The heme uptake systems by which bacterial pathogens acquire and utilize heme have recently been described. Such systems may utilize heme directly from the host's hemeproteins or via a hemophore that sequesters and transports heme to an outer membrane receptor and subsequently to the translocating proteins by which heme is further transported into the cell. However, little is known of the heme binding and release mechanisms that facilitate the uptake of heme into the pathogenic organism. As a first step toward elucidating the molecular level events that drive heme binding and release, we have undertaken a spectroscopic and mutational study of the first purified periplasmic heme-binding protein (PBP), ShuT from Shigella dysenteriae. On the basis of sequence identity, the ShuT protein is most closely related to the class of PBPs typified by the vitamin B(12) (BtuF) and iron-hydroxamate (FhuD) PBPs and is a monomeric protein having a molecular mass of 28.5 kDa following proteolytic processing of the periplasmic signaling peptide. ShuT binds one b-type heme per monomer with high affinity and bears no significant homology with other known heme proteins. The resonance Raman, MCD, and UV-visible spectra of WT heme-ShuT are consistent with a five-coordinate high spin heme having an anionic O-bound proximal ligand. Site-directed ShuT mutants of the absolutely conserved Tyr residues, Tyr-94 (Y94A) and Tyr-228 (Y228F), which are found in all putative periplasmic heme-binding proteins, were subjected to UV-visible, resonance Raman, and MCD spectroscopic investigations of heme coordination environment and rates of heme release. The results of these experiments confirmed Tyr-94 as the only axial heme ligand and Tyr-228 as making a significant contribution to the stability of heme-loaded ShuT, albeit without directly interacting with the heme iron.     

Computational prediction of heme-binding residues by exploiting residue interaction network

Computational identification of heme-binding residues is beneficial for predicting and designing novel heme proteins. Here we proposed a novel method for heme-binding residue prediction by exploiting topological properties of these residues in the residue interaction networks derived from three-dimensional structures. Comprehensive analysis showed that key residues located in heme-binding regions are generally associated with the nodes with higher degree, closeness and betweenness, but lower clustering coefficient in the network. HemeNet, a support vector machine (SVM) based predictor, was developed to identify heme-binding residues by combining topological features with existing sequence and structural features. The results showed that incorporation of network-based features significantly improved the prediction performance. We also compared the residue interaction networks of heme proteins before and after heme binding and found that the topological features can well characterize the heme-binding sites of apo structures as well as those of holo structures, which led to reliable performance improvement as we applied HemeNet to predicting the binding residues of proteins in the heme-free state. HemeNet web server is freely accessible at http://mleg.cse.sc.edu/hemeNet/.     

A novel approach for identifying the heme-binding proteins from mouse tissues

Heme is a key cofactor in aerobic life, both in eukaryotes and prokaryotes. Because of the high reactivity of ferrous protoporphyrin IX, the reactions of heme in cells are often carried out through heme-protein complexes. Traditionally studies of heme-binding proteins have been approached on a case by case basis, thus there is a limited global view of the distribution of heme-binding proteins in different cells or tissues. The procedure described here is aimed at profiling heme-binding proteins in mouse tissues sequentially by 1) purification of heme-binding proteins by heme-agarose, an affinity chromatographic resin; 2) isolation of heme-binding proteins by SDS-PAGE or two-dimensional electrophoresis; 3) identification of heme-binding proteins by mass spectrometry. In five mouse tissues, over 600 protein spots were visualized on 2DE gel stained by Commassie blue and 154 proteins were identified by MALDI-TOF, in which most proteins belong to heme related. This methodology makes it possible to globally c     

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.     

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.     

Human and rodent amyloid-β peptides differentially bind heme: Relevance to the human susceptibility to Alzheimer’s disease

Amyloid-β (Aβ) peptides are implicated in the neurodegeneration of Alzheimer’s disease (AD). We previously investigated the mechanism of neurotoxicity of Aβ and found that human Aβ (huAβ) binds and depletes heme, forming an Aβ-heme complex with peroxidase activity. Rodent Aβ (roAβ) is identical to huAβ, except for three amino acids within the proposed heme-binding motif (Site-H). We studied and compared heme-binding between roAβ and huAβ. Unlike roAβ, huAβ binds heme tightly (K_d = 140 ± 60 nM) and forms a peroxidase. The plot of bound (huAβ-heme) vs. unbound heme fits best to a two site binding hyperbola, suggesting huAβ possesses two heme-binding sites. Consistently, a second high affinity heme-binding site was identified in the lipophilic region (site-L) of huAβ (K_d = 210 ± 80nM). The plot of (roAβ-heme) vs. unbound heme, on the other hand, was different as it fits best to a sigmoidal binding curve, indicating different binding and lower affinity of roAβ for heme (K_d = 1 μM). The effect of heme-binding to site-H on heme-binding to site-L in roAβ and huAβ is discussed. While both roAβ and huAβ form aggregates equally, rodents lack AD-like neuropathology. High huAβ/heme ratio increases the peroxidase activity. These findings suggest that depletion of regulatory heme and formation of Aβ-heme peroxidase contribute to huAβ’s neurotoxicity in the early stages of AD. Phylogenic variations in the amino acid sequence of Aβ explain tight heme-binding to huAβ and likely contribute to the increased human susceptibility to AD.     

Control of DegP-dependent degradation of c-type cytochromes by heme and the cytochrome c maturation system in Escherichia coli

c-Type cytochromes are located partially or completely in the periplasm of gram-negative bacteria, and the heme prosthetic group is covalently bound to the protein. The cytochrome c maturation (Ccm) multiprotein system is required for transport of heme to the periplasm and its covalent linkage to the peptide. Other cytochromes and hemoglobins contain a noncovalently bound heme and do not require accessory proteins for assembly. Here we show that Bradyrhizobium japonicum cytochrome c550 polypeptide accumulation in Escherichia coli was heme dependent, with very low levels found in heme-deficient cells. However, apoproteins of the periplasmic E. coli cytochrome b562 or the cytosolic Vitreoscilla hemoglobin (Vhb) accumulated independently of the heme status. Mutation of the heme-binding cysteines of cytochrome c550 or the absence of Ccm also resulted in a low apoprotein level. These levels were restored in a degP mutant strain, showing that apocytochrome c550 is degraded by the periplasmic protease DegP. Introduction of the cytochrome c heme-binding motif CXXCH into cytochrome b562 (c-b562) resulted in a c-type cytochrome covalently bound to heme in a Ccm-dependent manner. This variant polypeptide was stable in heme-deficient cells but was degraded by DegP in the absence of Ccm. Furthermore, a Vhb variant containing a periplasmic signal peptide and a CXXCH motif did not form a c-type cytochrome, but accumulation was Ccm dependent nonetheless. The data show that the cytochrome c heme-binding motif is an instability element and that stabilization by Ccm does not require ligation of the heme moiety to the protein.     

Crystal structure of HugZ, a novel heme oxygenase from Helicobacter pylori

The crystal structure of a heme oxygenase (HO) HugZ from Helicobacter pylori complexed with heme has been solved and refined at 1.8 Å resolution. HugZ is part of the iron acquisition mechanism of H. pylori, a major pathogen of human gastroenteric diseases. It is required for the adaptive colonization of H. pylori in hosts. Here, we report that HugZ is distinct from all other characterized HOs. It exists as a dimer in solution and in crystals, and the dimer adopts a split-barrel fold that is often found in FMN-binding proteins but has not been observed in hemoproteins. The heme is located at the intermonomer interface and is bound by both monomers. The heme iron is coordinated by the side chain of His²⁴⁵ and an azide molecule when it is present in crystallization conditions. Experiments show that Arg¹⁶⁶, which is involved in azide binding, is essential for HugZ enzymatic activity, whereas His²⁴⁵, surprisingly, is not, implying that HugZ has an enzymatic mechanism distinct from other HOs. The placement of the azide corroborates the observed γ-meso specificity for the heme degradation reaction, in contrast to most known HOs that have α-meso specificity. We demonstrate through sequence and structural comparisons that HugZ belongs to a new heme-binding protein family with a split-barrel fold. Members of this family are widespread in pathogenic bacteria and may play important roles in the iron acquisition of these bacteria.     

Heme utilization by nontypeable Haemophilus influenzae is essential and dependent on Sap transporter function

Bacterial strategies of innate immune evasion and essential metabolic functions are critical for commensal-host homeostasis. Previously, we showed that Sap translocator function is necessary for nontypeable Haemophilus influenzae (NTHI) behaviors that mediate diseases of the human airway. Antimicrobial peptide (AP) lethality is limited by binding mediated by the Sap complex. SapA shares homology with the dipeptide-binding protein (DppA) and the heme-binding lipoprotein (HbpA), both of which have previously been shown to bind the iron-containing compound heme, whose acquisition is essential for Haemophilus survival. Computational modeling revealed conserved SapA residues, similarly modeled to mediate heme binding in HbpA. Here, we directly demonstrate that SapA bound heme and was essential for heme utilization by iron-starved NTHI. Further, the Sap translocator permease mediated heme transport into the bacterial cytoplasm, thus defining a heretofore unknown mechanism of intracytoplasmic membrane heme transport in Haemophilus. Since we demonstrate multiple ligand specificity for the SapA-binding protein, we tested whether APs would compete with heme for SapA binding. We showed that human β-defensins 2 and 3, human cathelicidin LL-37, human neutrophil protein 1, and melittin displaced heme bound to SapA, thus supporting a hierarchy wherein immune evasion supercedes even the needed iron acquisition functions of the Sap system.