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

Honsa, Erin S.; Owens, Cedric P.; Goulding, Celia W.; Maresso, Anthony W.

doi:10.1074/jbc.m112.430009

Cited by 24 publications

(41 citation statements)

References 56 publications

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“…As Hal N does not contain the characteristic heme-binding motif and has a phenylalanine in the fifth position (YDKEF) (53), we determined the structure of heme-Hal N to a 3.0-Å resolution to shed light on the mode of heme binding. The structure of Hal N consists of an immunoglobulin-like fold, as observed in multiple NEAT domain crystal structures (56,68,69,80,81), with eight ␤-strands arranged in two antiparallel ␤-sheets that form a ␤-sandwich (Fig. 5A).…”

Section: Resultsmentioning

confidence: 99%

“…All bacilli and staphylococcal NEAT domains studied thus far have a conserved heme-binding motif, YXXXY (56,68,69,80,81). In these structures, the first tyrosine coordinates with heme-iron as the axial ligand, and the second tyrosine hydrogen bonds (Hbonds) to the axial tyrosine, stabilizing the interaction.…”

Section: Resultsmentioning

confidence: 99%

“…We next purified each individual NEAT domain of IsdX1, IsdC, BslK, or Hal or all five NEAT domains as a polypeptide from IsdX2 from E. coli by GST affinity chromatography. Each preparation was assessed for purity by SDS-PAGE, the protein concentration was determined by using the Bradford method, and the ability to bind heme (an indication of a functional, folded protein domain) was assessed by using assays previously developed in our laboratory (68). Lipopolysaccharide (LPS) was removed by using a series of endotoxin-binding resins, and pure preparations were mixed with complete Freund's adjuvant (CFA).…”

Section: Resultsmentioning

confidence: 99%

“…Thus, Y112 may act as an alternate ligand for heme-iron or may be required for the extraction and transfer of heme from hemoglobin, possibly at the level of the NEAT-hemoglobin interaction, as observed for R54 in IsdX1 (56). Even though Hal N has a phenylalanine (F116) substitution at the second tyrosine within the highly conserved heme-binding motif (YXXXY), heme-iron is still coordinated by the first tyrosine (Y112), as observed for all other S. aureus and B. anthracis NEAT domains (56,68,69,80,81), and F116 -stacks with the heme pyrrole ring. Interestingly, the second NEAT domain of the proposed hemophore from Listeria monocytogenes (Hbp2 N2 ) also harbors an amino acid substitution at the second Tyr in the heme binding motif, to an alanine and not a phenylalanine, as observed for Hal N .…”

Section: Discussionmentioning

confidence: 99%

“…3SZ6) (56), B. anthracis IsdX2 N5 (PDB accession no. 4H8P) (68), and S. aureus IsdA (PDB accession no. 2ITE) (69), followed by additional rounds of molecular replacement and autobuilding in Phenix.MR-Rosetta (67).…”

Section: Methodsmentioning

confidence: 99%

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Progress toward the Development of a NEAT Protein Vaccine for Anthrax Disease

et al. 2016

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Bacillus anthracis is a sporulating Gram-positive bacterium that is the causative agent of anthrax and a potential weapon of bioterrorism. The U.S.-licensed anthrax vaccine is made from an incompletely characterized culture supernatant of a nonencapsulated, toxigenic strain (anthrax vaccine absorbed [AVA]) whose primary protective component is thought to be protective antigen (PA). AVA is effective in protecting animals and elicits toxin-neutralizing antibodies in humans, but enthusiasm is dampened by its undefined composition, multishot regimen, recommended boosters, and potential for adverse reactions. Improving next-generation anthrax vaccines is important to safeguard citizens and the military. Here, we report that vaccination with recombinant forms of a conserved domain (near-iron transporter [NEAT]), common in Gram-positive pathogens, elicits protection in a murine model of B. anthracis infection. Protection was observed with both Freund's and alum adjuvants, given subcutaneously and intramuscularly, respectively, with a mixed composite of NEATs. Protection correlated with an antibody response against the NEAT domains and a decrease in the numbers of bacteria in major organs. Anti-NEAT antibodies promote opsonophagocytosis of bacilli by alveolar macrophages. To guide the development of inactive and safe NEAT antigens, we also report the crystal structure of one of the NEAT domains (Hal) and identify critical residues mediating its heme-binding and acquisition activity. These results indicate that we should consider NEAT proteins in the development of an improved antianthrax vaccine.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Progress toward the Development of a NEAT Protein Vaccine for Anthrax Disease

et al. 2016

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NMR experiments redefine the hemoglobin binding properties of bacterial NEAr‐iron Transporter domains

et al. 2019

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Iron is a versatile metal cofactor that is used in a wide range of essential cellular processes. During infections, many bacterial pathogens acquire iron from human hemoglobin (Hb), which contains the majority of the body's total iron content in the form of heme (iron protoporphyrin IX). Clinically important Gram-positive bacterial pathogens scavenge heme using an array of secreted and cell-wall-associated receptors that contain NEAr-iron Transporter (NEAT) domains. Experimentally defining the Hb binding properties of NEAT domains has been challenging, limiting our understanding of their function in heme uptake. Here we show that solution-state NMR spectroscopy is a powerful tool to define the Hb binding properties of NEAT domains. The utility of this method is demonstrated using the NEAT domains from Bacillus anthracis and Listeria monocytogenes. Our results are compatible with the existence of at least two types of NEAT domains that are capable of interacting with either Hb or heme. These binding properties can be predicted from their primary sequences, with Hb-and heme-binding NEAT domains being distinguished by the presence of (F/Y)YH(Y/F) and S/YXXXY motifs, respectively. The results of this work should enable the functions of a wide range of NEAT domain containing proteins in pathogenic bacteria to be reliably predicted.Additional Supporting Information may be found in the online version of this article.Significance statement: During infections, many bacterial pathogens use NEAr-iron Transporter (NEAT) domains to acquire iron, an essential nutrient that is required for microbial growth. Here we show that NMR is a powerful tool to assess the hemoglobin binding properties of these domains. Our results resolve prior discrepancies in the literature and help to define the functions of a wide range of NEAT domains based on their amino acid primary sequences.

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