Dataset Information

The near-iron transporter (NEAT) domains of the anthrax hemophore IsdX2 require a critical glutamine to extract heme from methemoglobin.

ABSTRACT: 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.

SUBMITTER: Honsa ES

PROVIDER: S-EPMC3605663 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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The near-iron transporter (NEAT) domains of the anthrax hemophore IsdX2 require a critical glutamine to extract heme from methemoglobin.

Honsa Erin S ES Owens Cedric P CP Goulding Celia W CW Maresso Anthony W AW

The Journal of biological chemistry 20130130 12

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 ir ...[more]

PMID: 23364793

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Project description:Iron is essential for bacterial survival, being required for numerous biological processes. NEAr-iron Transporter (NEAT) domains have been studied in pathogenic Gram-positive bacteria to understand how their proteins obtain heme as an iron source during infection. While a 2002 study initially discovered and annotated the NEAT domain encoded by the genomes of several Gram-positive bacteria, there remains a scarcity of information regarding the conservation and distribution of NEAT domains throughout the bacterial kingdom, and whether these domains are restricted to pathogenic bacteria. This study aims to expand upon initial bioinformatics analysis of predicted NEAT domains, by exploring their evolution and conserved function. This information was used to identify new candidate domains in both pathogenic and nonpathogenic organisms. We also searched metagenomic datasets, specifically sequence from the Human Microbiome Project. Here, we report a comprehensive phylogenetic analysis of 343 NEAT domains, encoded by Gram-positive bacteria, mostly within the phylum Firmicutes, with the exception of Eggerthella sp. (Actinobacteria) and an unclassified Mollicutes bacterium (Tenericutes). No new NEAT sequences were identified in the HMP dataset. We detected specific groups of NEAT domains based on phylogeny of protein sequences, including a cluster of novel clostridial NEAT domains. We also identified environmental and soil organisms that encode putative NEAT proteins. Biochemical analysis of heme binding by a NEAT domain from a protein encoded by the soil-dwelling organism Paenibacillus polymyxa demonstrated that the domain is homologous in function to NEAT domains encoded by pathogenic bacteria. Together, this study provides the first global bioinformatics analysis and phylogenetic evidence that NEAT domains have a strong conservation of function, despite group-specific differences at the amino acid level. These findings will provide information useful for future projects concerning the structure and function of NEAT domains, particularly in pathogens where they have yet to be studied.

Project description: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 3(10)-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 3(10)-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.

Dataset Information

The near-iron transporter (NEAT) domains of the anthrax hemophore IsdX2 require a critical glutamine to extract heme from methemoglobin.

Publications

The near-iron transporter (NEAT) domains of the anthrax hemophore IsdX2 require a critical glutamine to extract heme from methemoglobin.

Similar Datasets

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