Solution Structure of the NEAT (NEAr Transporter) Domain from IsdH/HarA: the Human Hemoglobin Receptor in Staphylococcus aureus
Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, and the Molecular Biology Institute, University of California-Los Angeles, 611 Charles E. Young Drive, Los Angeles, CA 90095-1570, USA. Journal of Molecular Biology
(Impact Factor: 4.33).
08/2006; 360(2):435-47. DOI: 10.1016/j.jmb.2006.05.019
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.
Available from: Sarah Highlander
- "The non-heme binding NEAT domains of S. aureus (IsdB NEAT1 and IsdH NEAT1 and NEAT2) do not contain the canonical SXXXXY 310-helix sequence (FYHYAS, YYHFFS and FYHYAS, respectively), nor the YXXXY heme-binding sequence (EEKYD, ETNYD, and HEDYD, respectively). Since they lack both functional regions, these domains cannot scavenge heme from hemoglobin, or bind heme , , . Instead, they may serve as structural support for the heme-acquiring NEAT domains of the respective proteins. "
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ABSTRACT: 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.
PLoS ONE 08/2014; 9(8):e104794. DOI:10.1371/journal.pone.0104794 · 3.23 Impact Factor
Available from: onlinelibrary.wiley.com
- "The main differences are the loops on the protein surfaces. The structures of the holo-forms of IsdA-N1, IsdC-N1, IsdB-N2 and IsdH-N3 show overall Haemophore functions revisited 623 similar haem binding sites (Fig. 4) (Pilpa et al., 2006; Grigg et al., 2007a; Sharp et al., 2007; Villareal et al., 2008; Watanabe et al., 2008). The haem is located in a hydrophobic pocket and positioned on one b-strand (b-strand 8); it is covered by a 3–10 helix (N-terminal helix) that is, in some cases, less clearly defined in the apo form (Villareal et al., 2008) (see Fig. 4A and B). "
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ABSTRACT: Haem is the major iron source for bacteria that develop in higher organisms. In these hosts, bacteria have to cope with nutritional immunity imposed by the host, since haem and iron are tightly bound to carrier and storage proteins. Siderophores were the first recognized fighters in the battle for iron between bacteria and host. They are non-proteinaceus organic molecules having an extremely high affinity for Fe(3+) and able to extract it from host proteins. Haemophores, that display functional analogy with siderophores, were more recently discovered. They are a class of secreted proteins with a high affinity for haem; they are able to extract haem from host haemoproteins and deliver it to specific receptors that internalize haem. In the past few years, a wealth of data has accumulated on haem acquisition systems that are dependent on surface exposed/secreted bacterial proteins. They promote haem transfer from its initial source (in most cases, a eukaryotic haem binding protein) to the transporter that carries out the membrane crossing step. Here we review recent discoveries in this field, with particular emphasis on similar and dissimilar mechanisms in haemophores and siderophores, from the initial host source to the binding protein/receptor at the cell surface.
Molecular Microbiology 06/2012; 85(4):618-31. DOI:10.1111/j.1365-2958.2012.08136.x · 4.42 Impact Factor
Available from: Zehava Eichenbaum
- "The third NEAT domain of IsdH (IsdHN3) is also exclusively a heme-binding domain (Watanabe et al., 2008, Pilpa et al., 2009) and IsdBN2 binds heme as well (Tiedemann et al., 2008). In contrast, the two N-terminal NEAT domains of IsdH (HarA) have demonstrated binding to hemoglobin and hemoglobin/ haptoglobin, respectively (Dryla et al., 2003, Dryla et al., 2007, Pilpa et al., 2006). Secreted or cell wall anchored NEAT proteins which are central to heme acquisition pathways were identified in other Gram-positive pathogens including Listeria monocytogenes (Jin et al., 2006), Bacillus anthracis, (Gat et al., 2008, Maresso et al., 2006), and Bacillus cereus (Daou et al., 2009). "
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ABSTRACT: 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.
Molecular Microbiology 11/2010; 78(3):739-56. DOI:10.1111/j.1365-2958.2010.07367.x · 4.42 Impact Factor
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