Dataset Information

Bis-methionine ligation to heme iron in the streptococcal cell surface protein Shp facilitates rapid hemin transfer to HtsA of the HtsABC transporter.

ABSTRACT: The surface protein Shp of Streptococcus pyogenes rapidly transfers its hemin to HtsA, the lipoprotein component of the HtsABC transporter, in a concerted two-step process with one kinetic phase. The structural basis and molecular mechanism of this hemin transfer have been explored by mutagenesis and truncation of Shp. The heme-binding domain of Shp is in the amino-terminal region and is functionally active by itself, although inclusion of the COOH-terminal domain speeds up the process approximately 10-fold. Single alanine replacements of the axial methionine 66 and 153 ligands (Shp(M66A) and Shp(M153A)) cause formation of pentacoordinate hemin-Met complexes. The association equilibrium constants for hemin binding to wild-type, M66A, and M153A Shp are 5,300, 22,000, and 38 microM(-1), respectively, showing that the Met(153)-Fe bond is critical for high affinity binding and that Met(66) destabilizes hemin binding to facilitate its rapid transfer. Shp(M66A) and Shp(M153A) rapidly bind to hemin-free HtsA (apoHtsA), forming stable transfer intermediates. These intermediates appear to be Shp-hemin-HtsA complexes with one axial ligand from each protein and decay to the products with rate constants of 0.4-3 s(-1). Thus, the M66A and M153A replacements alter the kinetic mechanism and unexpectedly slow down hemin transfer by stabilizing the intermediates. These results, in combination with the structure of the Shp heme-binding domain, allow us to propose a "plug-in" mechanism in which side chains from apoHtsA are inserted into the axial positions of hemin in Shp to extract it from the surface protein and pull it into the transporter active site.

SUBMITTER: Ran Y

PROVIDER: S-EPMC2424027 | biostudies-literature | 2007 Oct

REPOSITORIES: biostudies-literature

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Bis-methionine ligation to heme iron in the streptococcal cell surface protein Shp facilitates rapid hemin transfer to HtsA of the HtsABC transporter.

Ran Yanchao Y Zhu Hui H Liu Mengyao M Fabian Marian M Olson John S JS Aranda Roman R Phillips George N GN Dooley David M DM Lei Benfang B

The Journal of biological chemistry 20070814 43

The surface protein Shp of Streptococcus pyogenes rapidly transfers its hemin to HtsA, the lipoprotein component of the HtsABC transporter, in a concerted two-step process with one kinetic phase. The structural basis and molecular mechanism of this hemin transfer have been explored by mutagenesis and truncation of Shp. The heme-binding domain of Shp is in the amino-terminal region and is functionally active by itself, although inclusion of the COOH-terminal domain speeds up the process approxima ...[more]

PMID: 17699155

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Project description:A large portion of biological iron is found in the form of an iron-protoporphyrin IX complex, or heme. In the human host environment, which is exceptionally poor in free iron, heme iron, particularly from hemoglobin, constitutes a major source of iron for invading microbial pathogens. Several fungi were shown to utilize free heme, and Candida albicans, a major opportunistic pathogen, is able both to capture free heme and to extract heme from hemoglobin using a network of extracellular hemophores. Human serum albumin (HSA) is the most abundant host heme-scavenging protein. Tight binding of heme by HSA restricts its toxic chemical reactivity and could diminish its availability as an iron source for pathogenic microbes. We found, however, that rather than inhibiting heme utilization, HSA greatly increases availability of heme as an iron source for C. albicans and other fungi. In contrast, hemopexin, a low-abundance but high-affinity heme-scavenging serum protein, does inhibit heme utilization by C. albicans However, inhibition by hemopexin is mitigated in the presence of HSA. Utilization of albumin-bound heme requires the same hemophore cascade as that which mediates hemoglobin-iron utilization. Accordingly, we found that the C. albicans hemophores are able to extract heme bound to HSA in vitro Since many common drugs are known to bind to HSA, we tested whether they could interfere with heme-iron utilization. We show that utilization of albumin-bound heme by C. albicans can be inhibited by the anti-inflammatory drugs naproxen and salicylic acid.IMPORTANCE Heme constitutes a major iron source for microorganisms and particularly for pathogenic microbes; to overcome the iron scarcity in the animal host, many pathogenic bacteria and fungi have developed systems to extract and take up heme from host proteins such as hemoglobin. Microbial heme uptake mechanisms are usually studied using growth media containing free heme or hemoglobin as a sole iron source. However, the animal host contains heme-scavenging proteins that could prevent this uptake. In the human host in particular, the most abundant serum heme-binding protein is albumin. Surprisingly, however, we found that in the case of fungi of the Candida species family, albumin promoted rather than prevented heme utilization. Albumin thus constitutes a human-specific factor that can affect heme-iron utilization and could serve as target for preventing heme-iron utilization by fungal pathogens. As a proof of principle, we identify two drugs that can inhibit albumin-stimulated heme utilization.

Dataset Information

Bis-methionine ligation to heme iron in the streptococcal cell surface protein Shp facilitates rapid hemin transfer to HtsA of the HtsABC transporter.

Publications

Bis-methionine ligation to heme iron in the streptococcal cell surface protein Shp facilitates rapid hemin transfer to HtsA of the HtsABC transporter.

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