title: 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.
authors: Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59718, USA.
authors: Yanchao
authors: Y
authors: Ran
authors: Hui
authors: H
authors: Zhu
authors: Mengyao
authors: M
authors: Liu
authors: Marian
authors: M
authors: Fabian
authors: John S
authors: JS
authors: Olson
authors: Roman
authors: R
authors: Aranda
authors: George N
authors: GN
authors: Phillips
authors: David M
authors: DM
authors: Dooley
authors: Benfang
authors: B
authors: Lei
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry*; D006427:Hemin / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013297:Streptococcus pyogenes / Q000378:metabolism*
chemical_list: D018528:ATP-Binding Cassette Transporters; D008565:Membrane Proteins; D006418:Heme; D006427:Hemin; D008715:Methionine; D000409:Alanine
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