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Mapping of the fibrinogen-binding site on the staphylocoagulase C-terminal repeat region

Fibrin (Fbn) deposits are a hallmark of staphylocoagulase (SC)-positive endocarditis. Binding of the N terminus of Staphylococcus aureus SC to host prothrombin triggers formation of an active SC·prothrombin∗ complex that cleaves host fibrinogen to Fbn. In addition, the C-terminal domain of the proto...

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Autores principales: Maddur, Ashoka A., Voehler, Markus, Panizzi, Peter, Meiler, Jens, Bock, Paul E., Verhamme, Ingrid M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8761706/
https://www.ncbi.nlm.nih.gov/pubmed/34915025
http://dx.doi.org/10.1016/j.jbc.2021.101493
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author Maddur, Ashoka A.
Voehler, Markus
Panizzi, Peter
Meiler, Jens
Bock, Paul E.
Verhamme, Ingrid M.
author_facet Maddur, Ashoka A.
Voehler, Markus
Panizzi, Peter
Meiler, Jens
Bock, Paul E.
Verhamme, Ingrid M.
author_sort Maddur, Ashoka A.
collection PubMed
description Fibrin (Fbn) deposits are a hallmark of staphylocoagulase (SC)-positive endocarditis. Binding of the N terminus of Staphylococcus aureus SC to host prothrombin triggers formation of an active SC·prothrombin∗ complex that cleaves host fibrinogen to Fbn. In addition, the C-terminal domain of the prototypical SC contains one pseudorepeat (PR) and seven repeats (R1 → R7) that bind fibrinogen/Fbn fragment D (frag D) by a mechanism that is unclear. Here, we define affinities and stoichiometries of frag D binding to C-terminal SC constructs, using fluorescence equilibrium binding, NMR titration, alanine scanning, and native PAGE. We found that constructs containing the PR and single repeats bound frag D with K(D) ∼50 to 130 nM and a 1:1 stoichiometry, indicating a conserved binding site bridging the PR and each repeat. NMR titration of PR–R7 with frag D revealed that residues 22 to 49, bridging PR and R7, constituted the minimal peptide (MP) for binding, corroborated by alanine scanning, and binding of labeled MP to frag D. MP alignment with the PR–R and inter-repeat junctions identified critical conserved residues. Full-length PR–(R1 → R7) bound frag D with K(D) ∼20 nM and a stoichiometry of 1:5, whereas constructs containing the PR and various three repeats competed with PR–(R1 → R7) for frag D binding, with a 1:3 stoichiometry. These findings are consistent with binding at PR–R and R–R junctions with modest inter-repeat sequence variability. CD of PR–R7 and PR–(R1 → R7) suggested a disordered flexible structure, allowing binding of multiple fibrin(ogen) molecules. Taken together, these results provide insights into pathogen localization on host fibrin networks.
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spelling pubmed-87617062022-01-20 Mapping of the fibrinogen-binding site on the staphylocoagulase C-terminal repeat region Maddur, Ashoka A. Voehler, Markus Panizzi, Peter Meiler, Jens Bock, Paul E. Verhamme, Ingrid M. J Biol Chem Research Article Fibrin (Fbn) deposits are a hallmark of staphylocoagulase (SC)-positive endocarditis. Binding of the N terminus of Staphylococcus aureus SC to host prothrombin triggers formation of an active SC·prothrombin∗ complex that cleaves host fibrinogen to Fbn. In addition, the C-terminal domain of the prototypical SC contains one pseudorepeat (PR) and seven repeats (R1 → R7) that bind fibrinogen/Fbn fragment D (frag D) by a mechanism that is unclear. Here, we define affinities and stoichiometries of frag D binding to C-terminal SC constructs, using fluorescence equilibrium binding, NMR titration, alanine scanning, and native PAGE. We found that constructs containing the PR and single repeats bound frag D with K(D) ∼50 to 130 nM and a 1:1 stoichiometry, indicating a conserved binding site bridging the PR and each repeat. NMR titration of PR–R7 with frag D revealed that residues 22 to 49, bridging PR and R7, constituted the minimal peptide (MP) for binding, corroborated by alanine scanning, and binding of labeled MP to frag D. MP alignment with the PR–R and inter-repeat junctions identified critical conserved residues. Full-length PR–(R1 → R7) bound frag D with K(D) ∼20 nM and a stoichiometry of 1:5, whereas constructs containing the PR and various three repeats competed with PR–(R1 → R7) for frag D binding, with a 1:3 stoichiometry. These findings are consistent with binding at PR–R and R–R junctions with modest inter-repeat sequence variability. CD of PR–R7 and PR–(R1 → R7) suggested a disordered flexible structure, allowing binding of multiple fibrin(ogen) molecules. Taken together, these results provide insights into pathogen localization on host fibrin networks. American Society for Biochemistry and Molecular Biology 2021-12-13 /pmc/articles/PMC8761706/ /pubmed/34915025 http://dx.doi.org/10.1016/j.jbc.2021.101493 Text en © 2021 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Research Article
Maddur, Ashoka A.
Voehler, Markus
Panizzi, Peter
Meiler, Jens
Bock, Paul E.
Verhamme, Ingrid M.
Mapping of the fibrinogen-binding site on the staphylocoagulase C-terminal repeat region
title Mapping of the fibrinogen-binding site on the staphylocoagulase C-terminal repeat region
title_full Mapping of the fibrinogen-binding site on the staphylocoagulase C-terminal repeat region
title_fullStr Mapping of the fibrinogen-binding site on the staphylocoagulase C-terminal repeat region
title_full_unstemmed Mapping of the fibrinogen-binding site on the staphylocoagulase C-terminal repeat region
title_short Mapping of the fibrinogen-binding site on the staphylocoagulase C-terminal repeat region
title_sort mapping of the fibrinogen-binding site on the staphylocoagulase c-terminal repeat region
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8761706/
https://www.ncbi.nlm.nih.gov/pubmed/34915025
http://dx.doi.org/10.1016/j.jbc.2021.101493
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