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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...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2021
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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. |
format | Online Article Text |
id | pubmed-8761706 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
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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