Fibrinogen substrate recognition by staphylocoagulase.(pro)thrombin complexes.

Panizzi P, Friedrich R, Fuentes-Prior P, Richter K, Bock PE, Bode W
J Biol Chem. 2006 281 (2): 1179-87

PMID: 16230339 · PMCID: PMC2291351 · DOI:10.1074/jbc.M507956200

Thrombin generation and fibrinogen (Fbg) clotting are the ultimate proteolytic reactions in the blood coagulation pathway. Staphylocoagulase (SC), a protein secreted by the human pathogen Staphylococcus aureus, activates prothrombin (ProT) without proteolysis. The SC.(pro)thrombin complex recognizes Fbg as a specific substrate, converting it directly into fibrin. The crystal structure of a fully active SC fragment containing residues 1-325 (SC-(1-325)) bound to human prethrombin 2 showed previously that SC inserts its Ile(1)-Val(2) N terminus into the Ile(16) pocket of prethrombin 2, inducing a functional active site in the cognate zymogen conformationally. Exosite I of alpha-thrombin, the Fbg recognition site, and proexosite I on ProT are blocked by domain 2 of SC-(1-325). In the present studies, active site-labeled fluorescent ProT analogs were used to quantitate Fbg binding to the SC-(1-325).ProT complex. Fbg binding and cleavage are mediated by expression of a new Fbg-binding exosite on the SC-(1-325).ProT complex, resulting in formation of an (SC-(1-325).ProT)(2).Fbg pentameric complex with a dissociation constant of 8-34 nm. In both crystal structures, the SC-(1-325).(pre)thrombin complexes form dimers, with both proteinases/zymogens facing each other over a large U-shaped cleft, through which the Fbg substrate could thread. On this basis, a molecular model of the pentameric (SC-(1-325).thrombin)(2).Fbg encounter complex was generated, which explains the coagulant properties and efficient Fbg conversion. The results provide new insight into the mechanism that mediates high affinity Fbg binding and cleavage as a substrate of SC.(pro)thrombin complexes, a process that is central to the molecular pathology of S. aureus endocarditis.

MeSH Terms (23)

Amino Acid Sequence Binding Sites Chromatography, Affinity Coagulase Crystallography, X-Ray Dimerization Endocarditis Enzyme Precursors Fibrinogen Humans Models, Chemical Models, Molecular Molecular Conformation Molecular Sequence Data Protein Binding Protein Conformation Protein Structure, Secondary Protein Structure, Tertiary Prothrombin Sequence Homology, Amino Acid Staphylococcus aureus Substrate Specificity Thrombin

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