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J. Biol. Chem., Vol. 281, Issue 2, 1179-1187, January 13, 2006

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Fibrinogen Substrate Recognition by Staphylocoagulase·(Pro)thrombin Complexes^*

Peter Panizzi12, Rainer Friedrich1, Pablo Fuentes-Prior¶, Klaus Richter||, Paul E. Bock3, and Wolfram Bode

From the Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, the Proteinase Research Group, Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany, the ^¶Cardiovascular Research Center, Institut Català de Ciències Cardiovasculars-Consejo Superior de Investigaciones Cientificas, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain, and the ^||Department of Biotechnology, Technical University Munich, D-85747 Garching, Germany

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 Ile¹-Val² N terminus into the Ile¹⁶ pocket of prethrombin 2, inducing a functional active site in the cognate zymogen conformationally. Exosite I of -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.

Received for publication, July 21, 2005 , and in revised form, October 14, 2005.

^* This work was supported by National Institutes of Health Grants HL038779 and HL071544 (to P. E. B.) and by the SPINE Project QLG2-CT-2002-00988 of the European Union and the Fonds der Chemischen Industrie (to W. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work.

² Supported in part by National Institutes of Health Training Grant HL07751.

³ To whom correspondence should be addressed: Dept. of Pathology, Vanderbilt University School of Medicine, C3321A Medical Center North, Nashville, TN 37232-2561. Tel.: 615-343-9863; Fax: 615-322-1855; E-mail: paul.bock{at}vanderbilt.edu.

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