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J. Biol. Chem., Vol. 281, Issue 2, 1188-1195, January 13, 2006

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Structural Basis for Reduced Staphylocoagulase-mediated Bovine Prothrombin Activation^*

Rainer Friedrich1, Peter Panizzi12, Shun-Ichiro Kawabata¶, Wolfram Bode, Paul E. Bock3, and Pablo Fuentes-Prior||

From the Proteinase Research Group, Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany, the Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2561, the ^¶Department of Biology, Kyushu University, Fukuoka 812-8581, Japan, and 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

Staphylocoagulase (SC) is a protein secreted by the human pathogen, Staphylococcus aureus, that activates human prothrombin (ProT) by inducing a conformational change. SC-bound ProT efficiently clots fibrinogen, thus bypassing the physiological blood coagulation pathway. The crystal structure of a fully active SC fragment, SC-(1-325), bound to human prethrombin 2 showed that the SC-(1-325) N terminus inserts into the Ile¹⁶ pocket of prethrombin 2, thereby inducing expression of a functional catalytic site in the cognate zymogen without peptide bond cleavage. As shown here, SC-(1-325) binds to bovine and human ProT with similar affinity but activates the bovine zymogen only very poorly. By contrast to the 2-fold difference in chromogenic substrate kinetic constants between human thrombin and the SC-(1-325)·human (pro)thrombin complexes, SC-(1-325)·bovine ProT shows a 3,500-fold lower k_cat/K_m compared with free bovine thrombin, because of a 47-fold increase in K_m and a 67-fold decrease in k_cat. The SC-(1-325)·bovine ProT complex is 5,800-fold less active compared with its human counterpart. Comparison of human and bovine fibrinogen as substrates of human and bovine thrombin and the SC-(1-325)·(pro)thrombin complexes indicates that the species specificity of SC-(1-325) cofactor activity is determined primarily by differences in conformational activation of bound ProT. These results suggest that the catalytic site in the SC-(1-325)·bovine ProT complex is incompletely formed. The current crystal structure of SC-(1-325)·bovine thrombin reveals that SC would dock similarly to the bovine proenzyme, whereas the bovine (pro)thrombin-characteristic residues Arg¹⁴⁴ and Arg¹⁴⁵ would likely interfere with insertion of the SC N terminus, thus explaining the greatly reduced activation of bovine ProT.

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

The atomic coordinates and structure factors (code 2A1D) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grant HL071544 (to P. E. B.) and SFB 469 of the Deutsche Forschungsgemeinschaft 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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