title: Structural basis for reduced staphylocoagulase-mediated bovine prothrombin activation.
abstract: 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 Ile(16) 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 approximately 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 approximately 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 Arg(144) and Arg(145) would likely interfere with insertion of the SC N terminus, thus explaining the greatly reduced activation of bovine ProT.
authors.affiliation: Proteinase Research Group, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Friedrich
authors.forename: Peter
authors.initials: P
authors.lastname: Panizzi
authors.forename: Shun-Ichiro
authors.initials: S
authors.lastname: Kawabata
authors.forename: Wolfram
authors.initials: W
authors.lastname: Bode
authors.forename: Paul E
authors.initials: PE
authors.lastname: Bock
authors.forename: Pablo
authors.initials: P
authors.lastname: Fuentes-Prior
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D001777:Blood Coagulation; D020134:Catalytic Domain; D002417:Cattle; D003030:Coagulase / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D004792:Enzyme Precursors / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011516:Prothrombin / Q000737:chemistry*; D013211:Staphylococcus aureus / Q000378:metabolism; D013917:Thrombin / Q000737:chemistry
chemical_list: D003030:Coagulase; D004792:Enzyme Precursors; D011516:Prothrombin; D001120:Arginine; D013917:Thrombin