Structural basis for reduced staphylocoagulase-mediated bovine prothrombin activation

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.