Specificity and affinity of the N-terminal residues in staphylocoagulase in binding to prothrombin.

In -caused endocarditis, the pathogen secretes staphylocoagulase (SC), thereby activating human prothrombin (ProT) and evading immune clearance. A previous structural comparison of the SC(1-325) fragment bound to thrombin and its inactive precursor prethrombin 2 has indicated that SC activates ProT by inserting its N-terminal dipeptide Ile-Val into the ProT Ile pocket, forming a salt bridge with ProT's Asp, thereby stabilizing the active conformation. We hypothesized that these N-terminal SC residues modulate ProT binding and activation. Here, we generated labeled SC(1-246) as a probe for competitively defining the affinities of N-terminal SC(1-246) variants preselected by modeling. Using ProT(R155Q,R271Q,R284Q) (ProT), a variant refractory to prothrombinase- or thrombin-mediated cleavage, we observed variant affinities between ∼1 and 650 nm and activation potencies ranging from 1.8-fold that of WT SC(1-246) to complete loss of function. Substrate binding to ProT caused allosteric tightening of the affinity of most SC(1-246) variants, consistent with zymogen activation through occupation of the specificity pocket. Conservative changes at positions 1 and 2 were well-tolerated, with Val-Val, Ile-Ala, and Leu-Val variants exhibiting ProT affinity and activation potency comparable with WT SC(1-246). Weaker binding variants typically had reduced activation rates, although at near-saturating ProT levels, several variants exhibited limiting rates similar to or higher than that of WT SC(1-246). The Ile pocket in ProT appears to favor nonpolar, nonaromatic residues at SC positions 1 and 2. Our results suggest that SC variants other than WT Ile-Val-Thr might emerge with similar ProT-activating efficiency.