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

In Staphylococcus aureus-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 Ile1-Val2 into the ProT Ile16 pocket, forming a salt bridge with ProT's Asp194, 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(R155,271,284Q) (ProT(QQQ)), 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 wild-type SC(1-246) to complete loss of function. Substrate binding to ProT(QQQ) 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 Val1-Val2, Ile1-Ala2, and Leu1-Val2 variants exhibiting ProT(QQQ) affinity and activation potency comparable to wild-type SC(1-246). Weaker binding variants typically had reduced activation rates, although at near-saturating ProT(QQQ) levels, several variants exhibited limiting rates similar to or higher than that of wild-type SC(1-246). The Ile16 pocket in ProT(QQQ) appears to favor non-polar, non-aromatic residues at SC positions 1 and 2. Our results suggest that SC variants other than wild-type Ile1-Val2-Thr3 might emerge with similar ProT-activating efficiency.