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

摘要: In Staphylococcus aureus-caused endocarditis, the pathogen secretes staphylocoagulase (SC), thereby activating human prothrombin (ProT) and evading immune clearance. A previous structural comparison of SC(1-325) fragment bound to thrombin its inactive precursor prethrombin 2 has indicated that SC activates ProT by inserting N-terminal dipeptide Ile1-Val2 into Ile16 pocket, forming a salt bridge with ProT's Asp194, stabilizing active conformation. We hypothesized these residues modulate binding activation. Here, we generated labeled SC(1-246) as probe for competitively defining affinities variants preselected modeling. Using ProT(R155Q,R271Q,R284Q) (ProTQQQ), variant refractory prothrombinase- or thrombin-mediated cleavage, observed between ∼1 650 nm activation potencies ranging from 1.8-fold WT complete loss function. Substrate ProTQQQ caused allosteric tightening affinity most variants, consistent zymogen through occupation specificity pocket. Conservative changes at positions 1 were well-tolerated, Val1-Val2, Ile1-Ala2, Leu1-Val2 exhibiting potency comparable SC(1-246). Weaker typically had reduced rates, although near-saturating levels, several exhibited limiting rates similar higher than The pocket in appears favor nonpolar, nonaromatic 2. Our results suggest other Ile1-Val2-Thr3 might emerge ProT-activating efficiency.