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

Abstract

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(R155Q,R271Q,R284Q) (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 WT 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 with WT 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 WT SC(1-246). The Ile¹⁶ pocket in ProT^QQQ appears to favor nonpolar, nonaromatic residues at SC positions 1 and 2. Our results suggest that SC variants other than WT Ile¹-Val²-Thr³ might emerge with similar ProT-activating efficiency.

Keywords: Staphylococcus aureus (S. aureus); affinity; clot formation; coagulation; coagulation factor; competitive equilibrium binding; endocarditis; equilibrium binding; fibrin; kinetics; ligand-binding protein; prothrombin; specificity; staphylocoagulase (SC); virulence factor.