Dissecting the molecular basis of reduced stability in staphylokinase variant 42D

Staphylokinase (SAK) is a highly fibrin-specific plasminogen activator with considerable potential as a thrombolytic agent. Among the three naturally occurring SAK variants, STAR and 42D exhibit comparable fibrinolytic activities but markedly different thermal stabilities. Although the reduced stability of 42D has been experimentally demonstrated, the molecular basis underlying this destabilization remains unclear. To identify residues responsible for the decreased stability of 42D, we systematically reconstructed the mutational transition from STAR to 42D by generating all corresponding single- and double-point mutants. Variants were characterized using differential scanning calorimetry (DSC), a fibrin matrix lysis assay, FoldX stability calculations, and molecular dynamics simulations. DSC measurements revealed that the G36R substitution is the principal determinant of the reduced thermal stability of 42D, decreasing the melting temperature by 11.9°C relative to STAR, whereas H43R exerts a compensatory stabilizing effect. FoldX calculations identified increased backbone conformational entropy penalties and van der Waals clashes as prominent energetic features associated with G36R-mediated destabilization. Molecular dynamics simulations demonstrated that G36R does not induce global unfolding but rather causes localized perturbations in secondary-structure organization. Importantly, the naturally occurring 42D variant exhibited a similar pattern of localized structural perturbations, supporting a major contribution of G36R to its reduced stability. Despite substantial differences in thermal stability, most variants retained fibrinolytic activities comparable to that of STAR, suggesting only a partial overlap between the determinants governing stability and function. Collectively, our results resolve the individual contributions of the three substitutions underlying the stability difference between STAR and 42D and identify G36R as the principal destabilizing mutation. These findings improve our understanding of sequence-stability relationships in SAK and may facilitate the rational engineering of thrombolytic proteins with enhanced developability and therapeutic potential.