Mapping the mutational landscape for streptokinase binding to plasminogen

Group A Streptococcus (GAS) expresses streptokinase (SK), a critical virulence factor that nonenzymatically activates the host's plasminogen (PLG) to an active form (PLG_SK) via a conformational change. PLG_SK then activates PLG to plasmin, resulting in fibrinolysis and facilitating bacterial dissemination. As a potent thrombolytic, SK has been used as a therapeutic agent.

GAS SK is highly specific for human PLG, and sequence variation among SKs from different GAS strains has been linked to differences in PLG binding and disease severity. The contributions of individual amino acids to these differences remain poorly understood.

We used phage display and deep mutational scanning to map how amino acid substitutions in SK from Group C Streptococcus (GCS), which shares high sequence homology with GAS, affect its ability to bind human PLG.

We demonstrate that SK, expressed as a fusion protein with the p3 coat protein of the M13 filamentous phage, retains its ability to bind human PLG. Deep mutational scanning mapped 71% of all possible single amino acid substitutions within SK, identifying specific regions where substitutions are likely to increase or decrease its affinity for PLG.

Our findings suggest a complex protein-protein interaction in which long-range protein dynamics influence the conformational activation of PLG to PLG_SK. These data lay the foundation for linking SK variation across GCS strains to differences in virulence, mapping the determinants of GCS SK's human specificity, and potentially contributing to the development of improved therapeutics for heart attack and stroke.