In silico engineering fibrin-directed streptokinase - kringle 2 fusion: Structural insights into fibrin interaction and plasminogen recognition.

Streptokinase (SK) is an effective plasminogen-activating fibrinolytic protein, but its lack of intrinsic fibrin specificity limits thrombus-directed fibrinolysis. We computationally evaluated an engineered SK-K2 fusion in which the fibrin-interacting Kringle 2 (K2) domain of human tissue-type plasminogen activator was appended to the SK C-terminus. Structural modeling showed high K2 confidence (mean pLDDT = 85.67), and all 22 K2 residues at the predicted fibrin interface had pLDDT values ≥70 (mean = 89.88). HDOCK ranked SK-K2-fibrin more favorably than native SK-fibrin (-276.71 vs. -249.24), while PRODIGY predicted a more favorable binding free energy (-14.9 vs. -13.3 kcal/mol). Although native SK showed a more favorable HDOCK score toward plasminogen, both plasminogen complexes had identical PRODIGY-predicted binding free energies (-11.2 kcal/mol) and closely comparable dissociation constants, indicating no marked computational loss in predicted plasminogen-binding characteristics after K2 fusion. PDBePISA showed direct K2 participation and a larger chain-matched fibrin interface for SK-K2 than native SK, with six hydrogen bonds and an Asp291-Arg515 salt bridge (2.966 Å) in the HDOCK-derived complex. An independent MOE workflow reproduced the fibrin-directed ranking, with SK-K2 showing a more favorable S score than native SK (-101.8501 vs. -93.9472). PDBePISA analysis of the independent MOE complex identified Arg515 in a second salt bridge, Arg515-Glu220 (3.369 Å), providing residue-level convergence across both docking models. These findings support the structural rationale for C-terminal K2 engineering of SK and prioritize SK-K2, particularly Arg515, for experimental evaluation of fibrin association, plasminogen activation, and clot-directed fibrinolysis.