Integrative experimental and computational approach for the development of novel plasminogen activators from truncated streptokinase fragments

Streptokinase (SK), a widely used thrombolytic agent, activates human plasminogen, which in turn dissolves blood clots. However, its clinical application is limited because it elicits immunogenic responses in patients. The present study aims to develop novel plasminogen activators derived from truncated fragments of SK that retain activity comparable to the full-length SK enzyme. Based on the structure-function relationships and the previous literature, ten fragments of SK were designed by systematically truncating the N- and C-terminal regions and the central β-domain. These fragments included variants lacking N-terminal residues (amino acids 1-24 or 1-37), C-terminal truncations (beyond amino acid 300) and segments focused on the central domain (starting at amino acid 130 or ending at 173). Fragments were expressed in E. coli and their plasminogen activation activity was evaluated. The experimental results indicated that fragments containing an intact N1 domain retained a high level of plasminogen activation activity (fragments 1-300, 1-173), while deletion of key N-terminal residues significantly reduced it. C-terminal fragments partially compensated for N-terminal loss (fragment 130-414), but overall activity remained lower. Molecular modelling studies of protein-protein complex interface analyses revealed that the N1 domain (residues 1-173) of SK constitutes the principal binding interface with HPlg, with specific N-terminal residues serving as interaction hotspots. Residue-level contact mapping correlated strongly with experimental activity, underscoring the importance of precise interface residues rather than overall domain-level contact coverage. The study identifies SK fragments 1-300, 1-173 and 130-414 as having substantial activity comparable to full-length SK, suggesting that these constructs could serve as the basis for novel thrombolytic drug candidates pending further immunogenicity evaluation. These interesting findings provide both structural and functional insights for the rational design of improved streptokinase-derived therapeutics.