Coiled Coil Region of Streptokinase γ-Domain Is Essential for Plasminogen Activation

The specific functions of the amino acid residues in the streptokinase (SK) γ-domain were analyzed by studying the interactions of human plasminogen (HPlg) and SK mutants prepared by charge-to-alanine mutagenesis. SK with mutations of groups of amino acids outside the coiled coil region of SK γ-domain, SKK278A,K279A,E281A,K282A, and SKD360A,R363A had similar HPlg activator activities as wild-type SK. However, significant changes of the functions of SK with mutations within the coiled coil region were observed. Both SKD322A,R324A,D325A and SKR330A,D331A,K332A,K334A had decreased amounts of complex formation with microplasminogen and failed to activate HPlg. SKD328A,R330A had a 21-fold reduced catalytic efficiency for HPlg activation. The studies of SK with single amino acid mutation to Ala demonstrate that Arg324, Asp325, Lys332, and Lys334 play important roles in the formation of a HPlg·SK complex. On the other hand, amino acid residues Asp322, Asp328, and Arg330of SK are involved in the virgin enzyme induction. Potential contact between Lys332 of SK and Glu623 of human microplasmin and strong interactions between Asp328 and Lys330, Asp331 and Lys334, and Asp322 and Lys334 of SK are noticed. These interactions are important in maintaining a coiled coil conformation. Therefore, we conclude that the coiled coil region of SK γ-domain, SK(Leu314-Ala342), plays very important roles in HPlg activation by participating in virgin enzyme induction and stabilizing the activator complex.