Coiled coil region of streptokinase gamma-domain is essential for plasminogen activation

The specific functions of the amino acid residues in the streptokinase (SK) gamma -domain were analyzed by studying the interactions of human plasmino gen (HPlg) and SK mutants prepared by charge-to-alanine mutagenesis. SK wit h mutations of groups of amino acids outside the coiled coil region of SK g amma -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 amoun ts of complex formation with microplasminogen and failed to activate HPlg. SKD328A,R330A had a 21-fold reduced catalytic efficiency for HPlg activatio n. The studies of SK with single amino acid mutation to Ala demonstrate tha t Arg(324), Asp(325), Lys(332), and Lys(334) play important roles in the fo rmation of a HPlg SK complex. On the other hand, amino acid residues Asp(32 2), Asp(328), and Arg(330) of SK are involved in the virgin enzyme inductio n. Potential contact between Lys(332) of SK and Glu(623) of human microplas min and strong interactions between Asp(328) and Lys(330), Asp(331) and Lys (334), and Asp(322) and Lys(334) of SK are noticed. These interactions are important in maintaining a coiled coil conformation, Therefore, we conclude that the coiled coil region of SK gamma -domain, SK(Leu(314)-Ala(342)), pl ays very important roles in HPlg activation by participating in virgin enzy me induction and stabilizing the activator complex.