Domain Interactions between Streptokinase and Human Plasminogen

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Abstract
Plasmin (Pm), the main fibrinolytic protease in the plasma, is derived from its zymogen plasminogen (Plg) by cleavage of a peptide bond at Arg561−Val562. Streptokinase (SK), a widely used thrombolytic agent, is an efficient activator of human Plg. Both are multiple-domain proteins that form a tight 1:1 complex. The Plg moiety gains catalytic activity, without peptide bond cleavage, allowing the complex to activate other Plg molecules to Pm by conventional proteolysis. We report here studies on the interactions between individual domains of the two proteins and their roles in Plg activation. Individually, all three SK domains activated native Plg. While the SK α domain was the most active, its activity was uniquely dependent on the presence of Pm. The SK γ domain also induced the formation of an active site in PlgR561A, a mutant that resists proteolytic activation. The α and γ domains together yielded synergistic activity, both in Plg activation and in PlgR561A active site formation. However, the synergistic activity of the latter was dependent on the correct N-terminal isoleucine in the α domain. Binding studies using surface plasmon resonance indicated that all three domains of SK interact with the Plg catalytic domain and that the β domain additionally interacts with Plg kringle 5. These results suggest mechanistic steps in SK-mediated Plg activation. In the case of free Plg, complex formation is initiated by the rapid and obligatory interaction between the SK β domain and Plg kringle 5. After binding of all SK domains to the catalytic domain of Plg, the SK α and γ domains cooperatively induce the formation of an active site within the Plg moiety of the activator complex. Substrate Plg is then recognized by the activator complex through interactions predominately mediated by the SK α domain.