Streptokinase triggers conformational activation of plasminogen through specific interactions of the amino-terminal sequence and stabilizes the active zymogen conformation

Cleavage of Arg(561)-Val(562) in plasminogen (Pg) generates plasmin (Pm) through a classical activation mechanism triggered by an insertion of the new amino terminus into a binding pocket in the Pg catalytic domain. Streptokinase (SK) circumvents this process and activates Pg through a unique nonproteolytic mechanism postulated to be initiated by the intrusion of IIe(1) of SK in place of Val(562). This hypothesis was evaluated in equilibrium binding and kinetic studies of Pg activation with an SK mutant lacking IIe(1) (SK2-414). SK2-414 retained the affinity of native SK for fluorescein-labeled [Lys]Pg and [Lys]Pm but induced no detectable conformational activation of Pg. The activity of SK2-414 was partially restored by the peptides SK1-2, Sg(1-5), SK1-10, and SK1-15, whereas Pg(562-569) peptides were much less effective. Active site-specific fluorescence labeling demonstrated directly that the active catalytic site was formed on the Pg zymogen by the combination of SK1-10 and Sk(2-414), whereas sequence-scrambled SK1-10 was inactive. The characterization of SK1-10 containing single Ala substitutions demonstrated the sequence specificity of the interaction. SK1-10 did not restore activity to the further truncated mutant SK55-414, which was correlated with the loss of binding affinity of SK55-414 for labeled [Lys]Pm but not for [Lys]Pg. The studies support a mechanism for conformational activation in which the insertion of II1 of SK into the Pg amino-terminal binding cleft occurs through sequence-specific interactions of the first 10 SK residues. This event and the preferentially higher affinity of SK2-414 for the activated proteinase domain of Pm are thought to function cooperatively to trigger the conformational change and stabilize the active zymogen conformation.