Plasminogen Substrate Recognition by the Streptokinase-Plasminogen Catalytic Complex Is Facilitated by Arg253, Lys256, and Lys257 in the Streptokinase β-Domain and Kringle 5 of the Substrate

Streptokinase (SK) conformationally activates the central zymogen of the fibrinolytic system, plasminogen (Pg). The SK·Pg* catalytic complex binds Pg as a specific substrate and cleaves it into plasmin (Pm), which binds SK to form the SK·Pm complex that propagates Pm generation. Catalytic complex formation is dependent on lysine-binding site (LBS) interactions between a Pg/Pm kringle and the SK COOH-terminal Lys414. Pg substrate recognition is also LBS-dependent, but the kringle and SK structural element(s) responsible have not been identified. SK mutants lacking Lys414 with Ala substitutions of charged residues in the SK β-domain 250-loop were evaluated in kinetic studies that resolved conformational and proteolytic Pg activation. Activation of [Lys]Pg and mini-Pg (containing only kringle 5 of Pg) by SK with Ala substitutions of Arg253, Lys256, and Lys257 showed decreases in the bimolecular rate constant for Pm generation, with nearly total inhibition for the SK Lys256/Lys257 double mutant. Binding of bovine Pg (BPg) to the SK·Pm complex containing fluorescently labeled Pm demonstrated LBS-dependent assembly of a SK·labeled Pm·BPg ternary complex, whereas BPg did not bind to the complex containing the SK Lys256/Lys257 mutant. BPg was activated by SK·Pm with a Km indistinguishable from the KD for BPg binding to form the ternary complex, whereas the SK Lys256/Lys257 mutant did not support BPg activation. We conclude that SK residues Arg253, Lys256, and Lys257 mediate Pg substrate recognition through kringle 5 of the [Lys]Pg and mini-Pg substrates. A molecular model of the SK·kringle 5 complex identifies the putative interactions involved in LBS-dependent Pg substrate recognition.