Streptokinase Triggers Conformational Activation of Plasminogen through Specific Interactions of the Amino-terminal Sequence and Stabilizes the Active Zymogen Conformation

Paul D Boxrud,Ingrid M.A Verhamme,William P Fay,Paul E Bock

doi:10.1074/jbc.m101966200

Abstract

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 Ile(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 Ile(1) (SK(2--414)). SK(2--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 SK(2--414) was partially restored by the peptides SK(1--2), SK(1--5), SK(1--10), and SK(1--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 SK(1--10) and SK(2--414), whereas sequence-scrambled SK(1-10) was inactive. The characterization of SK(1--10) containing single Ala substitutions demonstrated the sequence specificity of the interaction. SK(1--10) did not restore activity to the further truncated mutant SK(55-414), which was correlated with the loss of binding affinity of SK(55--414) for labeled [Lys]Pm but not for [Lys]Pg. The studies support a mechanism for conformational activation in which the insertion of Ile(1) 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 SK(2--414) for the activated proteinase domain of Pm are thought to function cooperatively to trigger the conformational change and stabilize the active zymogen conformation.

Highlights

Teolytic cleavage of plasminogen (Pg) at Arg561-Val562 through a mechanism that is similar for most serine proteinase zymogens [1, 2]
The results support a mechanism of conformational activation in which the amino-terminal sequence of SK beginning with Ile1 interacts in a sequence-specific manner with the amino-terminal binding cleft of Pg and through extended binding sites on the SK2–4141⁄7Pg complex to induce a transition toward the active conformation
Binding of SK2–414 to Pg and Pm—The binding of SK2–414 to fluorescent analogs of Pg and Pm that were labeled at the catalytic site with ATA-FFR-CH2Cl and 5-(iodoacetamido)fluorescein [18, 19] ([5F]FFR-[Lys]Pg and [5F]FFR-[Lys]Pm) was evaluated to determine whether the affinities for Pg and Pm were affected by the mutation

Summary

Introduction

Teolytic cleavage of plasminogen (Pg) at Arg561-Val562 through a mechanism that is similar for most serine proteinase zymogens [1, 2]. The unresolved roles of SK binding to Pg and Pm and interactions of the SK amino-terminal sequence with the Pg aminoterminal binding site in the conformational activation mechanism were evaluated in the present fluorescence and kinetic studies of Pg interactions with an SK mutant lacking Ile1 (SK2–414).

Results

Conclusion