Abstract
The NH(2) terminus (residues 1-59) of streptokinase (SK) is a molecular switch that permits fibrin-independent plasminogen activation. Targeted mutations were made in recombinant (r) SK1-59 to identify structural interactions required for this process. Mutagenesis established the functional roles of Phe-37and Glu-39, which were projected to interact with microplasmin in the activator complex. Mutation of Leu-42 (rSK1-59(L42A)), a conserved residue in the SK fibronectin motif that lacks interactions with microplasmin, strongly reduced plasminogen activation (k(cat) decreased 50-fold) but not amidolysis (k(cat) decreased 1.5-fold). Otherwise rSK1-59(L42A) and native rSK1-59 were indistinguishable in several parameters. Both displayed saturable and specific binding to Glu-plasminogen or the remaining SK fragment (rSKDelta59). Similarly rSK1-59 and rSK1-59(L42A) bound simultaneously to two different plasminogen molecules, indicating that both plasminogen binding sites were intact. However, when bound to SKDelta59, rSK1-59(L42A) was less effective than rSK1-59 in restructuring the native conformation of the SK A domain, as detected by conformation-dependent monoclonal antibodies. In the light of previous studies, these data provide evidence that SK1-59 contributes to fibrin-independent plasminogen activation through 1) intermolecular interactions with the plasmin in the activator complex, 2) binding interactions with the plasminogen substrate, and 3) intramolecular interactions that structure the A domain of SK for Pg substrate processing.
Highlights
EXPERIMENTAL PROCEDURESThe SK gene was cloned from Streptococcus equisimilis, expressed in bacteria via the pMALc vector (New England Biolabs, Beverly, MA) and purified as described in detail [3]
Plasminogen (Pg)1 activators cleave the zymogen Pg to the enzyme plasmin [1]
Mutagenesis established the functional roles of Phe-37and Glu-39, which were projected to interact with microplasmin in the activator complex
Summary
The SK gene was cloned from Streptococcus equisimilis, expressed in bacteria via the pMALc vector (New England Biolabs, Beverly, MA) and purified as described in detail [3]. The effect of rSK1–59 (native or mutant) on the activation of bovine Pg (300 nM) by the stoichiometric complex of rSK⌬59 and human plasmin was studied in a similar fashion. Competitive Binding of Native and Mutant rSK1–59 to Glu-Pg— Wells of microtiter plates were coated with Glu-Pg (5 g/ml). Simultaneous Binding of rSK1–59 to Two Pg Molecules Was Assessed in a “Sandwich” Binding Assay—Microtiter plates were coated with or without Glu-Pg (5 g/ml), and nonspecific binding sites were blocked with 1% BSA. Bound mAb was detected by 125I goat anti-mouse Ab followed by ␥-counting The binding of these anti-SK mAbs to native rSK or the isolated rSK A, B, or C domains was assessed in a similar binding assay
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