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Originally published In Press as doi:10.1074/jbc.M003963200 on August 28, 2000

J. Biol. Chem., Vol. 275, Issue 48, 37686-37691, December 1, 2000
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Leucine 42 in the Fibronectin Motif of Streptokinase Plays a Critical Role in Fibrin-independent Plasminogen Activation*

Lin Liu, Irina Y. Sazonova, Ryan B. Turner, Shakeel A. Chowdhry, Judy Tsai, Aiilyan K. Houng, and Guy L. ReedDagger §

From the Harvard School of Public Health, Boston, Massachusetts 02115 and Dagger  Massachusetts General Hospital, Boston, Massachusetts 02114

The NH2 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 (rSK1-59L42A), a conserved residue in the SK fibronectin motif that lacks interactions with microplasmin, strongly reduced plasminogen activation (kcat decreased 50-fold) but not amidolysis (kcat decreased 1.5-fold). Otherwise rSK1-59L42A and native rSK1-59 were indistinguishable in several parameters. Both displayed saturable and specific binding to Glu-plasminogen or the remaining SK fragment (rSKDelta 59). Similarly rSK1-59 and rSK1-59L42A bound simultaneously to two different plasminogen molecules, indicating that both plasminogen binding sites were intact. However, when bound to SKDelta 59, rSK1-59L42A 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.

* This work was supported in part by National Institutes of Health Grant HL-57314 (to G. L. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Cardiovascular Biology Laboratory, HSPH II-127, 677 Huntington Ave., Boston, MA 02115. Tel.: 617-432-4992; Fax: 617-432-0031; E-mail: reed@cvlab.harvard.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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