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J. Biol. Chem., Vol. 282, Issue 27, 19502-19509, July 6, 2007

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Maspin Binds to Urokinase-type and Tissue-type Plasminogen Activator through Exosite-Exosite Interactions^*

Maher Al-Ayyoubi, Bradford S. Schwartz, and Peter G. W. Gettins¹

From the Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607 and the Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Maspin is a member of the serpin family with a reactive center loop that is incompatible with proteinase inhibition by the serpin conformational change mechanism. Despite this there are reports that maspin might regulate uPA-dependent processes in vivo. Using exogenous and endogenous fluorescence, we demonstrate here that maspin can bind uPA and tPA in both single-chain and double-chain forms, with K_d values between 300 and 600 nM. Binding is at an exosite on maspin close to, but outside of, the reactive center loop and is therefore insensitive to mutation of Arg³⁴⁰ within the reactive center loop. The binding site on tPA does not involve the proteinase active site, with the result that maspin can bind to S195A tPA that is already complexed to plasminogen activator inhibitor-1. The ability of maspin to bind these proteinases without involvement of the reactive center loop leaves the latter free to engage in additional, as yet unidentified, maspin-protein interactions that may serve to regulate the properties of the exosite-bound proteinase. This may help to reconcile apparently conflicting studies that demonstrate the importance of the reactive center loop in certain maspin functions, despite the inability of maspin to directly inhibit tPA or uPA catalytic activity in in vitro assays through engagement between its reactive center loop and the active site of the proteinase.

Received for publication, March 21, 2007 , and in revised form, May 10, 2007.

^* This work was supported by Grant R37 HL49234 (to P. G. W. G.) from the National Institutes of Health. The Microcal VP isothermal titration calorimeter was funded by shared instrumentation Grant S10 RR15958 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: 900 S. Ashland, M/C 669, Chicago, IL 60607. Tel.: 312-996-5534; Fax: 312-413-0353; E-mail: pgettins{at}uic.edu.

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