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J. Biol. Chem., Vol. 282, Issue 11, 8446-8453, March 16, 2007

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Multiple WASP-interacting Protein Recognition Motifs Are Required for a Functional Interaction with N-WASP^*

Francis C. Peterson, Qing Deng, Markus Zettl, Kenneth E. Prehoda^¶, Wendell A. Lim^||, Michael Way^**, and Brian F. Volkman¹

From the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, Laboratory of Molecular Biology, Medical Research Council, Cambridge CB2 2QH, United Kingdom, ^¶Department of Chemistry, University of Oregon, Eugene, Oregon 97403-1229, ^||Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143-2240, and ^**Cell Motility Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom

The WASP-interacting protein (WIP) targets WASP/WAVE proteins through a constitutive interaction with an amino-terminal enabled/VASP homology (EVH1) domain. Parallel investigations had previously identified two distinct N-WASP binding motifs corresponding to WIP residues 451-461 and 461-485, and we determined the structure of a complex between WIP-(461-485) and the N-WASP EVH1 domain (Volkman, B. F., Prehoda, K. E., Scott, J. A., Peterson, F. C., and Lim, W. A. (2002) Cell 111, 565-576). The present results show that, when combined, the WIP-(451-485) sequence wraps further around the EVH1 domain, extending the interface observed previously. Specific contacts with three WIP epitopes corresponded to regions of high sequence conservation in the verprolin family. A central polyproline motif occupied the canonical binding site but in a reversed orientation relative to other EVH1 complexes. This interaction was augmented in the amino- and carboxyl-terminal directions by additional hydrophobic contacts involving WIP residues 454-459 and 475-478, respectively. Disruption of any of the three WIP epitopes reduced N-WASP binding in cells, demonstrating a functional requirement for the entire binding domain, which is significantly longer than the polyproline motifs recognized by other EVH1 domains.

Received for publication, October 23, 2006 , and in revised form, December 27, 2006.

The atomic coordinates and structure factors (code 2IFS) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

Coordinates and related data (accession number 15020) have been deposited in the Biological Magnetic Resonance Bank.

^* This work was supported by National Institutes of Health Grants GM55940 and GM62583 (to W. A. L.), the Packard Foundation (to W. A. L.), and Cancer Research UK (to M. W.). 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: Dept. of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-456-8400; Fax: 414-456-6510; E-mail: bvolkman{at}mcw.edu.

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