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,Brian F Volkman

doi:10.1074/jbc.m609902200

Abstract

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.

Highlights

Platelet and immune deficiency first identified in 1937
Interaction, perhaps controlling Wiskott-Aldrich syndrome (WAS) protein (WASP) stability in the cell [6]. This residue was not implicated in the WASP-interacting protein (WIP)-WASP interaction but is immediately carboxyl-terminal to the WIP461–485 sequence that binds the N-WASP enabled/VASP homology-1 (EVH1) domain
To examine the structural role of residues adjacent to the WIP-(461– 485) WASP binding domain, we purified a series of 15N-labeled fusion proteins containing WIP fragments corresponding to residues 461– 485, 451– 485, or 451– 493 linked to the amino terminus of the N-WASP EVH1 domain

Summary

Introduction

Platelet and immune deficiency first identified in 1937. The WAS protein (WASP), expressed in cells of hematopoietic lineage, and its ubiquitously expressed neuronal homolog (N-WASP) encode multidomain proteins that stimulate actin polymerization in a phospholipid- and GTPase-dependent manner [2,3,4]. Multiple WIP Epitopes Specify WASP Targeting line motif alone was insufficient for WIP/N-WASP interaction They found that a 10-residue segment of WIP (451ESRFYFHPISD461) immediately amino-terminal to the LPPPEP sequence was by itself sufficient to bind the EVH1 domain. This interaction depended on conserved aromatic side chains (Phe454 and Phe456) but required no proline-rich sequence whatsoever. Results from different groups have suggested that elements both within and adjacent to the WIP-(461– 485) sequence are required for functional WIPWASP interactions in vivo To clarify these apparently discordant results, we determined the structure of a complex between the EVH1 domain of N-WASP and an extended WIP sequence that encompasses both of the previously defined interaction domains. Binding measurements in cells demonstrated the essential role for multiple WIP epitopes conserved in the verprolin family

Methods

Results

Conclusion