Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites.

Baoyu Chen,Lynda K Doolittle,Sheng Yang,Wenmin Xing,Hui-Ting Chou,Lisa Henry,Michael K Rosen,Chad A Brautigam,Thomas Walz

doi:10.7554/elife.29795

Abstract

The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly.

Highlights

Dynamic rearrangements of the actin cytoskeleton play central roles in cellular processes, ranging from cell migration and adhesion to endocytosis and intracellular vesicle trafficking (Skau and Waterman, 2015)
We reported crystal structures of an inhibited WAVE Regulatory Complex (WRC) assembly containing all five subunits, but lacking the disordered C terminus and SH3 domain of Abi2 and the proline-rich region of WAVE1 (Chen et al, 2010), and of this assembly bound to a WRC Interacting Receptor Sequence (WIRS) motif peptide derived from the adhesion receptor, protocadherin 10 (Chen et al, 2014a)
We sought to determine the crystal structure of an intermolecular complex of the WRC bound to Rac1

Summary

Introduction

Dynamic rearrangements of the actin cytoskeleton play central roles in cellular processes, ranging from cell migration and adhesion to endocytosis and intracellular vesicle trafficking (Skau and Waterman, 2015). In many of these processes, actin dynamics are spatially and temporally controlled by members of the Wiskott-Aldrich Syndrome Protein (WASP) family. These proteins integrate a diverse array of upstream signals and transmit them through their conserved VCA sequence to the Arp2/3 complex, which, in turn, nucleates actin filaments to create branched actin networks at membranes (Campellone and Welch, 2010; Padrick and Rosen, 2010). Multiple signals, including binding to ligands (e.g. Rho family GTPases, phosphoinositide lipids and membrane receptors) and covalent modifications (e.g. phosphorylation and ubiquitination), often act cooperatively to relieve inhibition and concomitantly recruit WASP proteins to their sites of action at membranes (Chen et al, 2014a, 2010; Hao et al, 2013; Lebensohn and Kirschner, 2009; Padrick and Rosen, 2010; Prehoda et al, 2000; Torres and Rosen, 2003; 2006)

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