Journal Article

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

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

2017

Overview

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. Our cells contain a network of filaments made up of a protein called actin. Just like the skeleton that supports our body, the actin ‘cytoskeleton’ gives a cell its shape and strength. Actin filaments are also critical for many other processes including enabling cells to move and divide. The assembly of actin filaments must be properly controlled so that they are formed at the right time and place within the cell. A complex of proteins known as the WAVE Regulatory Complex (WRC) promotes the assembly of actin filaments. The complex contains a region called the VCA, which is able to bind to and activate another protein to make the new actin filaments. The WRC regulates filament assembly by controlling the availability of the VCA in a way that is similar to opening and closing a safe box. When new actin filaments are not needed, the safe box is closed and the VCA is not available. However, when cells need to make new actin filaments, the WRC is opened to release the VCA region so that it is able to bind to the filament-producing protein. Previous studies have shown that a protein called Rac1 acts as a key to open the WRC and trigger actin filament assembly. But it remains unclear how this works. A major obstacle to studying this process is that Rac1 and the WRC only weakly interact with each other, which makes it difficult to capture the interaction under experimental conditions. To overcome this obstacle, Chen et al. tethered a Rac1 molecule to the WRC in order to make the interaction more stable. A technique called cryo-electron microscopy was used to study the three-dimensional shape of this Rac1-WRC complex. Unexpectedly, Rac1 was attached to a different part of the WRC than the site predicted by previous studies. Further experiments showed that Rac1 needs to bind to both of these sites at the same time in order to open the WRC and release VCA, similar to using two keys to open one safe box for increased security. Some cancers, neurological disorders and other diseases can be caused by defects in WRC and Rac1 activity. Therefore, these findings could lead to new ways to treat these conditions in human patients.

• Publisher: eLife Sciences Publications Ltd,eLife Sciences Publications, Ltd
• Subject: Actin / actin assembly / Actins - metabolism / Adaptor Proteins, Signal Transducing - metabolism / Arp2/3 complex / Binding Sites / Biophysics / Carrier Proteins - genetics / Carrier Proteins - metabolism / Carrier Proteins - ultrastructure / Cell adhesion & migration / Cryoelectron Microscopy / Cytoskeletal Proteins - metabolism / DNA Mutational Analysis / Electron microscopy / Endangered & extinct species / Guanosine triphosphatases / Humans / Membranes / Mutagenesis / Mutation / Phosphorylation / Polymerization / Protein Binding / Protein Multimerization / Proteins / Proteins - metabolism / rac1 GTP-Binding Protein - metabolism / rac1 GTP-Binding Protein - ultrastructure / Rac1 protein / Rho family GTPase / Sedimentation & deposition / signal integration / signal transduction / Structural Biology and Molecular Biophysics / WASP family protein / Wiskott-Aldrich Syndrome Protein Family - metabolism