Structures reveal a key mechanism of WAVE Regulatory Complex activation by Rac1 GTPase

Abstract

Rho-family GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3-mediated actin polymerization in many essential processes. Rac1 binds to WRC at two distinct sites—the A and D sites. Precisely how Rac1 binds and how the binding triggers WRC activation remain unknown. Here we report WRC structures by itself, and when bound to single or double Rac1 molecules, at ∼3 Å resolutions by cryogenic-electron microscopy. The structures reveal that Rac1 binds to the two sites by distinct mechanisms, and binding to the A site, but not the D site, drives WRC activation. Activation involves a series of unique conformational changes leading to the release of sequestered WCA (WH2- central-acidic) polypeptide, which stimulates the Arp2/3 complex to polymerize actin. Together with biochemical and cellular analyses, the structures provide a novel mechanistic understanding of how the Rac1-WRC-Arp2/3-actin signaling axis is regulated in diverse biological processes and diseases.

Arf GTPase activates the WAVE Regulatory Complex through a novel binding site

“…We found that in the absence of Rac1, the Arf1-WRC interaction was weak, with a dissociate constant K D ∼23 µM ( Figure 1C , black). By contrast, in the presence of 100 µM Rac1, which should saturate both A and D sites of the WRC (Chen et al, 2017; Ding et al, 2022), Arf1 binding affinity was increased nearly 30 fold (K D ∼ 0.66 µM, Figure 1C , orange). The enhanced binding was not an artifact of high concentration of free Rac1 included in the assay, as a mutant Rac1, in which the entire Switch I motif critical for WRC binding was removed (herein referred to as Rac1 Dead ; Figure S1A ), could not promote Arf1 binding at the same concentration ( Figure 1C , blue).…”

“…Therefore, we asked which Rac1 binding event was key to promoting Arf1 binding. To answer this question, we first used single amino acid mutations to specifically disrupt the A or D site from binding to Rac1 (Chen et al, 2017, 2010; Ding et al, 2022) ( Figure 2A , cartoon). When Rac1 binding to the A site was disrupted by Sra1 C179R , Rac1 binding to the D site still enhanced Arf1 binding, but to a lower extent than the WT WRC ( Figure 2A , lane 6, 7).…”

“…As an alternative strategy to validate the contribution of the D site to Arf1 binding, we stabilized Rac1 binding to the D site by tethering it to the C-terminus of Sra1 (which we refer to as ΔWRC230 D-Rac1 ) (Ding et al, 2022) or the C-terminus of WAVE1 that lacked the WCA (which we named ΔWRC230 WAVE1-Rac1 ) (Chen et al, 2017) ( Figure 2C , cartoon). These constructs stabilize D site Rac1 binding, which had allowed us to solve cryo-EM structures of the WRC with Rac1 bound to the D site (Chen et al, 2017; Ding et al, 2022). We found that, without free Rac1, both ΔWRC230 D-Rac1 and ΔWRC230 WAVE1-Rac1 were able to enhance Arf1 binding to the level of the WT WRC enhanced by free Rac1 ( Figure 2C , lane 4, 5, 7).…”

“…This indicates that Rac1 binding to the A site should also play a role, which might have eluded detection in the assays described above due to the low affinity of the A site for Rac1. The potential cooperativity between A and D sites could further reduce A site binding when the D site is disrupted (Chen et al, 2017; Ding et al, 2022). To examine the contribution of the A site more directly, we stabilized Rac1 binding to the A site by inserting a Rac1 between Y423/S424 in a non-conserved surface loop near the A site (termed ΔWRC230 A-Rac1 ; Figure 2D , cartoon).…”

“…To examine the contribution of the A site more directly, we stabilized Rac1 binding to the A site by inserting a Rac1 between Y423/S424 in a non-conserved surface loop near the A site (termed ΔWRC230 A-Rac1 ; Figure 2D , cartoon). This strategy had allowed us to determine the cryo-EM structure of the WRC with Rac1 bound to the A site and D site simultaneously (Ding et al, 2022). We found that, without free Rac1, tethering Rac1 to the A site mildly promoted Arf1 binding ( Figure 2D , lane 3 vs. 5).…”