Role and structural mechanism of WASP-triggered conformational changes in branched actin filament nucleation by Arp2/3 complex

Abstract

<p id="d10158480e180">Assembly of actin filaments is tightly regulated to orchestrate basic cellular processes such as motility, division, and differentiation. To control when and where actin filaments assemble, cells rely on actin filament nucleators including the Arp2/3 (Actin-related proteins 2/3) complex, a seven-subunit protein assembly that nucleates branched actin filaments to create dendritic actin networks. Activity of the Arp2/3 complex is controlled by WASP (Wiskott–Aldrich syndrome protein) proteins, which bind directly to it to activate nucleation. To understand how WASP proteins activate the complex, we used chemical cross-linking to engineer an Arp2/3 complex that is locked into an “on” state without WASP. In addition, we used biochemical experiments to determine how WASP proteins stimulate the on state. These results have important implications for understanding how cells control the actin cytoskeleton. </p><p class="first" id="d10158480e183">The Arp2/3 (Actin-related proteins 2/3) complex is activated by WASP (Wiskott–Aldrich syndrome protein) family proteins to nucleate branched actin filaments that are important for cellular motility. WASP recruits actin monomers to the complex and stimulates movement of Arp2 and Arp3 into a “short-pitch” conformation that mimics the arrangement of actin subunits within filaments. The relative contribution of these functions in Arp2/3 complex activation and the mechanism by which WASP stimulates the conformational change have been unknown. We purified budding yeast Arp2/3 complex held in or near the short-pitch conformation by an engineered covalent cross-link to determine if the WASP-induced conformational change is sufficient for activity. Remarkably, cross-linked Arp2/3 complex bypasses the need for WASP in activation and is more active than WASP-activated Arp2/3 complex. These data indicate that stimulation of the short-pitch conformation is the critical activating function of WASP and that monomer delivery is not a fundamental requirement for nucleation but is a specific requirement for WASP-mediated activation. During activation, WASP limits nucleation rates by releasing slowly from nascent branches. The cross-linked complex is inhibited by WASP’s CA region, even though CA potently stimulates cross-linking, suggesting that slow WASP detachment masks the activating potential of the short-pitch conformational switch. We use structure-based mutations and WASP–Arp fusion chimeras to determine how WASP stimulates movement toward the short-pitch conformation. Our data indicate that WASP displaces the autoinhibitory Arp3 C-terminal tail from a hydrophobic groove at Arp3′s barbed end to destabilize the inactive state, providing a mechanism by which WASP stimulates the short-pitch conformation and activates Arp2/3 complex. </p>