Identification of an ATP-controlled allosteric switch that controls actin filament nucleation by Arp2/3 complex

Abstract

Nucleation of branched actin filaments by Arp2/3 complex is tightly regulated to control actin assembly in cells. Arp2/3 complex activation involves conformational changes brought about by ATP, Nucleation Promoting Factor (NPF) proteins, actin filaments and NPF-recruited actin monomers. To understand how these factors promote activation, we must first understand how the complex is held inactive in their absence. Here we demonstrate that the Arp3 C-terminal tail is a structural switch that prevents Arp2/3 complex from adopting an active conformation. The interaction between the tail and a hydrophobic groove in Arp3 blocks movement of Arp2 and Arp3 into an activated filament-like (short pitch) conformation. Our data indicate ATP binding destabilizes this interaction via an allosteric link between the Arp3 nucleotide cleft and the hydrophobic groove, thereby promoting the short-pitch conformation. Our results help explain how Arp2/3 complex is locked in an inactive state without activators and how autoinhibition is relieved.

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

“…Deletion of the entire Arp3 C-terminal tail in either budding or fission yeast Arp2/3 complexes stimulates the short-pitch conformation and increases NPF-independent activity (41). Furthermore, mutations that disrupt interactions of the tip of the tail with the barbed-end groove also stimulate the short-pitch conformation, supporting a model in which release of the tip from the barbed-end groove allosterically relieves autoinhibition by destabilizing the splayed Arp2-Arp3 interface (41) (Fig. 8A).…”

“…We also analyzed low-resolution EM models of the Arp2/3 complex at a branch junction to identify WASP-CA interactions that could stabilize the short-pitch conformation (24). These analyses, in conjunction with results from a recent study (41), led us to hypothesize that interactions of WASP-C with the Arp3 barbed-end groove are critical for stimulating the short-pitch conformation. We recently found that the Arp3 C terminus is an autoregulatory switch important for holding the complex inactive in the absence of NPFs.…”

“…Our data indicate that this mechanism must work coordinately with other activation triggers. For example, in a recent study, we showed that ATP bound to the Arp3 nucleotide cleft allosterically disrupts interactions between the Arp3 tail and the barbed-end groove, providing another mechanism to destabilize the splayed conformation (41). Structural features other than the Arp3 C-terminal tail also must be involved in triggering activation.…”

Structure of the nucleation‐promoting factorSPIN90 bound to the actin filament nucleator Arp2/3 complex

“…WASP proteins comprise the largest family of NPFs and are characterized by a C‐terminal region called VCA (Campellone & Welch, ). VCA binds and recruits actin monomers to Arp2/3 complex (Rohatgi et al , ; Marchand et al , ; Padrick et al , ; Boczkowska et al , ), making interactions that drive the two actin‐related subunits, Arp2 and Arp3 into filament‐like (short pitch) arrangement (Boczkowska et al , ; Xu et al , ; Hetrick et al , ; Rodnick‐Smith et al , ,b). Adoption of the short‐pitch conformation is a critical activation step, and stimulating this structural change is the main function of WASP and actin monomers recruited by WASP (Rodnick‐Smith et al , ).…”

“…Binding to this site likely allows SPIN90 to trigger conformational changes normally stimulated by filaments, since it causes structural changes that move Arp3 toward a “flattened” conformation, an activating conformational change that occurs in actin upon incorporation into filaments (Oda et al , ). Further, while the SPIN90‐binding site is distinct from the two binding sites for WASP (Padrick et al , ; Ti et al , ; Boczkowska et al , ; Jurgenson & Pollard, ; Rodnick‐Smith et al , ; Luan et al , ), it binds along a long alpha helix in the ARPC4 subunit that bends to rotate Arp2 into the short‐pitch conformation in some models for activation (Robinson et al , ; Dalhaimer & Pollard, ). Therefore, our data support a model in which SPIN90 binds to a different site on Arp2/3 complex than WASP to allosterically stimulate the same activating conformational change as WASP.…”