The actin cytoskeleton is a dynamic filamentous network essential for processes such as cell migration, environmental sensing, shape maintenance, and vesicular trafficking. Actin filament assembly begins with a rate-limiting nucleation step, which is strongly accelerated by Arp2/3 complex. While Arp2/3 complex is indispensable for branched actin network formation, it remains inactive without stimulation by nucleation promoting factors (NPFs). Precise spatiotemporal control of actin nucleation is therefore achieved through the regulation of these NPFs. The Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) is the principal endosomal NPF, driving the recycling of internalized proteins back to the cell surface to maintain membrane protein homeostasis and regulate signaling. WASH functions as part of a large, multi-subunit WASH regulatory complex (SHRC). Despite its central role, the mechanistic regulation of WASH within SHRC has remained poorly understood due to the lack of high-resolution structural information of the complex or any of its subunits. To address this, we performed high-resolution electron cryo-microscopy (cryo-EM) structural studies to resolve the architecture and conformational dynamics of SHRC. We obtained structures of SHRC in multiple states, revealing that WASH intrinsically toggles between nucleation-competent and inactive forms. This intrinsic basal activity distinguishes WASH from canonical NPFs. Biochemical assays validated these structural findings, while molecular dynamics simulations provided mechanistic and thermodynamic insights into SHRC inhibition and activation. Our results establish a structural and mechanistic framework for SHRC regulation, setting the stage for future studies of disease-associated mutations and additional cellular factors that modulate WASH/SHRC activity.
Pathri et al. (Sun,) studied this question.