BPS2026 - Atomistic mechanism of large-scale conformational transition in Arp2/3 complex

The actin related protein, Arp2/3, is a large complex of seven subunits that nucleates actin filament branches on the sides of preexisting actin filaments and generates forces for cell and organelle movements. In its native state, Arp2/3 is in a splayed/inactive conformation. Activation of Arp2/3 involves large conformational changes that place the actin-like subunits Arp2 and Arp3 in a short-pitch conformation, enabling nucleation of branched actin filaments. Conversion from splayed/inactive to short-pitch/active conformation does not occur spontaneously, it is promoted by nucleation promoting factors (NPFs) and by actin filament. In this work, we elucidate the molecular mechanism of Arp2/3 activation. Further, we investigate the role of NPF Neuronal Wiskott-Aldrich syndrome protein (N-WASp) and actin monomer in promoting and stabilizing the transition state during the conversion to short-pitch conformation. Using metadynamics and umbrella free energy sampling simulations, we measure a free-energy barrier of 20 ± 6 kcal/mol during the conversion from splayed to short-pitch conformation that results from a clash between the D-loop of Arp2 and Arp3. Equilibrium atomistic molecular dynamics simulations showed that binding the CA motif of the nucleation promoting factor N-WASp to splayed Arp2/3 complex shifts it toward the short-pitch active conformation and opens a binding site for an actin monomer on Arp3 and actin monomer stabilizes a transition state of Arp2/3 complex. Taken together, our observations along with prior experimental work provide mechanistic insights into the pathway for actin filament branch formation.