Transition State of Arp2/3 Complex Activation by Actin-Bound Dimeric Nucleation-Promoting Factor

Abstract

Arp2/3 complex generates branched actin networks that drive fundamental processes such as cell motility and cytokinesis. The complex comprises seven proteins, including actin-related proteins (Arps) 2 and 3 and five scaffolding proteins (ArpC1–ArpC5) that mediate interactions with a pre-existing (mother) actin filament at the branch junction. Arp2/3 complex exists in two main conformations, inactive with the Arps interacting end-to-end and active with the Arps interacting side-by-side like subunits of the short-pitch helix of the actin filament. Several cofactors drive the transition toward the active state, including ATP binding to the Arps, WASP-family nucleation-promoting factors (NPFs), actin monomers, and binding of Arp2/3 complex to the mother filament. The precise contribution of each cofactor to activation is poorly understood. We report the 3.32-Å resolution cryo-electron microscopy structure of a transition state of Arp2/3 complex activation with bound constitutively dimeric NPF. Arp2/3 complex-binding region of the NPF N-WASP was fused C-terminally to the α and β subunits of the CapZ heterodimer. One arm of the NPF dimer binds Arp2 and the other binds actin and Arp3. The conformation of the complex is intermediate between those of inactive and active Arp2/3 complex. Arp2, Arp3, and actin also adopt intermediate conformations between monomeric (G-actin) and filamentous (F-actin) states, but only actin hydrolyzes ATP. In solution, the transition complex is kinetically shifted toward the short-pitch conformation and has higher affinity for F-actin than inactive Arp2/3 complex. The results reveal how all the activating cofactors contribute in a coordinated manner toward Arp2/3 complex activation.

Liquid-like condensates mediate competition between actin branching and bundling

“…Therefore, enrichment of these proteins in VASP droplets is likely driven by nonspecific or weak binding interactions, which can appear amplified in concentrated droplet systems ( 19 , 25 ). Since both Arp2/3 and the VCA domain of WASP natively participate in electrostatic interactions in the cell, we anticipate that these residues may contribute to nonspecific electrostatic interactions with VASP ( 26 – 29 ). While addition of Arp2/3 had little effect on VASP droplets, addition of the VCA fragment resulted in VASP droplets with a distribution of larger diameters in comparison to droplets consisting of VASP alone ( Fig.…”

“…This effect, which suggests that VCA reinforces condensation of VASP, could result from non-specific interactions between acidic residues in the VCA fragment with basic residues in VASP ( 16 , 28 – 31 ). Furthermore, the seemingly larger impact of VCA on VASP droplets compared to Arp2/3 could be attributed to the excess VCA relative to Arp2/3, which we used to mimic previous in vitro actin branching assays ( 27 – 31 ). Importantly, VASP droplets formed with Arp2/3 and VCA remained liquid-like, fusing rapidly upon contact ( Fig.…”