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

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 ß 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.

Mechanism of synergistic activation of Arp2/3 complex by cortactin and WASP-family proteins.

Cortactin coactivates Arp2/3 complex synergistically with WASP-family nucleation-promoting factors (NPFs) and stabilizes branched networks by linking Arp2/3 complex to F-actin. It is poorly understood how cortactin performs these functions. We describe the 2.89 Å resolution cryo-EM structure of cortactin's N-terminal domain (Cort1-76) bound to Arp2/3 complex. Cortactin binds Arp2/3 complex through an inverted Acidic domain (D20-V29), which targets the same site on Arp3 as the Acidic domain of NPFs but with opposite polarity. Sequences N- and C-terminal to cortactin's Acidic domain do not increase its affinity for Arp2/3 complex but contribute toward coactivation with NPFs. Coactivation further increases with NPF dimerization and for longer cortactin constructs with stronger binding to F-actin. The results suggest that cortactin contributes to Arp2/3 complex coactivation with NPFs in two ways, by helping recruit the complex to F-actin and by stabilizing the short-pitch (active) conformation, which are both byproducts of cortactin's core function in branch stabilization.

Nucleation, stabilization, and disassembly of branched actin networks.

Arp2/3 complex is an actin filament nucleation and branching machinery conserved in all eukaryotes from yeast to human. Arp2/3 complex branched networks generate pushing forces that drive cellular processes ranging from membrane remodeling to cell and organelle motility. Several molecules regulate these processes by directly inhibiting or activating Arp2/3 complex and by stabilizing or disassembling branched networks. Here, we review recent advances in our understanding of Arp2/3 complex regulation, including high-resolution cryoelectron microscopy (cryo-EM) structures that illuminate the mechanisms of Arp2/3 complex activation and branch formation, and novel cellular pathways of branch formation, stabilization, and debranching. We also identify major gaps in our understanding of Arp2/3 complex inhibition and branch stabilization and disassembly.

Molecular mechanism of Arp2/3 complex inhibition by Arpin.

Positive feedback loops involving signaling and actin assembly factors mediate the formation and remodeling of branched actin networks in processes ranging from cell and organelle motility to mechanosensation. The Arp2/3 complex inhibitor Arpin controls the directional persistence of cell migration by interrupting a feedback loop involving Rac-WAVE-Arp2/3 complex, but Arpin's mechanism of inhibition is unknown. Here, we describe the cryo-EM structure of Arpin bound to Arp2/3 complex at 3.24-Å resolution. Unexpectedly, Arpin binds Arp2/3 complex similarly to WASP-family nucleation-promoting factors (NPFs) that activate the complex. However, whereas NPFs bind to two sites on Arp2/3 complex, on Arp2-ArpC1 and Arp3, Arpin only binds to the site on Arp3. Like NPFs, Arpin has a C-helix that binds at the barbed end of Arp3. Mutagenesis studies in vitro and in cells reveal how sequence differences within the C-helix define the molecular basis for inhibition by Arpin vs. activation by NPFs.