Molecular mechanism of Arp2/3 complex inhibition by Arpin

Fred E Fregoso,Austin Zimmet,Trevor Van Eeuwen,Gleb Simanov,Roberto Dominguez,Malgorzata Boczkowska,Grzegorz Rebowski,Alexis M Gautreau

doi:10.1038/s41467-022-28112-2

Abstract

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.

Highlights

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
A positive feedback loop regulated by the small GTPase Rac and involving Arp2/3 complex activation by the nucleation-promoting factors (NPFs) WAVE controls the assembly of branched networks at the cell cortex to drive cell motility[9]
Arpin was discovered through a bioinformatics search for proteins containing a C-terminal A domain analogous to that of NPFs (Fig. 1a), and when it was revealed that Arpin inhibited Arp2/3 complex it was assumed to be through competition with NPFs10

Summary

Introduction

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. Branched actin networks nucleated by Arp2/3 complex are abundant near the cell periphery and around most intracellular membranous organelles, where they drive a host of cellular processes such as cell motility, vesicular trafficking, membrane scission, and mechanosensation[2,3]. Several proteins regulate these processes by activating or inhibiting Arp2/ 3 complex and by stabilizing or disassembling branched networks. A positive feedback loop regulated by the small GTPase Rac and involving Arp2/3 complex activation by the NPF WAVE controls the assembly of branched networks at the cell cortex to drive cell motility[9]. Contrary to the initially proposed model of Arpin inhibition through direct competition with NPFs, the structuralfunctional data presented here support a model in which Arpin competes only with NPF binding to Arp[3] and locks Arp2/3 complex in the inactive conformation through interactions of the C-helix with the C-terminal inhibitory tail of Arp[3]

Methods

Results

Conclusion