The Arp2/3 complex, a core component of the actin filament network, mediates the formation of branching points upon activation by nucleation-promoting factors (NPFs), which is essential for diverse cellular processes such as lamellipodial protrusion and directional migration. Arpin, a conserved inhibitor of the Arp2/3 complex, localizes to the lamellipodial leading edge, where it directly binds to the Arp2/3 complex at multiple sites to antagonize NPF binding. While low-resolution electron microscopy studies have proposed the existence of two Arpin-binding sites on the Arp2/3 complex, the stoichiometry of this interaction remains controversial. In addition, the lack of high-resolution structural information on Arpin has impeded mechanistic insights into how it modulates Arp2/3 complex activity and actin filament branching. Here, we report the 1.65-Å crystal structure of Arpin’s N-terminal globular domain, which reveals a previously uncharacterized homodimeric architecture—an observation validated by complementary biophysical analyses. This dimeric assembly enables Arpin to engage the two binding sites on the Arp2/3 complex simultaneously. Mutational disruption of the homodimerization interface substantially reduced its Arp2/3-binding affinity and abrogated its ability to suppress lamellipodial branching and cellular directional migration, indicating positive cooperativity between the two Arpin protomers. Guided by these structural insights, we engineered dimeric Arpin mimics, including a dual-tailed peptide variant that retained robust Arp2/3-binding activity. Subsequent AI-assisted modification further enhanced the inhibitory potency of the peptide. Our findings uncover the structural basis of Arpin’s inhibitory mechanism, delineating a critical role for homodimerization in cooperative Arp2/3 inhibition.
DOI：10.1093/procel/pwag021