Small Molecules CK-666 and CK-869 Inhibit Actin-Related Protein 2/3 Complex by Blocking an Activating Conformational Change

Highlights

• CK-666 blocks formation of short pitch dimer by stabilizing the splayed conformation

• CK-869 disrupts the Arp2-Arp3 short pitch interface required for activation

• Actin monomers stimulate the short pitch conformation

• Neither inhibitor actively disassembles branched actin filaments

Summary

Actin-related protein 2/3 (Arp2/3) complex is a seven-subunit assembly that nucleates branched actin filaments. Small molecule inhibitors CK-666 and CK-869 bind to Arp2/3 complex and inhibit nucleation, but their modes of action are unknown. Here, we use biochemical and structural methods to determine the mechanism of each inhibitor. Our data indicate that CK-666 stabilizes the inactive state of the complex, blocking movement of the Arp2 and Arp3 subunits into the activated filament-like (short pitch) conformation, while CK-869 binds to a serendipitous pocket on Arp3 and allosterically destabilizes the short pitch Arp3-Arp2 interface. These results provide key insights into the relationship between conformation and activity in Arp2/3 complex and will be critical for interpreting the influence of the inhibitors on actin filament networks in vivo.