Interaction of WASP/Scar proteins with actin and vertebrate Arp2/3 complex

Abstract

The Wiskott–Aldrich-syndrome protein (WASP) regulates polymerization of actin by the Arp2/3 complex. Here we show, using fluorescence anisotropy assays, that the carboxy-terminal WA domain of WASP binds to a single actin monomer with a K_d of 0.6 μM in an equilibrium with rapid exchange rates. Both WH-2 and CA sequences contribute to actin binding. A favourable ΔH of −10 kcal mol⁻¹ drives binding. The WA domain binds to the Arp2/3 complex with a K_d of 0.9 μM; both the C and A sequences contribute to binding to the Arp2/3 complex. Wiskott–Aldrich-syndrome mutations in the WA domain that alter nucleation by the Arp2/3 complex over a tenfold range without affecting affinity for actin or the Arp2/3 complex indicate that there may be an activation step in the nucleation pathway. Actin filaments stimulate nucleation by producing a fivefold increase in the affinity of WASP-WA for the Arp2/3 complex.