Activation of the Arp2/3 complex by the Listeria ActA protein - ActA bindstwo actin monomers and three subunits of the Arp2/3 complex

ActA is a bacterially encoded protein that enables Listeria monocytogenes t o hijack the host cell actin cytoskeleton. It promotes Arp2/3-dependent act in nucleation, but its interactions with cellular components of the nucleat ion machinery are not well understood. Here we show that two domains of Act A (residues 85-104 and 121-138) with sequence similarity to WASP homology 2 domains bind two actin monomers with submicromolar affinity. ActA binds Ar p2/3 with a K-d of 0.6 muM and competes for binding with the WASP family pr oteins N-WASP and Scar1. By chemical cross-linking, ActA, N-WASP, and Scar1 contact the same three subunits of the Arp2/3 complex, p40, Arp2, and Arp3 . Interestingly, profilin competes with ActA for binding of Arp2/3, but act ophorin (cofilin) does not. The minimal Arp2/3-binding site of ActA (residu es 144-170) is C-terminal to both actin-binding sites and shares sequence h omology with Arp2/3-binding regions of WASP family proteins. The maximal ac tivity at saturating concentrations of ActA is identical to the most active domains of the WASP family proteins. We propose that ActA and endogenous W ASP family proteins promote Arp2/3-dependent nucleation by similar mechanis ms and require simultaneous binding of Arp2 and Arp3.