Abstract
ActA is a bacterially encoded protein that enables Listeria monocytogenes to hijack the host cell actin cytoskeleton. It promotes Arp2/3-dependent actin nucleation, but its interactions with cellular components of the nucleation machinery are not well understood. Here we show that two domains of ActA (residues 85-104 and 121-138) with sequence similarity to WASP homology 2 domains bind two actin monomers with submicromolar affinity. ActA binds Arp2/3 with a K(d) of 0.6 microm and competes for binding with the WASP family proteins 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 actophorin (cofilin) does not. The minimal Arp2/3-binding site of ActA (residues 144-170) is C-terminal to both actin-binding sites and shares sequence homology with Arp2/3-binding regions of WASP family proteins. The maximal activity at saturating concentrations of ActA is identical to the most active domains of the WASP family proteins. We propose that ActA and endogenous WASP family proteins promote Arp2/3-dependent nucleation by similar mechanisms and require simultaneous binding of Arp2 and Arp3.
Highlights
ActA is a bacterially encoded protein that enables Listeria monocytogenes to hijack the host cell actin cytoskeleton
In the present study we find that ActA binds two actin monomers with submicromolar affinity using domains with some sequence similarity to WASP homology 2 (WH2) domains from endogenous nucleation promoting factors
The binding site for Arp2/3 is located C-terminal to the actin-binding sites, and it does not contain the characteristic cluster of acidic residues, it shares limited sequence homology with the Arp2/3-binding domains found in WASP family proteins
Summary
ActA is a bacterially encoded protein that enables Listeria monocytogenes to hijack the host cell actin cytoskeleton It promotes Arp2/3-dependent actin nucleation, but its interactions with cellular components of the nucleation machinery are not well understood. We propose that ActA and endogenous WASP family proteins promote Arp2/3-dependent nucleation by similar mechanisms and require simultaneous binding of Arp and Arp. By chemical cross-linking and polarization anisotropy, we show that ActA and the WASP family proteins hScar and N-WASP contact the same subunits on Arp2/3 and compete for the same binding site. This binding site is much more extensive than previously thought and includes both of the actin-related proteins, Arp and Arp. In vitro the maximal activity of full-length ActA is nearly identical to the endogenous nucleation promoting factor N-WASP
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