Zalevsky J, Grigorova I, Mullins R D
Department of Cellular and Molecular Pharmacology, University of California School of Medicine, San Francisco, California 94129, USA.
J Biol Chem. 2001 Feb 2;276(5):3468-75. doi: 10.1074/jbc.M006407200. Epub 2000 Oct 11.
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.
肌动蛋白激活蛋白（ActA）是一种细菌编码的蛋白质，它使单核细胞增生李斯特菌能够操控宿主细胞的肌动蛋白细胞骨架。它促进依赖于Arp2/3的肌动蛋白成核作用，但其与成核机制的细胞成分之间的相互作用尚未完全清楚。在这里，我们表明，ActA的两个与WASP同源2结构域具有序列相似性的结构域（第85 - 104位氨基酸残基和第121 - 138位氨基酸残基）以亚微摩尔亲和力结合两个肌动蛋白单体。ActA与Arp2/3的解离常数（K(d)）为0.6微摩尔，并与WASP家族蛋白N-WASP和Scar1竞争结合。通过化学交联，ActA、N-WASP和Scar1与Arp2/3复合物的相同三个亚基p40、Arp2和Arp3接触。有趣的是，肌动蛋白单体结合蛋白（profilin）与ActA竞争Arp2/3的结合，但肌动蛋白解聚因子（actophorin，即cofilin）则不然。ActA的最小Arp2/3结合位点（第144 - 170位氨基酸残基）位于两个肌动蛋白结合位点的C末端，并且与WASP家族蛋白的Arp2/3结合区域具有序列同源性。ActA饱和浓度下的最大活性与WASP家族蛋白的最活跃结构域相同。我们提出，ActA和内源性WASP家族蛋白通过类似机制促进依赖于Arp2/3的心核作用，并且需要同时结合Arp2和Arp3。
