Actin binding to the central domain of WASP/Scar proteins plays a critical role in the activation of the arp2/3 complex
Actin binding to the central domain of WASP/Scar proteins plays a critical role in the activation of the arp2/3 complex
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DOI：
10.1074/jbc.m507470200
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发表时间：
2006-04-14
影响因子：
4.8
通讯作者：
Mullins, RD
中科院分区：
文献类型：
--
作者：
Kelly, AE;Kranitz, H;Mullins, RD
Arp2/3复合体在运动细胞的前沿形成并交叉连接肌动蛋白细丝，其活性受到WASP/SCAR蛋白的C-末端区域的刺激，称为VCA结构域。VCA结构域包含一个与单体肌动蛋白和中心肌动蛋白结合的异丙氨酸同源序列(V)和一个结合Arp2/3复合体的酸性序列(A)。在这里，我们证明了C结构域与单体肌动蛋白(K-d=10muM)的亲和力高于与Arp2/3复合体(K-d>；200muM)的亲和力。核磁共振波谱显示，肌动蛋白与C结构域的N端一半结合，并且V和C结构域都可以独立地同时结合肌动蛋白，表明它们与不同的位点相互作用。C结构域肌动蛋白结合界面上保守的疏水残基的突变会破坏Arp2/3复合体的激活，但不会改变复合体的亲和力。通过化学交联，C结构域与Arp2/3复合体的p40亚单位相互作用，通过荧光偏振各向异性，肌动蛋白与Arp2/3复合体的结合是相互排斥的。我们的结果表明，肌动蛋白和Arp2/3结合对C结构域的功能都是重要的，但C结构域并不在两者之间形成静态桥梁。我们提出了一个激活Arp2/3复合体的模型，其中C结构域首先通过诱导必要的构象变化来启动复合体，然后通过将肌动蛋白单体带到启动的复合体附近来启动核组装。
The Arp2/3 complex nucleates and cross-links actin filaments at the leading edge of motile cells, and its activity is stimulated by C-terminal regions of WASP/Scar proteins, called VCA domains. VCA domains contain a verprolin homology sequence (V) that binds monomeric actin and central (C) and acidic sequences (A) that bind the Arp2/3 complex. Here we show that the C domain binds to monomeric actin with higher affinity (K-d = 10 mu M) than to the Arp2/3 complex (K-d > 200 mu M). Nuclear magnetic resonance spectroscopy reveals that actin binds to the N-terminal half of the C domain and that both the V and C domains can bind actin independently and simultaneously, indicating that they interact with different sites. Mutation of conserved hydrophobic residues in the actin-binding interface of the C domain disrupts activation of the Arp2/3 complex but does not alter affinity for the complex. By chemical cross-linking the C domain interacts with the p40 subunit of the Arp2/3 complex and, by fluorescence polarization anisotropy, the binding of actin and the Arp2/3 complex are mutually exclusive. Our results indicate that both actin and Arp2/3 binding are important for C domain function but that the C domain does not form a static bridge between the two. We propose a model for activation of the Arp2/3 complex in which the C domain first primes the complex by inducing a necessary conformational change and then initiates nucleus assembly by bringing an actin monomer into proximity of the primed complex.