An actin-filament-binding interface on the Arp2/3 complex is critical for nucleation and branch stability
An actin-filament-binding interface on the Arp2/3 complex is critical for nucleation and branch stability
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DOI：
10.1073/pnas.0911668107
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发表时间：
2010-05-04
影响因子：
11.1
通讯作者：
Welch, Matthew D.
中科院分区：
文献类型：
--
作者：
Goley, Erin D.;Rammohan, Aravind;Welch, Matthew D.
Arp 2/3复合物从现有细丝的侧面聚合新的肌动蛋白细丝，形成Y分支网络，这对肌动蛋白介导的力的产生至关重要。因此，Arp 2/3复合物与肌动蛋白丝侧的结合对其肌动蛋白成核和分支活性至关重要。虽然Arp 2/3复合物在细丝分支的模型已提出基于电子显微镜，该模型尚未使用独立的方法进行验证，预测肌动蛋白结合残基的功能的重要性还没有得到广泛的测试。使用分子动力学和蛋白质-蛋白质对接模拟的组合，我们推导出一个独立的结构模型的Arp 2/3复合物的两个亚基之间的相互作用，是关键的肌动蛋白结合，ARPC 2和ARPC 4，和侧的肌动蛋白丝。这个模型与以前的电子显微镜结果非常一致。互补诱变实验揭示了ARPC 2和ARPC 4中的许多残基，这些残基是整个复合物的生化活性所需的。功能关键的残基聚集在一起，并确定了一个表面，预测蛋白质-蛋白质对接被掩埋在与肌动蛋白的相互作用。此外，在这个接口的关键残基是至关重要的肌动蛋白成核和Y-分支，高亲和力的F-肌动蛋白结合，和Y-分支稳定性，表明Arp 2/3复合物的F肌动蛋白的亲和力独立调节分支的形成和稳定性。我们的研究结果突出了计算和实验相结合的方法来研究蛋白质-蛋白质相互作用的效用，并为进一步阐明F-actin结合在Arp 2/3复合物激活和功能中的作用提供了基础。
The Arp2/3 complex polymerizes new actin filaments from the sides of existing filaments, forming Y-branched networks that are critical for actin-mediated force generation. Binding of the Arp2/3 complex to the sides of actin filaments is therefore central to its actin-nucleating and branching activities. Although a model of the Arp2/3 complex in filament branches has been proposed based on electron microscopy, this model has not been validated using independent approaches, and the functional importance of predicted actin-binding residues has not been extensively tested. Using a combination of molecular dynamics and protein-protein docking simulations, we derived an independent structural model of the interaction between two subunits of the Arp2/3 complex that are key to actin binding, ARPC2 and ARPC4, and the side of an actin filament. This model agreed remarkably well with the previous results from electron microscopy. Complementary mutagenesis experiments revealed numerous residues in ARPC2 and ARPC4 that were required for the biochemical activity of the entire complex. Functionally critical residues clustered together and defined a surface that was predicted by protein-protein docking to be buried in the interaction with actin. Moreover, key residues at this interface were crucial for actin nucleation and Y-branching, high-affinity F-actin binding, and Y-branch stability, demonstrating that the affinity of Arp2/3 complex for F actin independently modulates branch formation and stability. Our results highlight the utility of combining computational and experimental approaches to study protein-protein interactions and provide a basis for further elucidating the role of F-actin binding in Arp2/3 complex activation and function.