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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Visualizing Arp2/3 complex activation mediated by binding of ATP and WASp using structural mass spectrometry
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Visualizing Arp2/3 complex activation mediated by binding of ATP and WASp using structural mass spectrometry

机译：使用结构质谱法可视化由ATP和WASp结合介导的Arp2 / 3复合物激活

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摘要

Actin-related protein (Arp) 2/3 complex nucleates new branches in actin filaments playing a key role in controlling eukaryotic cell motility. This process is tightly regulated by activating factors: ATP and WASp-family proteins. However, the mechanism of activation remains largely hypothetical. We used radiolytic protein footprint-ing with mass spectrometry in solution to probe the effects of nucleotide- and WASp-binding on Arp2/3. These results represent two significant advances in such footprinting approaches. First, Arp2/3 is the most complex macromolecular assembly yet examined; second, only a few picomoles of Arp2/3 was required for individual experiments. In terms of structural biology of Arp 2/3, we find that ATP binding induces conformational changes within Arp2/3 complex in Arp3 (localized in peptide segments 5-18, 212-225, and 318-327) and Arp2 (within peptide segment 300-316). These data are consistent with nucleotide docking within the nucleotide clefts of the actin-related proteins promoting closure of the cleft of the Arp3 subunit. However, ATP binding does not induce conformational changes in the other Arp subunits. Arp2/3 complex binds to WASp within the C subdomain at residue Met 474 and within the A subdomain to Trp 500. Our data suggest a bivalent attachment of WASp to Arp3 (within peptides 162-191 and 318-329) and Arp2 (within peptides 66-80 and 87-97). WASp-depen-dent protections from oxidation within peptides 54-65 and 80-91 of Arp3 and in peptides 300-316 of Arp2 suggest domain rearrangements of Arp2 and Arp3 resulting in a closed conformational state consistent with an "actin-dimer" model for the active state.
机译：肌动蛋白相关蛋白（Arp）2/3复合物使肌动蛋白丝中的新分支成核，在控制真核细胞运动中起关键作用。此过程受激活因子：ATP和WASp家族蛋白的调控。但是，激活机制在很大程度上仍是假设的。我们在溶液中使用质谱分析了放射性蛋白质足迹，以研究核苷酸和WASp结合对Arp2 / 3的影响。这些结果代表了这种足迹方法的两个重大进展。首先，Arp2 / 3是迄今为止最复杂的大分子组装体。其次，单个实验只需要几个皮摩尔的Arp2 / 3。在Arp 2/3的结构生物学方面，我们发现ATP结合诱导Arp3（位于肽段5-18、212-225和318-327中）和Arp2（位于肽段内）的Arp2 / 3复合物中的构象变化300-316）。这些数据与肌动蛋白相关蛋白的核苷酸裂口内的核苷酸对接相一致，从而促进了Arp3亚基裂口的闭合。但是，ATP结合不会诱导其他Arp亚基的构象变化。 Arp2 / 3复合物在残基Met 474的C子域和Trp 500的A子域内与WASp结合。我们的数据表明WASp与Arp3（在肽162-191和318-329中）和Arp2（在肽内）具有二价结合。 66-80和87-97）。 WASp依赖于Arp3的肽54-65和80-91以及Arp2的肽300-316的氧化保护表明Arp2和Arp3的结构域重排导致与“肌动蛋白二聚体”模型一致的闭合构象状态活动状态。

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