Structure of Arp2/3 complex at a branched actin filament junction resolved by single-particle cryo-electron microscopy.
Structure of Arp2/3 complex at a branched actin filament junction resolved by single-particle cryo-electron microscopy.
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DOI：
10.1073/pnas.2202723119
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发表时间：
2022-05-31
影响因子：
11.1
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中科院分区：
文献类型：
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Arp2/3复合体的肌动蛋白细丝成核必须由WASP家族蛋白等激活剂触发。由于缺乏处于激活状态的Arp2/3复合体的高分辨率结构，了解WASP蛋白如何激活Arp2/3复合体一直是一个重大挑战。我们确定了Wasp激活的Arp2/3复合体在分支连接的高分辨(∼3.9o)结构，并使用生化、细胞生物学和分子动力学模拟来了解Wasp介导的激活机制。这项工作详细展示了完全激活的Arp2/3复合体、有核子肌动蛋白细丝和母肌动蛋白细丝之间的联系，并为了解Arp2/3复合体在激活过程中如何刺激构象重排提供了重要的见解。Arp2/3复合体使分支的肌动蛋白细丝成核，这些肌动蛋白细丝提供推动细胞过程的力量，如板脂突起和内吞作用。Arp2/3复合体本质上是非活性的，多类成核促进因子(NPF)刺激其成核活性。当被WASP家族NPF激活时，该复合体必须与预先存在的(母)肌动蛋白细丝结合以完成成核过程，确保WASP介导的激活产生分支而不是线性肌动蛋白细丝。肌动蛋白细丝如何参与激活目前还不清楚，这在很大程度上是由于缺乏结合在细丝一侧的激活的Arp2/3复合体的高分辨率结构。在这里，我们给出了Arp2/3复合体在分支结处的3.9°低温电子显微镜结构。该结构揭示了Arp2/3复合体与母肌动蛋白细丝一侧之间的联系，这可能会刺激亚单位扁平化，这种构象变化允许复合体中与肌动蛋白相关的蛋白质亚基(Arp2和Arp3)模仿丝状肌动蛋白亚基。相反，复合体的下半部分与母丝之间的有限接触表明，钳制扭曲是在激活状态下观察到的第二个主要构象变化，不受肌动蛋白细丝的刺激，这可能解释了为什么在WASP介导的激活过程中，肌动蛋白细丝是必需的，但不足以触发成核。结合生化和活细胞成像数据以及分子动力学模拟，该结构揭示了Arp2/3复合体与肌动蛋白细丝相互作用的关键特征，以及细胞内分支肌动蛋白细丝网络的调控组装。
Actin filament nucleation by Arp2/3 complex must be triggered by activators like WASP family proteins. Understanding how WASP proteins activate Arp2/3 complex has been a major challenge due to a lack of high-resolution structures of the complex in an activated state. We determined a high-resolution (∼3.9 Å) structure of the WASP-activated Arp2/3 complex at a branch junction and used biochemical, cell biological, and molecular dynamic simulations to understand the mechanism of WASP-mediated activation. This work shows in detail the contacts between the fully activated Arp2/3 complex, the nucleated daughter actin filament, and the mother actin filament and provides important insights into how conformational rearrangements in the Arp2/3 complex are stimulated during activation. Arp2/3 complex nucleates branched actin filaments that provide pushing forces to drive cellular processes such as lamellipodial protrusion and endocytosis. Arp2/3 complex is intrinsically inactive, and multiple classes of nucleation promoting factors (NPFs) stimulate its nucleation activity. When activated by WASP family NPFs, the complex must bind to the side of a preexisting (mother) filament of actin to complete the nucleation process, ensuring that WASP-mediated activation creates branched rather than linear actin filaments. How actin filaments contribute to activation is currently not understood, largely due to the lack of high-resolution structures of activated Arp2/3 complex bound to the side of a filament. Here, we present the 3.9-Å cryo-electron microscopy structure of the Arp2/3 complex at a branch junction. The structure reveals contacts between Arp2/3 complex and the side of the mother actin filament that likely stimulate subunit flattening, a conformational change that allows the actin-related protein subunits in the complex (Arp2 and Arp3) to mimic filamentous actin subunits. In contrast, limited contact between the bottom half of the complex and the mother filament suggests that clamp twisting, a second major conformational change observed in the active state, is not stimulated by actin filaments, potentially explaining why actin filaments are required but insufficient to trigger nucleation during WASP-mediated activation. Along with biochemical and live-cell imaging data and molecular dynamics simulations, the structure reveals features critical for the interaction of Arp2/3 complex with actin filaments and regulated assembly of branched actin filament networks in cells.
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