Clathrin-coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the β2 subunit of AP2, and several binding sites have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of β2 hinge-appendage (β2HA). We find that the β2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, β2-appendage cross-links two adjacent terminal domains from different triskelia. Functional analysis of β2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the “sandwich site” on the appendage. We propose that β2-appendage binding to more than one triskelion is a key feature of the system and likely explains why assembly is driven by AP2.

中文翻译：

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多模态适配器-网格蛋白触点驱动涂层囊泡组件

网格蛋白包被的小坑是通过 AP2 复合物识别膜和货物以及随后招募网格蛋白 triskelia 而形成的。尚未探索 AP2 在初始网格蛋白募集之外的涂层凹坑组装中的作用。网格蛋白与 AP2 的 β2 亚基结合，并且已经确定了几个结合位点，但我们对这些相互作用的结构了解不完整，并且在内吞过程中它们的功能重要性尚不清楚。在这里，我们分析了在 β2 铰链附件 (β2HA) 存在下组装的网格蛋白笼的冷冻电镜结构。我们发现 β2 附件至少在笼子的两个位置结合，表明多模式结合是网格蛋白-AP2 相互作用的基本特性。在一个位置，β2-附属物交叉连接来自不同三棱藻的两个相邻末端结构域。β2HA-网格蛋白相互作用的功能分析表明，内吞需要两个网格蛋白相互作用位点：铰链上的网格蛋白盒基序和附属物上的“三明治位点”。我们认为β2-附件与多个triskelion 的结合是该系统的一个关键特征，并且可能解释了为什么组装是由AP2 驱动的。