Dynamic interactions between clathrin and locally structured elements in a disordered protein mediate clathrin lattice assembly.
Dynamic interactions between clathrin and locally structured elements in a disordered protein mediate clathrin lattice assembly.
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DOI：
10.1016/j.jmb.2010.09.044
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发表时间：
2010-11-26
影响因子：
5.6
通讯作者：
Lafer EM
中科院分区：
文献类型：
--
作者：
Zhuo Y;Ilangovan U;Schirf V;Demeler B;Sousa R;Hinck AP;Lafer EM
网格蛋白晶格的组装是由装配/接头蛋白介导的，其包含结合脂质或膜结合货物蛋白的结构域，以及招募网格蛋白的网格蛋白结合结构域（CBDs）。在这里，我们描述了网格蛋白与网格蛋白组装蛋白AP180的大片段CBD之间的相互作用。突变，核磁共振化学位移和分析性超离心分析使我们能够在该片段中精确定义两个网格蛋白结合位点，每个位点都与网格蛋白重链（TD）的n端结构域弱结合。这两个网格蛋白结合位点的位置与先前鉴定的网格蛋白结合元件的序列匹配预测一致，并且通过扩展，表明完整的AP180 CBD包含约12个简并重复序列，每个重复序列包含一个网格蛋白结合位点。序列和圆二色性分析表明，AP180 CBD主要是非结构化的，我们的核磁共振分析证实，这在很大程度上是AP180片段的情况。出乎意料的是，与许多蛋白质在与它们的结合伙伴相互作用时发生结合偶联折叠不同，AP180片段在其结合和自由状态下同样是非结构化的。相反，我们发现该片段在游离和与网格蛋白结合时，在两个网格蛋白结合位点都表现出局部的β turn样结构。这些观察结果被纳入到一个模型中，在这个模型中，多个预先结构化的网格蛋白结合元素的弱结合规律地分散在一个很大程度上非结构化的CBD中，允许网格蛋白有效地招募到内噬位点和网格蛋白晶格的动态组装。
Assembly of clathrin lattices is mediated by assembly/adaptor proteins which contain domains that bind lipids or membrane bound cargo proteins, and clathrin binding domains (CBDs) that recruit clathrin. Here we characterize the interaction between clathrin and a large fragment of the CBD of the clathrin assembly protein AP180. Mutational, NMR chemical shift, and analytical ultracentrifugation analyses allowed us to precisely define two clathrin binding sites within this fragment, each of which is found to bind weakly to the N-terminal domain of the clathrin heavy chain (TD). The locations of the two clathrin binding sites are consistent with predictions from sequence alignments of previously identified clathrin binding elements and, by extension, indicate that the complete AP180 CBD contains ~12 degenerate repeats, each containing a single clathrin binding site. Sequence and circular dichroism analyses have indicated that the AP180 CBD is predominantly unstructured and our NMR analyses confirm that this is largely the case for the AP180 fragment characterized here. Unexpectedly, unlike the many proteins which undergo binding coupled folding upon interaction with their binding partners, the AP180 fragment is similarly unstructured in its bound and free states. Instead, we find that this fragment exhibits localized β turn-like structures at the two clathrin binding sites both when free and bound to clathrin. These observations are incorporated into a model in which weak binding by multiple, pre-structured clathrin binding elements regularly dispersed throughout a largely unstructured CBD allows efficient recruitment of clathrin to endocytic sites and dynamic assembly of the clathrin lattice.
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