STRUCTURAL BASIS FOR CLATHRIN FUNCTION

笼蛋白包裹的囊泡(CCV)介导受体结合的营养物质、激素和蛋白质的内吞作用，以及溶酶体和分泌颗粒的生物发生所需的跨高尔基体网络中的受体分选。三棱柱形状的网状蛋白分子自组装成多面体蛋白涂层是CCVS受体隔离的驱动力。哺乳动物clathrin triskelion由LCA和LCB两种不同类型的三条重链和三条轻链组成，并带有剪接变体。轻链亚基调节网状蛋白的组装和分解，受体通过接头分子与聚合的网状蛋白格子相连，这也调节了网状蛋白的自组装。这项建议旨在通过解析分子筛的晶体结构来确定分子自组装的分子基础，作为基于结构的突变的基础。在这笔赠款的第一个资助期，我们结晶并解决了笼状蛋白三倍体近端结构域的结构。这揭示了一个称为笼状重链重复(CHCR)的结构基序，根据序列图谱，我们预测它可能解释了笼状重链三聚相互作用。CHCR在clathrin triskelion腿的线性部分重复，终止于连接到末端结构域的柔性连接区，其结构最近由Kirchhausen和Harison的小组解决。在与酵母液泡的膜运输有关的其他蛋白质中也发现了CHCR。我们建议完成其余部分的结构测定(三聚化结构域、远端腿段和几种形式的与笼状蛋白重链结合的笼状蛋白轻链亚基)，并将测试CHCR结构基序的一般功能。这种对网状蛋白轻链的分析将有助于阐明它们是如何发挥调节作用的，并有助于理解它们的不同功能。随着网状蛋白片段的结构被确定，它们将被适合于由Pearse和他的同事制作的21埃分辨率的组装网状蛋白模型，以确定网状蛋白片段如何在多面体晶格中相互作用。还将分析自组装亚片段的晶体形式，以建立组装相互作用。最后，将通过基于结构的突变对亚基和组装相互作用模型进行检验。了解参与CCV形成的分子调控与细胞生长控制、通过受体和配体下调维持动态平衡以及产生免疫应答的膜信号通路有关。

Clathrin-coated vesicles (CCVs) mediate endocytosis of receptor- bound nutrients, hormones and proteins destined for degradation, as well as receptor sorting in the trans-Golgi network required for biogenesis of lysosomes and secretory granules. Self- assembly of the triskelion-shaped clathrin molecule into a polyhedral protein coat is the driving force for receptor sequestration by CCVs. The mammalian clathrin triskelion comprises three heavy chains and three light chains of two different types, LCa and LCb, with splicing variants. The light chain subunits regulate clathrin assembly and disassembly, and receptors become associated with the polymerized clathrin lattice via adaptor molecules, which also regulate clathrin self- assembly. This proposal aims to define the molecular basis for clathrin self-assembly by resolution of the crystallographic structure of clathrin, as a foundation for structure-based mutagenesis. During the first funding period of this grant, we crystallized and solved the structure of the proximal domain of the clathrin triskelion. This revealed a structural motif, termed clathrin heavy chain repeat (CHCR), which we predict, by sequence profile, may account for clathrin heavy chain trimerization interactions. The CHCRs are repeated throughout the linear portion of the clathrin triskelion leg, terminating at the flexible linker region attached to the terminal domain, whose structures were recently solved by the groups of Kirchhausen and Harrison. The CHCR was also found in other proteins involved in membrane traffic to the yeast vacuole. We propose to complete the structural determination of the remainder of the clathrin triskelion leg (the trimerization domain, the distal leg segment and several forms of the clathrin light chain subunits bound to the clathrin heavy chain) and will test the general function of the CHCR structural motif. This analysis of clathrin light chains will help elucidate how they exert regulation and contribute to understanding their differential function. As the structures of the clathrin segments are determined, they will be fit into a 21 angstrom resolution model of assembled clathrin, produced by Pearse and colleagues, to determine how clathrin segments interact in the polyhedral lattice. Crystal forms of self-assembled sub-fragments will also be analyzed to establish assembly interactions. Lastly, the models of subunit and assembly interactions will be tested by structure-based mutagenesis. Understanding the molecular control of clathrin polymerization involved in formation of CCVs is relevant to cell growth control, to maintenance of homeostasis by receptor and ligand downregulation, and to membrane traffic pathways generating an immune response.