STRUCTURAL BASIS FOR CLATHRIN FUNCTION

网格蛋白包被囊泡（CCVs）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生物生成所需的反式高尔基网络中的受体分选。三螺旋形网格蛋白分子自组装成多面体蛋白外壳是ccv隔离受体的驱动力。哺乳动物网格蛋白三叉链由LCa和LCb两种不同类型的3条重链和3条轻链组成，并具有剪接变体。轻链亚基调节网格蛋白的组装和拆卸，受体通过接头分子与聚合的网格蛋白晶格结合，接头分子也调节网格蛋白的自组装。本课题旨在通过解析网格蛋白的晶体结构来确定网格蛋白自组装的分子基础，为基于结构的诱变奠定基础。在该基金的第一个资助期内，我们结晶并解决了网格蛋白三旋翼近端结构域的结构。这揭示了一个结构基序，称为网格蛋白重链重复（CHCR），我们预测，通过序列谱，可以解释网格蛋白重链三聚化相互作用。chcr在网格蛋白三叉腿的线性部分重复，终止于连接末端结构域的柔性连接体区域，其结构最近由Kirchhausen和Harrison小组解决。在参与酵母液泡膜运输的其他蛋白质中也发现了CHCR。我们建议完成剩余的网格蛋白三叉腿的结构确定（三聚域，远端腿段和与网格蛋白重链结合的网格蛋白轻链亚基的几种形式），并将测试CHCR结构基序的一般功能。这种对网格蛋白轻链的分析将有助于阐明它们如何发挥调控作用，并有助于理解它们的差异功能。在确定网格蛋白片段的结构后，它们将被装入Pearse及其同事制作的组装网格蛋白的21埃分辨率模型中，以确定网格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，亚基和组装相互作用的模型将通过基于结构的诱变进行测试。了解参与ccv形成的网格蛋白聚合的分子控制与细胞生长控制、通过受体和配体下调维持体内平衡以及产生免疫反应的膜交通途径有关。

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.