DETERMINANTS OF CLATHRIN STRUCTURE AND FUNCTION

网格蛋白结构和功能的决定因素

• 批准号: 6726377
• 负责人: JOEL A YBE
• 金额: $ 25.16万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6726377
• 关键词: X ray crystallography; clathrin; cryoelectron microscopy; cysteine; molecular assembly /self assembly; polymerization; protein biosynthesis; protein folding; protein structure function; structural biology

DESCRIPTION (provided by applicant): Clathrin coated vesicles (CCVs) carry out receptor-mediated endocytosis (RME) at the plasma membrane, where they drive the uptake of receptor bound nutrients, hormones and proteins targeted for degradation. Clathrin is shaped like a "three-legged" pinwheel to fit its function of forming honeycomb lattices around transport vesicles. The long-term goal of this proposal is to define and validate productive atomic contacts between lattice-bound clathrin molecules to elucidate the fundamental principles of how clathrin function is regulated in cells. The first specific aim is to determine the crystal structure of the clathrin trimerization domain, where the three filamentous legs meet, to complete the atomic level reconstruction of the entire clathrin molecule. Trimerization domain crystals are highly ordered, diffracting between 1.7-2.3 A at Argonne and at the Advanced Light Source. Selenomethionine crystals have been prepared to solve the phasing problem by MAD analysis, while heavy metal replacement and "cryo-halide" soaking methods are being performed as alternative phasing strategies. The X-ray model of trimerization domain will then be docked into the 21 A cryo-EM image of the clathrin barrel (with Barbara Pearse, Corrine Smith and Alan Roseman) to identify productive molecular contacts between the trimerization domain and segments of other clathrin molecules in the lattice. Revealed interactions will be tested biochemically and validated in living cells. The second aim is to explore how cysteines in the trimerization domain stabilize this structure by using structural information from Aim 1 to design mutants to test specific linkages. The third aim explores whether there is a relationship between the cysteines in the trimerization domain and those in light chain subunit to understand why these two groups of cysteines impact regulation of clathrin assembly. The experimental approach is to determine whether light chains can confer structural stability to trimerization domain constructs weakened by mutations made in Aim 2. The final aim of this proposal is to test the possibility that light chain control of lattice formation might involve the transfer of the light chain EED Ph switch between an "active" and "inactive" position on the proximal domain of clathrin. The approach is to use light chain fragments and molecule mimics to ferret out these putative sites and to use X-ray crystallography to try to capture the orientation of light chain fragments bound to the proximal region. Understanding the structural basis for clathrin function is central to defining the role of RME malfunction in hypercholesterolemia and heart disease, as well as understanding the mechanism of virus infection.

描述（由申请人提供）：网格蛋白涂层的囊泡（CCV）在质膜上携带受体介导的内吞作用（RME），在那里它们驱动靶向降解的受体结合养分，激素和蛋白质的吸收。网状蛋白的形状像“三足”的风车，适合其在运输囊泡周围形成蜂窝晶格的功能。该提案的长期目标是定义和验证晶状体结合的网格蛋白分子之间的生产性原子接触，以阐明细胞中网状蛋白功能如何调节网状蛋白功能的基本原理。第一个具体目的是确定三丝腿相遇的网格蛋白三聚体域的晶体结构，以完成整个网格蛋白分子的原子水平重建。修剪域晶体是高度排序的，在Argonne和高级光源处的1.7-2.3 A之间的衍射。已经准备好通过MAD分析来解决阶段问题的准备，而重金属替代品和“冷冻卤化物”浸泡方法正在作为替代性相位策略进行。然后，修剪域的X射线模型将停靠到21中，即在lattice中其他网状蛋白分子的三聚体和其他网状分子之间的生产性分子接触中，网状蛋白枪管的冷冻EM图像（与Barbara Pearse，Corrine Smith和Alan Roseman一起）。揭示的相互作用将在生物化学上进行测试并在活细胞中进行验证。第二个目的是探索通过使用AIM 1到设计突变体的结构信息来测试特定链接的结构信息，探索三聚域中的半胱氨酸如何稳定该结构。第三个目的探讨了三聚体中的半胱氨酸与轻链亚基中的半胱氨酸之间是否存在关系，以了解为什么这两组半胱氨酸会影响网格蛋白组装的调节。实验方法是确定光链是否可以赋予结构稳定性，以通过AIM 2中的突变削弱构造的结构稳定性。该建议的最终目的是测试晶格形成的轻链控制的可能性可能涉及光链EED EED pH开关之间的“活性”和“活性”和“非主动性”位置的转移。该方法是使用轻链碎片和分子模拟物来消除这些假定的位点，并使用X射线晶体学来捕获与近端区域结合的轻链碎片的方向。了解网格蛋白功能的结构基础对于定义RME故障在高胆固醇血症和心脏病中的作用以及了解病毒感染的机制至关重要。