Structural characterization of dynamin GTPases

描述（由申请人提供）：本实验室的长期目标是了解细胞骨架蛋白质所采用的详细结构机制，以产生力并介导与其各种分子内或分子间蛋白质靶点的相互作用。对于ATP驱动的分子马达，以及GTP驱动的G蛋白家族的分子开关，构象状态由核苷酸邻磷酸的存在或不存在决定。一个有趣的问题是：蛋白质是如何感知如此微小的构象变化，然后放大它们，有时放大几个数量级，以实现其特定的细胞功能？该提案旨在探索发动蛋白家族中的结构/功能关系，该家族由参与诸如内吞作用、囊泡运输、线粒体形态维持和病毒抗性等功能的大GTP酶组成。虽然有证据表明发动蛋白通过力产生机制驱动膜裂变，但关于它是否主动产生力，或者更确切地说是内吞作用的调节剂，存在很多争议。我们的五年计划的目标是回答三个主要问题：1）什么结构特征使发动蛋白能够在胞吞囊泡的颈部周围形成聚合物环？2)调节发动蛋白GT3活性的结构基础是什么？3)当发动蛋白通过GTP水解循环时，它的构象发生了什么变化？我们将通过解决一个或多个来自发动蛋白超家族的蛋白质在不同核苷酸状态下的高分辨率X射线晶体结构，以及识别和结构表征发动蛋白突变体来研究这些问题。

DESCRIPTION (provided by applicant): The long-term goal of this laboratory is to understand the detailed structural mechanisms employed by cytoskeletal proteins in order to produce force and mediate interactions with their various intra- or intermolecular protein targets. For ATP-driven molecular motors, as well as the GTP-driven G-protein family of molecular switches, conformational states are governed by the presence or absence of the nucleotide o-phosphate. An intriguing question is: how proteins can sense such small conformational changes and then amplify them, sometimes by several orders of magnitude, in order to achieve their specific cellular function? This proposal aims to explore structure/function relationships in the dynamin protein family, which consists of large GTPases involved in functions such as endocytosis, vesicle trafficking, maintenance of mitochondrial morphology, and viral resistance. Although there is evidence that dynamin drives membrane fission via a force-generating mechanism, there is much debate as to whether it actively generates force, or rather acts a regulator of endocytosis. Our goal for the five-year project is to answer three major questions: 1) What structural features enable dynamin to form polymeric rings around the necks of endocytotic vesicles? 2) What is the structural basis for regulation of dynamin's GTPase activity? 3) What conformational changes take place in dynamin as it cycles through its GTP hydrolysis cycle? We will investigate these questions by solving the high-resolution X-ray crystal structure of one or more proteins from the dynamin superfamily in different nucleotide states, as well as identifying and structurally characterizing dynamin mutants.