Structural characterization of dynamin GTPases

描述(由申请人提供)：该实验室的长期目标是了解细胞骨架蛋白所采用的详细结构机制，以便产生力并调节与其各种分子内或分子间蛋白质靶标的相互作用。对于ATP驱动的分子马达，以及GTP驱动的G蛋白分子开关家族，构象状态由o-磷酸核苷酸的存在或不存在决定。一个耐人寻味的问题是：蛋白质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？本研究旨在探索Dynamin蛋白家族的结构/功能关系，该家族由大的GTP酶组成，参与内吞作用、囊泡运输、维持线粒体形态和病毒抗性等功能。尽管有证据表明，动力素通过一种力量产生机制驱动膜分裂，但对于它是否主动产生力量，或者更确切地说，它是一种内吞作用的调节者，仍有很多争论。我们这个为期五年的项目的目标是回答三个主要问题：1)什么结构特征使Dynamin能够在内吞囊泡的颈部形成聚合环？2)Dynamin GTP酶活性调节的结构基础是什么？3)Dynamin在其GTP水解循环过程中发生了什么构象变化？我们将通过解决Dynamin超家族中一个或多个蛋白质在不同核苷酸状态下的高分辨率X射线晶体结构，以及鉴定和结构特征Dynamin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.