Structural Basis of Actin Cytoskeleton Dynamics

肌动蛋白细胞骨架动力学的结构基础

• 批准号: 7912112
• 负责人: ROBERTO DOMINGUEZ
• 金额: $ 7.19万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-09 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7912112
• 关键词: ADP-G-actin; ATP-G-actin; Actin-Binding Protein; Actinin; Actins; Address; Adenylate Cyclase; Affinity; Amino Acid Motifs; Amino Acids; Binding; Binding Sites; Biochemical; Cardiovascular system; Cell Shape; Cell division; Cell physiology; Collaborations; Complement; Complex; Cytoskeletal Proteins; Cytoskeleton; Disease; Dissociation; Electron Microscopy; Eukaryotic Cell; F-Actin; G Actin; Gelsolin; Goals; Homologous Protein; Human; Hydrolysis; Inflammatory; Intracellular Transport; Investigation; Link; Maps; Microfilaments; Mitosis; Molecular; Muscle Contraction; Nature; Neurodegenerative Disorders; Phosphatidylinositol 4,5-Diphosphate; Process; Protein Family; Proteins; Regulation; Roentgen Rays; Role; Signal Transduction; Structure; Thymosin; Wiskott-Aldrich Syndrome; X-Ray Crystallography; actin depolymerizing factor; alpha Actinin; base; calponin; cofactor; cofilin; crosslink; destrin; in vivo; link protein; numb protein

DESCRIPTION (provided by applicant): The rapid assembly and disassembly of the actin cytoskeleton is central to many cellular functions, including mitosis, cell division, intracellular transport, and the control of cell shape and polarity. Alterations of cytoskeletal proteins are linked to diseases, including cardiovascular and neurodegenerative disorders. The hydrolysis of ATP by actin drives the transition between its monomeric form (G-actin) and filamentous form (F-actin). F-actin is asymmetric, undergoing net association of ATP-actin to the "barbed" end and dissociation of ADP-actin from the "pointed" end. This dynamic process, known as actin filament treadmilling, is regulated in vivo by a plethora of actin-binding proteins (ABPs). ABPs have evolved relatively few actin-binding motifs, including the WASP homology domain-2 (WH2), actin-depolymerizing factor-homology (ADF-H), and calponin-homology (CH) motifs. The long-term goal of this proposal is to understand the structural basis for the interactions of these motifs with G- and F-actin and their role in the regulation of the cytoskeleton. A unifying hypothesis is proposed that holds that ADF-H, WH2, gelsolin, and actin itself, which are generally unrelated, present common structural features that allow them to share a common binding site on actin, consisting of a hydrophobic pocket at the interface between subdomains 1 and 3. In contrast, the CH domain, which is a classical F-actin-binding motif, is not expected to bind in this pocket. Specific aim 1 studies the X-ray structures of complexes of WH2 domains from various disease-related proteins with actin. Specific aim 2 deals with the study of twinfilin, which is composed of two ADF-H domains in tandem. Specific aim 3 focuses on the study of the crystal structure of the CH pair from alpha-actinin, its regulation by PIP2, and its interaction with F-actin. Crystals are available in aims 1 and 3, and micro-crystals are also available in aim 2. The crystallographic and biochemical studies are being complemented by EM studies of twinfilin and a-actinin decorated F-actin, in collaboration with W.J. Lehman at BU. This proposal offers a comprehensive approach to study three of the most important actin-binding motifs in nature. By studying these motifs in parallel we will establish common and distinctive features of their structures and interactions with G- and F-actin that determine their functions in the regulation of the cytoskeleton.

描述（由申请人提供）：肌动蛋白细胞骨架的快速组装和拆卸对于许多细胞功能至关重要，包括有丝分裂、细胞分裂、细胞内运输以及细胞形状和极性的控制。细胞骨架蛋白的改变与疾病有关，包括心血管和神经退行性疾病。肌动蛋白对 ATP 的水解驱动其单体形式（G-肌动蛋白）和丝状形式（F-肌动蛋白）之间的转变。 F-肌动蛋白是不对称的，经历ATP-肌动蛋白与“带刺”端的净结合以及ADP-肌动蛋白与“尖”端的解离。这种动态过程被称为肌动蛋白丝跑步，在体内受到大量肌动蛋白结合蛋白（ABP）的调节。 ABP 进化出相对较少的肌动蛋白结合基序，包括 WASP 同源结构域 2 (WH2)、肌动蛋白解聚因子同源 (ADF-H) 和钙调蛋白同源 (CH) 基序。该提案的长期目标是了解这些基序与 G- 和 F-肌动蛋白相互作用的结构基础及其在细胞骨架调节中的作用。 提出了一个统一的假设，认为 ADF-H、WH2、凝溶胶蛋白和肌动蛋白本身通常不相关，但具有共同的结构特征，使它们能够在肌动蛋白上共享一个共同的结合位点，由子结构域 1 和 3 之间界面处的疏水口袋组成。相比之下，CH 结构域是经典的 F-肌动蛋白结合基序，预计不会在该口袋中结合。具体目标 1 研究各种疾病相关蛋白的 WH2 结构域与肌动蛋白复合物的 X 射线结构。具体目标 2 涉及 twinfilin 的研究，它由两个串联的 ADF-H 结构域组成。具体目标 3 重点研究 α-肌动蛋白 CH 对的晶体结构、PIP2 对其的调节及其与 F-肌动蛋白的相互作用。目标 1 和 3 中提供晶体，目标 2 中也提供微晶体。与 BU 的 W.J. Lehman 合作，对 twinfilin 和 a-肌动蛋白修饰的 F-肌动蛋白的电镜研究补充了晶体学和生化研究。该提案提供了一种综合方法来研究自然界中三个最重要的肌动蛋白结合基序。通过并行研究这些基序，我们将确定它们结构的共同和独特特征以及与 G 和 F 肌动蛋白的相互作用，从而确定它们在细胞骨架调节中的功能。