Structural Basis of Actin Cytoskeleton Dynamics

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。肌动蛋白水解ATP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“倒刺”端的净结合和adp -肌动蛋白与“尖”端的解离。这一动态过程被称为肌动蛋白丝跑步，在体内受大量肌动蛋白结合蛋白（ABPs）的调节。ABPs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）和钙钙蛋白同源（CH）基序。这项建议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。

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.