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是许多细胞功能的核心，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“倒刺”末端的净缔合和ADP-肌动蛋白与“尖”末端的解离。这种动态过程，被称为肌动蛋白丝的微磨，在体内由大量的肌动蛋白结合蛋白（ABP）调节。ABP已经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（WH 2）、肌动蛋白解聚因子同源性（ADF-H）和钙调蛋白同源性（CH）基序。这个提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。 一个统一的假设提出，认为ADF-H，WH 2，凝溶胶蛋白，和肌动蛋白本身，这通常是不相关的，目前共同的结构特征，使他们能够共享一个共同的结合位点的肌动蛋白，由一个疏水口袋之间的接口子域1和3。相比之下，CH结构域，这是一个经典的F-肌动蛋白结合基序，预计不会结合在这个口袋里。具体目标1研究来自各种疾病相关蛋白质的WH 2结构域与肌动蛋白的复合物的X射线结构。具体目标2涉及twinfilin的研究，twinfilin由两个ADF-H结构域串联组成。具体目标3侧重于研究α-辅肌动蛋白的CH对的晶体结构，PIP 2对其的调节，以及其与F-肌动蛋白的相互作用。晶体可在目标1和3中获得，微晶也可在目标2中获得。与BU的W. J. Lehman合作，对Twinfilin和α-辅肌动蛋白修饰的F-肌动蛋白进行了EM研究，以补充晶体学和生物化学研究。这一建议提供了一个全面的方法来研究自然界中最重要的三个肌动蛋白结合基序。通过平行研究这些图案，我们将建立共同的和独特的功能，它们的结构和相互作用的G-和F-肌动蛋白，确定其功能的调节细胞骨架。

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.