Structural Basis of Actin Cytoskeleton Dynamics

描述(申请人提供)：肌动蛋白细胞骨架的快速组装和分解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包括心血管和神经退行性疾病在内的疾病有关。肌动蛋白对三磷酸腺苷的水解促使其单体形式(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，经历了ATP-肌动蛋白的净结合和ADP-肌动蛋白从“尖端”的解离。这个动态的过程被称为肌动蛋白细丝踏板，在体内受到过多的肌动蛋白结合蛋白(ABPs)的调节。ABPS进化出的肌动蛋白结合基序相对较少，包括WASP同源结构域2(WH2)、肌动蛋白解聚因子同源(ADF-H)和钙蛋白同源(CH)基序。这个建议的长期目标是了解这些基序与G-肌动蛋白和F-肌动蛋白相互作用的结构基础，以及它们在细胞骨架调控中的作用。 提出了一个统一的假设，认为ADF-H、WH2、明胶蛋白和肌动蛋白本身通常是不相关的，它们呈现共同的结构特征，使它们能够共享肌动蛋白上的共同结合部位，该结合部位由亚区1和3之间的界面上的疏水口袋组成。相反，作为经典的F-肌动蛋白结合基序的CH结构域不被期望结合在这个口袋中。具体目的1研究各种疾病相关蛋白的WH2结构域与肌动蛋白的复合体的X射线结构。特定目的2涉及双丝蛋白的研究，它由两个ADF-H结构域串联组成。具体目标3重点研究α-肌动蛋白的CH对的晶体结构，PIP2对它的调控，以及它与F-肌动蛋白的相互作用。AIM 1和AIM 3中有晶体，AIM 2中也有微晶体。与BU的W.J.Lehman合作，对孪丝蛋白和α-肌动蛋白修饰的F-肌动蛋白的EM研究正在补充结晶学和生化研究。这一提议提供了一种全面的方法来研究自然界中三个最重要的肌动蛋白结合基序。通过平行研究这些基序，我们将建立它们的结构和与G-和F-肌动蛋白相互作用的共同和独特的特征，这决定了它们在细胞骨架调节中的功能。

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.