Structural Dynamics of Vinculin in Adhesion Junctions

描述(申请人提供)：细胞-基质(焦点粘连)和细胞-细胞(粘连连接)粘连复合体的形成将细胞表面的信号连接到肌动蛋白细胞骨架，这些直接的细胞迁移、细胞生长和生存以及正常发育所需的形态变化。来自焦点粘连或粘着连接的信号分别由整合素或钙粘附素跨膜受体引导，它们与肌动蛋白细胞骨架的连接需要激活细胞骨架蛋白vinculin，该蛋白与直接与这些受体相互作用的蛋白质，如talin，a-actinin，a-catenin，以及控制细胞迁移的机械部件结合。在GM071596的上一个资助周期中，我们定义了vinculin的闭合、非活性构象的结构，它由五个松散排列的螺旋束结构域组成，通过其N末端七螺旋束(VH1)结构域和五螺旋束尾(Vt)结构域的疏水作用而以闭合钳形构象持有。我们的研究还定义了伴随着vinculin激活的原子变化，其中VH1结构域经历了显著的结构变化，从远处取代了Vt结构域，并释放了vinculin的结构域以允许与其伙伴结合。最后，我们证明了talin和a-actinin是可以激活vinculin的生理触发器，它们必须首先经历结构改变才能与vinculin结合并激活vinculin，从而确定黏附信号涉及结构改变的连锁反应。虽然这些令人惊讶和令人兴奋的进展定义了控制vinculin激活的机制和结构变化，但关于激活的vinculin如何与其众多伙伴结合，或者它如何在整个细胞中指导如此多样化的过程，人们知之甚少。在这里，我们建议以正面的方式解决这些重要的问题，并通过解决激活的纽蛋白在与三个伙伴的复合体中的晶体结构来控制细胞黏附和细胞迁移，以及在新生的连接处局部产生黏附成分来进行功能研究。此外，我们还将解决Metavinculin的晶体结构，Metavinculin是纽蛋白的一种亚型，仅在肌肉组织中表达，我们还将定义Metavinculin功能所需的相互作用。总而言之，拟议的研究将解决激活的纽蛋白和Metavinculin如何指导其不同的功能，并将为针对它们的相互作用治疗涉及纽蛋白或Metavinculin的疾病奠定基础，特别是转移性癌症、缺血和肌病。与公共健康相关的细胞需要在其细胞表面形成不同的黏附复合体，以便与邻近细胞或细胞外环境形成接触，而蛋白纽蛋白在将这些黏附复合体连接到肌动蛋白细胞骨架以及指导细胞迁移机制方面发挥着至关重要的作用。这些连接的形成需要纽蛋白从封闭的、非活性的构象转变为激活的状态，R01 GM071596支持的研究定义了失活和活化的纽蛋白的结构，并揭示了其激活的机制。然而，对于激活的纽蛋白与其在细胞中的结合伙伴的相互作用基本上一无所知，我们在R01 GM071596的这一修订的竞争更新应用中的新研究将确定纽蛋白与其三个伙伴的复合体的结构和功能，这些复合体在黏附复合体、细胞迁移和黏附连接成分的局部产生中发挥重要作用。最后，我们还将定义Metavinculin的结构和功能，Metavinculin是纽蛋白的一种异构体，在肌肉组织的形成和功能中发挥重要作用。

DESCRIPTION (provided by applicant): The formation of cell-matrix (focal adhesions) and cell-cell (adherens junctions) adhesion complexes links signals at the cell surface to the actin cytoskeleton, and these direct cell migration, cell growth and survival, and the morphological changes that are needed for proper development. Signaling from focal adhesions or adherens junctions is directed by integrin or cadherin transmembrane receptors, respectively, and their links to the actin cytoskeleton require activation of the cytoskeletal protein vinculin, which binds to proteins that directly interact with these receptors, such as talin, a-actinin, and a-catenin, as well as to components of the machinery that controls cell migration. During the past funding cycle of GM071596 we defined the structure of the closed, inactive conformation of vinculin, which is compromised of five, loosely-packed helical bundle domains that are held in a closed-clamp conformation via extensive hydrophobic interactions of its N-terminal seven-helical bundle (Vh1) domain with its five-helical bundle tail (Vt) domain. Our studies also defined the atomic changes that accompany vinculin activation, where the Vh1 domain undergoes remarkable structural changes that displace Vt domain from a distance, and which release the domains of vinculin to allow binding to its partners. Finally, we demonstrated that talin and a-actinin are physiological triggers that can activate vinculin, and that they must first undergo structural alterations to bind to and activate vinculin, establishing that adhesion signaling involves a chain reaction of structural alterations. While these surprising and exciting advances defined the mechanism and structural alterations that control vinculin activation, very little is known regarding how activated vinculin binds to its numerous partners, or how it directs such diverse processes throughout the cell. Here we propose to address these important questions in a head-on fashion, together with functional studies by solving the crystal structures of activated vinculin in complex with three partners that control cell adhesion and cell migration, and the localized production of adhesion components at nascent junctions. Further, we will solve the crystal structure of metavinculin, an isoform of vinculin that is exclusively expressed in muscle tissue, and we will also define the interactions that are required for metavinculin function. Collectively, the proposed studies will resolve how activated vinculin and metavinculin direct their diverse functions, and they will lay the foundation for targeting their interactions for the treatment of diseases having vinculin or metavinculin involvement, in particular metastatic cancer, ischemia, and myopathies. PUBLIC HEALTH RELEVANCE Cells require distinct adhesion complexes at their cell surface to form contacts with their neighbors or with the extracellular environment, and the protein vinculin plays essential roles in linking these adhesion complexes to the actin cytoskeleton, and in directing the cell migration machinery. The formation of these links requires that vinculin transition from its closed, inactive conformation to its activated state, and the studies supported by R01 GM071596 defined the structure of inactive and activated vinculin, and revealed its mechanism of activation. However, essentially nothing is known regarding the interactions of activated vinculin with its binding partners in the cell, and our new studies in this revised competitive renewal application of R01 GM071596 will define the structure and function of vinculin in complex with three of its partners that play essential roles in adhesion complexes, in cell migration, and in the localized production of components of adhesion junctions. Finally, we will also define the structure and function of metavinculin, an isoform of vinculin that plays essential roles in the formation and function of muscle tissue.