Structural Dynamics of Vinculin in Adhesion Junctions

描述（由申请人提供）：细胞-基质（局灶黏附）和细胞-细胞（黏附连接）黏附复合物的形成将细胞表面的信号与肌动蛋白细胞骨架联系起来，这些直接细胞迁移，细胞生长和存活，以及正常发育所需的形态变化。来自局点黏附或黏附连接的信号分别由整合素或钙粘蛋白跨膜受体引导，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，后者结合直接与这些受体相互作用的蛋白质，如talin， a-actin和a-catenin，以及控制细胞迁移的机制组件。在GM071596的过去的资助周期中，我们定义了血管蛋白的封闭，无活性构象的结构，它是由五个松散排列的螺旋束结构域组成的，这些结构域通过其n端七螺旋束（Vh1）结构域与五螺旋束尾（Vt）结构域的广泛疏水相互作用而保持在封闭钳形结构中。我们的研究还定义了伴随血毒蛋白激活的原子变化，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，并释放了血毒蛋白的结构域，使其能够与其伴侣结合。最后，我们证明了talin和a-actin是激活血管蛋白的生理触发器，并且它们必须首先经历结构改变才能与血管蛋白结合并激活血管蛋白，从而建立了粘附信号涉及结构改变的连锁反应。虽然这些令人惊讶和激动人心的进展定义了控制血管蛋白激活的机制和结构改变，但对于活化的血管蛋白如何与众多伴侣结合，或如何在整个细胞中指导如此多样化的过程，我们知之甚少。在这里，我们建议以正面的方式解决这些重要的问题，以及通过解决激活的血管蛋白与控制细胞粘附和细胞迁移的三个伙伴复合物的晶体结构的功能研究，以及新生连接处粘附成分的局部生产。此外，我们将解决metavinculin的晶体结构，这是一种在肌肉组织中表达的同种异构体，我们还将定义metavinculin功能所需的相互作用。总的来说，拟议的研究将解决活化的血管蛋白和元血管蛋白如何指导它们的不同功能，并将为靶向它们的相互作用为治疗有血管蛋白或元血管蛋白参与的疾病，特别是转移性癌症、缺血和肌病奠定基础。细胞需要在其细胞表面形成不同的粘附复合物，以与邻近细胞或细胞外环境形成接触，而在将这些粘附复合物与肌动蛋白细胞骨架连接并指导细胞迁移机制方面，蛋白质毒蛋白起着至关重要的作用。这些链接的形成需要血管蛋白从封闭、失活的构象转变为激活状态，R01 GM071596支持的研究明确了失活和活化的血管蛋白结构，揭示了其活化机制。然而，关于活化的血管蛋白与细胞中结合伙伴的相互作用，我们基本上一无所知，我们在R01 GM071596的修订竞争性更新应用中的新研究将定义血管蛋白及其三种伙伴的复合物的结构和功能，这些伙伴在粘附复合物、细胞迁移和粘附连接成分的局部生产中发挥重要作用。最后，我们还将定义元血管蛋白的结构和功能，这是一种在肌肉组织的形成和功能中起重要作用的血管蛋白的异构体。

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.