Structural Dynamics of Vinculin in Adhesion Junctions

描述（由申请人提供）：细胞-基质（粘着斑）和细胞-细胞（粘附连接）粘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，这些信号指导细胞迁移、细胞生长和存活以及正常发育所需的形态学变化。来自粘着斑或粘着连接的信号传导分别由整联蛋白或钙粘蛋白跨膜受体指导，并且它们与肌动蛋白细胞骨架的连接需要细胞骨架蛋白粘着斑蛋白的活化，粘着斑蛋白结合与这些受体直接相互作用的蛋白质，如talin、α-辅肌动蛋白和α-连环蛋白，以及控制细胞迁移的机制的组分。在GM 071596的过去的资助周期中，我们定义了黏着斑蛋白的封闭、无活性构象的结构，黏着斑蛋白由五个松散堆积的螺旋束结构域组成，这些螺旋束结构域通过其N端七螺旋束（Vh 1）结构域与其五螺旋束尾（Vt）结构域的广泛疏水相互作用而保持在闭合钳构象中。我们的研究还定义了伴随黏着斑蛋白激活的原子变化，其中Vh 1结构域经历了显着的结构变化，从远处取代Vt结构域，并释放黏着斑蛋白的结构域以允许与其伴侣结合。最后，我们证明了talin和α-辅肌动蛋白是可以激活黏着斑蛋白的生理触发因子，并且它们必须首先经历结构改变才能结合并激活黏着斑蛋白，从而确定粘附信号传导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黏着斑蛋白激活的机制和结构改变，但关于激活的黏着斑蛋白如何与其众多伴侣结合，或者它如何在整个细胞中指导这些不同的过程，我们知之甚少。在这里，我们建议解决这些重要的问题，在一个正面的方式，连同功能研究，通过解决激活黏着斑蛋白的晶体结构与三个合作伙伴，控制细胞粘附和细胞迁移，并在新生路口的粘附成分的本地化生产复杂。此外，我们将解决metavinculin的晶体结构，这是一种专门在肌肉组织中表达的vinculin亚型，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黏着斑蛋白和后黏着斑蛋白如何指导其不同的功能，并且它们将为靶向它们的相互作用以治疗具有黏着斑蛋白或后黏着斑蛋白参与的疾病，特别是转移性癌症、缺血和肌病奠定基础。公共卫生相关性细胞需要在其细胞表面形成不同的粘附复合物，以与其邻居或细胞外环境形成接触，并且黏着斑蛋白在将这些粘附复合物连接到肌动蛋白细胞骨架以及指导细胞迁移机制中起重要作用。这些连接的形成需要黏着斑蛋白从其封闭的非活性构象转变为其活化状态，R 01 GM 071596支持的研究定义了非活性和活化黏着斑蛋白的结构，并揭示了其活化机制。然而，基本上没有什么是已知的关于活化黏着斑蛋白与其结合伙伴在细胞中的相互作用，我们在这个修订的竞争性更新申请R 01 GM 071596的新研究将定义黏着斑蛋白的结构和功能，在复合物中发挥重要作用的三个合作伙伴，在粘附复合物，细胞迁移，并在局部生产的组件的粘附连接。最后，我们还将定义后黏着斑蛋白的结构和功能，黏着斑蛋白的一种亚型，在肌肉组织的形成和功能中起着至关重要的作用。

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.