Structure and Function of vinculin in focal adhesions

描述（由申请方提供）：白藜芦醇是一种120 kDa的胞质蛋白，在细胞迁移和胚胎发生中起关键作用。这是一个多方面的粘附分子的范例，由自抑制调节。我们最近确定了原子分辨率的120 kDa全长黏着斑蛋白的晶体结构。在此基础上，我们将在体外和细胞中进行深入的结构-功能研究。黏着斑蛋白在细胞中的功能需要与大量其他蛋白质结合，但只有在少数情况下，我们才能清楚地了解相互作用的性质。黏着斑蛋白头部和尾部结构域之间的紧密关联调节许多这些相互作用。一种可能的调节机制是头尾复合物中的空间闭塞，其在头尾解离后减轻，但也有变构调节的良好证据。在这项资助的第一个4年资助期内，我们确定了黏着斑蛋白尾部和全长分子的结构。我们开始了基于结构的尾巴在其与其主要配体-F-肌动蛋白，酸性磷脂和桩蛋白的相互作用的功能研究。我们将集中精力研究黏着斑蛋白尾部与F-肌动蛋白、尾部与磷脂和桩蛋白（与Sharon坎贝尔博士合作）之间的相互作用，以及黏着斑蛋白头部与其配体之间的相互作用。我们将建立分子的封闭和开放形式的图片，结合配体上的黏着斑蛋白结构域发生了什么构象变化，从而调节结合的机制。这项工作的主要成果将是体外配体结合和调节的结构基础。这些信息将用于设计实验，以测试这些结合和调节功能在细胞中的作用，这将与大卫克里奇利和苏珊克雷格博士合作完成。

DESCRIPTION (provided by applicant): Vinculin is a 120 kDa cytosolic protein that plays a critical role in cell migration and embryogenesis. It is the paradigm of a multifaceted adhesion molecule that is regulated by autoinhibition. We recently determined the crystal structure of the 120 kDa full-length vinculin at atomic resolution. Building upon this structure, we will pursue an in-depth structure-function study in vitro and in cells. The function of vinculin in cells requires binding to a large number of other proteins, but in only a few cases do we have a clear picture of the nature of the interaction. A tight association between vinculin head and tail domains regulates many of these interactions. One likely mechanism of regulation is steric occlusion in the head-tail complex that is relieved upon head-tail dissociation, but there is also good evidence for allosteric regulation. During the first 4-year funding period of this grant, we determined the structure of the vinculin tail and the full-length molecule. We initiated structure-based functional studies of the tail in its interactions with its major ligands - F-actin, acidic phospholipids and paxillin. We will focus our efforts on the interactions between the vinculin tail and F-actin, the tail with phospholipids and paxillin (in collaboration with Dr. Sharon Campbell), and interactions of the vinculin head with its ligands. We will build up pictures of the closed and open forms of the molecule, what conformational changes occur to vinculin domains on binding ligand, and hence the mechanisms of regulated binding. The primary output of this work will be the structural basis of ligand binding and regulation in vitro. This information will then be used to design experiments to test the role of these binding and regulatory functions in cells, which will be done in collaborations with Drs. David Critchley and Susan Craig.