Structure and Function of vinculin in focal adhesions

描述（由申请人提供）：Vinculin是一种120 kDa的细胞质蛋白，在细胞迁移和胚胎发生中起关键作用。它是一个由自抑制调节的多面粘附分子的范例。我们最近在原子分辨率上确定了全长120 kDa的血管蛋白的晶体结构。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。血管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，我们对这种相互作用的性质有了清晰的了解。血管蛋白头部和尾部结构域之间的紧密联系调节了许多这些相互作用。一种可能的调节机制是头尾复合体的位阻闭塞，这种闭塞在头尾分离时得到缓解，但也有很好的证据表明它具有变构调节作用。在这项资助的前4年资助期内，我们确定了vinculin尾部的结构和全长分子。我们开始了基于结构的尾部功能研究，研究其与主要配体- f -肌动蛋白，酸性磷脂和帕西林的相互作用。我们将重点研究血管蛋白尾部与f -肌动蛋白之间的相互作用，尾部与磷脂和帕西林之间的相互作用（与Sharon Campbell博士合作），以及血管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，结合配体上的血管蛋白结构域发生的构象变化，以及调节结合的机制。这项工作的主要成果将是配体结合和体外调控的结构基础。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，这将与博士合作。大卫·克里奇利和苏珊·克雷格。

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.