STRUCTURAL DYNAMICS OF VINCULIN IN ADHESION JUNCTIONS

描述（由申请人提供）：白蛋白是一种高度保守的蛋白质，在形成局灶（细胞-基质）和粘附（细胞-细胞）连接后指导肌动蛋白细胞骨架的正确组装中发挥重要作用。曲马多蛋白通过结合关键支架蛋白来协调细胞骨架的变化，所述支架蛋白包括talin，其在粘着斑中结合整联蛋白受体，以及结合α-辅肌动蛋白和α-连环蛋白，其在粘着连接中结合钙粘蛋白。以往的生物化学研究表明，黏着斑蛋白的激活主要是通过切断黏着斑蛋白的头部（Vh）和尾部（Vt）结构域的分子内缔合来完成的。这被认为是通过酸性磷脂与Vt结构域的结合而发生的，这将展开Vt，留下刚性Vh结构域自由地与其结合伴侣缔合。在我们的初步研究中，为了建立支配粘着斑蛋白激活的基本原理，我们解决了无活性的人Vh：Vt复合物和与Vh结合的塔林粘着斑蛋白结合位点（塔林VBS3）的晶体结构。Vh：Vt复合物的晶体结构表明，在其静止状态下，黏着斑蛋白通过存在于Vh和Vt中的两亲性α-螺旋束的相互作用保持在闭合构象中。更重要的是，塔林结合的VH的晶体结构已经证明了一种新的和意想不到的控制水平，从而塔林与VH的结合触发了VH的显著改变，产生了一种全新的螺旋束结构，消除了Vt的结合位点。这些发现支持了一个模型，其中：1。Talin在黏着斑蛋白激活中起直接和积极的作用;和2. Vh是一个动态的、灵活的结构域，作为一个分子开关，控制粘附细胞与粘着斑连接中的细胞骨架重排。为了支持该模型，特定目标#1中的实验将定义天然全长粘着斑蛋白和与talin中存在的粘着斑蛋白结合位点结合的Vh的晶体结构。黏着斑蛋白结合域的塔林和其他黏着斑蛋白结合蛋白是高度相关的，并预测以相互排斥的方式结合VH。因此，在具体目标#2中，我们将通过解决Vh结构域和粘着斑蛋白与其他粘着斑蛋白结合蛋白复合的结构，测试Vh结构域作为分子开关的模型，该分子开关引导粘着斑与粘着斑连接。最后，全长人类黏着斑蛋白的成功结晶现在使我们能够确定与黏着斑蛋白相互作用的其他关键结合伙伴的晶体结构。因此，在具体目标#3中，我们将确定黏着斑蛋白与蛋白质结合的晶体结构，所述蛋白质与连接Vh和Vt的富含脯氨酸的结构域结合。希望拟议的实验将查明正常细胞中适当的细胞骨架组装所需的相互作用，以及这些相互作用在癌症或其他疾病状态下如何被破坏。

DESCRIPTION (provided by applicant): Vinculin is a highly conserved protein that plays essential roles in directing the proper assembly of the actin cytoskeleton following the formation of focal (cell-matrix) and adherens (cell-cell) junctions. Vinculin orchestrates changes in the cytoskeleton by binding to key scaffold proteins, including talin, which binds to integrin receptors in focal adhesions, and to a-actinin and a-catenin, which bind to cadherins in adherens junctions. Previous biochemical studies have suggested that activation of vinculin is largely accomplished through severing the intramolecular association of vinculin's head (Vh) and tail (Vt) domains. This was thought to occur through the binding of acidic phospholipids to the Vt domain, which would unfurl Vt, leaving a rigid Vh domain free to associate with its binding partners. To establish the underlying principles governing vinculin activation in our Preliminary Studies we solved the crystal structures of the inactive human Vh:Vt complex, and of a talin vinculin binding site (talin VBS3) bound to Vh. The crystal structure of the Vh:Vt complex reveals that, in its resting state, vinculin is held in a closed conformation through interactions of amphipathic a-helical bundles present in Vh and Vt. More importantly, the crystal structure of talin-bound Vh has demonstrated a novel and unexpected level of control, whereby binding of talin to Vh triggers striking alterations in Vh, creating an entirely new helical bundle structure that abolishes the binding site for Vt. These findings support a model in which: 1. Talin plays a direct and active role in vinculin activation; and 2. Vh is a dynamic, flexible, domain that functions as a molecular switch which controls cytoskeletal rearrangements in adherens versus focal adhesion junctions. In support of this model, experiments in Specific Aim #1 will define the crystal structure of native, full-length vinculin and of Vh bound to vinculin binding sites present in talin. The vinculin binding domains of talin and of other vinculin-binding proteins are highly related and are predicted to bind Vh in a mutually exclusive manner. Therefore in Specific Aim #2 we will test the model that the Vh domain functions as a molecular switch that directs focal adhesions versus adherens junctions, by solving the structure of the Vh domain, and of vinculin, in complex with other vinculin binding proteins. Finally, the successful crystallization of full-length human vinculin now allows us to also determine the crystal structures of other key binding partners that interact with vinculin. Thus in Specific Aim #3 we will determine the crystal structures of vinculin bound to proteins that bind to its Proline-rich domain that links Vh and Vt. It is hoped that the proposed experiments will pinpoint the interactions that are required for proper cytoskeletal assembly in normal cells, and how these interactions may be disrupted in cancer or other disease states.