STRUCTURAL DYNAMICS OF VINCULIN IN ADHESION JUNCTIONS

描述(申请人提供)：纽蛋白是一种高度保守的蛋白质，在形成焦点(细胞-基质)和粘连(细胞-细胞)连接后，在指导肌动蛋白细胞骨架的正确组装方面发挥重要作用。纽蛋白通过与关键的支架蛋白结合来协调细胞骨架的变化，包括在局部粘连中与整合素受体结合的Talin，以及在粘连连接中与钙粘附素结合的α-肌动蛋白和α-连环蛋白。以往的生化研究表明，纽蛋白的激活在很大程度上是通过切断纽蛋白的头(Vh)和尾(Vt)结构域的分子内关联来完成的。这被认为是通过酸性磷脂与Vt结构域结合而发生的，这将展开Vt，留下刚性的VH结构域与其结合伙伴自由结合。为了在我们的初步研究中建立支配纽蛋白激活的基本原理，我们解决了不活跃的人VH：VT复合体的晶体结构，以及与VH结合的Talin纽蛋白结合位点(Talin VBS3)的晶体结构。VH：VT复合体的晶体结构表明，在其静止状态下，通过VH和Vt中存在的两亲性α-螺旋束的相互作用，纽蛋白以封闭的构象持有。更重要的是，Talin结合的VH的晶体结构显示了一种新的和意想不到的控制水平，由此Talin与VH的结合触发了VH的显著变化，创建了一种全新的螺旋束结构，取消了Vt的结合部位。这些发现支持这样一个模型：1.Talin在纽蛋白的激活中发挥直接和积极的作用；以及2.VH是一个动态的、灵活的结构域，其功能是一个分子开关，控制粘附素与焦点黏附连接中的细胞骨架重排。为了支持这一模型，特定目标#1的实验将定义天然全长纽蛋白的晶体结构，以及VH与他林中存在的纽蛋白结合部位的结合。Talin和其他vinculin结合蛋白的纽蛋白结合域高度相关，并被预测以相互排斥的方式结合VH。因此，在特定的目标#2中，我们将测试VH结构域作为分子开关的作用，通过解决VH结构域和纽蛋白与其他纽蛋白结合蛋白的复合体的结构，引导焦点粘连而不是粘连连接。最后，全长人纽蛋白的成功结晶现在使我们能够确定与纽蛋白相互作用的其他关键结合伙伴的晶体结构。因此，在特定的目标#3中，我们将确定纽蛋白与蛋白质结合的晶体结构，这些蛋白质结合到连接VH和Vt的富含Pro的区域。人们希望，拟议的实验将准确定位正常细胞中正确组装细胞骨架所需的相互作用，以及这些相互作用在癌症或其他疾病状态下可能会如何被破坏。

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.