The Molecular Basis of Cadherin Adhesion

描述(申请人提供)：钙粘附素是所有软组织中重要的细胞表面蛋白，调节细胞与细胞之间的黏附。它们通过指导组织特异性的细胞分离和分选，在发育和形态发生中发挥关键作用。一个主要的、尚未解决的问题涉及钙粘附素黏附和结合选择性的分子基础。人们曾多次尝试从晶体结构上阐明钙粘蛋白功能的结构基础，但尽管有大量的生化和结构研究，但功能粘附域的特性和钙粘附素结合的机制仍然存在争议。 在这项工作中，我们建议通过直接探索这些蛋白质的黏附功能的实验方法来直接解决这个问题。分子力探测器提供了实现这一目标的手段。表面力装置(SFA)和单分子动态力光谱(DFS)都提供了一套强大的、互补的工具来独特地确定钙粘附素粘附性和选择性的分子机制。通过直接测量与SFA的距离依赖的钙粘蛋白相互作用势，我们将获得关于钙粘蛋白对接排列的唯一信息，从而获得介导结合的蛋白质区域。这些信息不能通过任何其他现有技术直接获得，这将有助于识别粘连的胞外结构域片段，从而直接检验关于功能钙粘附素结构域的几个关键假说。另一方面，拟议的DFS测量将揭示单个钙粘附素键的微妙细节，这些细节可能在总体平均的SFA测量中不明显，但仍可能控制钙粘附素的识别。这些后一种测量将检验钙粘附素键特征的差异决定钙粘附素特异性的假设。 我们将通过确定钙粘附素结构变化对蛋白质间潜力和钙粘附素键强度的影响，直接测试这些力测量所建议的钙粘附素结合和识别模型。后者将在钙粘素结构和功能之间提供直接联系。总之，这些拟议的研究将以前所未有的详细程度提供对钙粘附素功能的全面分析，揭示潜在的黏附机制及其结构起源。

DESCRIPTION (provided by applicant): Cadherins are essential cell surface proteins that mediate cell-cell adhesion in all soft tissues. They play critical roles in development and morphogenesis, by directing tissue-specific cell segregation and sorting. A major, unresolved question concerns the molecular basis of cadherin adhesion and binding selectivity. There have been several attempts to elucidate the structural basis of cadherin function from crystal structures, but despite numerous biochemical and structural studies, the identities of the functional adhesive domains and the mechanisms of cadherin binding remain controversial. In this work, we propose to directly address this issue with experimental approaches that directly probe the adhesive function of these proteins. Molecular force probes provide the means to achieve this. Both the surface force apparatus (SFA) and single molecule dynamic force spectroscopy (DFS) provide a powerful, complementary set of tools for uniquely determining the molecular mechanisms of cadherin adhesion and selectivity. By directly measuring the distance-dependent cadherin interaction potentials with the SFA, we will obtain unique information regarding the cadherin docking alignments, and hence, the protein regions that mediate binding. This information, which can't be directly obtained by any other current techniques, will facilitate the identification of the adhesive ectodomain segments, and thereby directly test several key hypotheses concerning the identities of the functional cadherin domains. On the other hand, the proposed DFS measurements will reveal subtle details of single cadherin bonds that may not be evident from the population-averaged, SFA measurements, but may nevertheless control cadherin recognition. These latter measurements will test the hypothesis that differences in the characteristics of cadherin bonds determine cadherin specificity. We will directly test models for cadherin binding and recognition suggested by these force measurements, by determining the impact of cadherin structure variations on both the interprotein potentials and the cadherin bond strengths. The latter will provide a direct link between cadherin architecture and function. Together, these proposed investigations will provide a comprehensive analysis of cadherin function at an unprecedented level of detail, revealing both the underlying adhesive mechanisms and their structural origins.