Allosteric Activation of Vinculin by Talin [data]

The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single molecule magnetic tweezer experiments, Molecular Dynamics simulations, actin bundling assays, and adhesion assembly experiments in live cells, we here discover a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding which reinforces the binding to talin at a rate of 0.03 s−1. This allosteric transition can compete with force-induced dissociation of vinculin from talin only at 7-10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The ‘allosteric vinculin mutant’ is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere.

踝蛋白-黏着斑蛋白轴（talin-vinculin axis）是细胞黏着斑（cellular focal adhesions）中关键的机械感应组分。目前，踝蛋白与黏着斑蛋白如何响应外力并相互调控的机制仍未阐明。本研究结合单分子磁镊实验、分子动力学模拟（Molecular Dynamics simulations）、肌动蛋白束化测定以及活细胞黏着斑组装实验，发现黏着斑蛋白内部存在双向变构网络，可作为调控踝蛋白-黏着斑蛋白相互作用的关键因子。我们直接观测到黏着斑蛋白结合踝蛋白后的构象成熟过程，该过程以0.03 s⁻¹的速率增强其与踝蛋白的结合亲和力。该变构转变仅在7~10皮牛（pN）的外力条件下，才能与黏着斑蛋白从踝蛋白上的力诱导解离过程相抗衡。通过突变模拟该变构激活过程，可获得不依赖外力即可束化肌动蛋白并定位于黏着斑的黏着斑蛋白突变体。由此可见，该变构开关将踝蛋白-黏着斑蛋白相互作用与黏着斑组装限定在中等外力水平范围内。该‘变构激活黏着斑蛋白突变体’是一种极具价值的分子工具，可用于进一步解析黏着斑及其他区域的机械与生化信号传导通路。