Salt bridges gate α-catenin activation at intercellular junctions.
Salt bridges gate α-catenin activation at intercellular junctions.
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DOI：
10.1091/mbc.e17-03-0168
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发表时间：
2018-01-15
影响因子：
3.3
通讯作者：
Leckband D
中科院分区：
文献类型：
--
作者：
Barrick S;Li J;Kong X;Ray A;Tajkhorshid E;Leckband D
分子动力学模拟、平衡结合测量和荧光成像揭示了关键的盐桥对细胞间粘附中α-连环蛋白机械激活的影响。模拟揭示了α-catenin在实验荧光和平衡结合数据下可能的构象变化。钙粘蛋白复合物在连接处传导力波动，激活强化细胞间接触的信号。α-连环蛋白是钙粘蛋白复合物中的一种力传感器，在低张力下被自动抑制。增加的力触发构象变化，暴露出肌动蛋白结合蛋白的隐位点。本研究验证了力传感核心内盐桥调节α-连环蛋白激活的预测。基于荧光共振能量转移（FRET）的α-catenin构象传感器研究表明，盐桥突变R551A和D503N均能增强活细胞中α-catenin的激活，但R551A的影响更大。在再退火细胞-细胞连接处的动态力载荷下，R551A突变体比野生型α-连环蛋白结合更多的血管蛋白。体外结合测量量化了R551A突变对活性α-连环蛋白构象和自抑制α-连环蛋白构象之间自由能差的影响。对核心力传感区进行了2 μs恒力定向分子动力学模拟，揭示了盐桥突变体如何改变α-catenin的构象，并鉴定出一种新型的承重盐桥。这些结果揭示了决定这种关键纳米机器的力传导机制和力灵敏度的关键结构特征。
Molecular dynamics simulations, equilibrium binding measurements, and fluorescence imaging reveal the influence of a key salt bridge in the mechanical activation of α-catenin at intercellular adhesions. Simulations reveal possible α-catenin conformational changes underlying experimental fluorescence and equilibrium binding data. Cadherin complexes transduce force fluctuations at junctions to activate signals that reinforce stressed intercellular contacts. α-Catenin is an identified force transducer within cadherin complexes that is autoinhibited under low tension. Increased force triggers a conformational change that exposes a cryptic site for the actin-binding protein vinculin. This study tested predictions that salt bridges within the force-sensing core modulate α-catenin activation. Studies with a fluorescence resonance energy transfer (FRET)-based α-catenin conformation sensor demonstrated that each of the salt-bridge mutations R551A and D503N enhances α-catenin activation in live cells, but R551A has a greater impact. Under dynamic force loading at reannealing cell–cell junctions, the R551A mutant bound more vinculin than wild-type α-catenin. In vitro binding measurements quantified the impact of the R551A mutation on the free-energy difference between the active and autoinhibited α-catenin conformers. A 2-μs constant-force, steered molecular dynamics simulation of the core force-sensing region suggested how the salt-bridge mutants alter the α-catenin conformation, and identified a novel load-bearing salt bridge. These results reveal key structural features that determine the force-transduction mechanism and the force sensitivity of this crucial nanomachine.