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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Cadherin interaction probed by atomic force microscopy
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Cadherin interaction probed by atomic force microscopy

机译：钙离子相互作用的原子力显微镜探测

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摘要

Single molecule atomic force microscopy was used to characterize structure, binding strength (unbinding force). and binding kinetics of a classical cadherin, vascular endothelial (VE)-cadherin, secreted by transfected Chinese hamster ovary cells as cis-dimerized full- length external domain fused to Fc-portion of human IgG. In physiological buffer, the external domain of VE-cadherin dimers is a ≈20-nm-long rod-shaped molecule that collapses and dissociates into monomers (V-shaped structures) in the absence of Ca~2+. Trans-interaction of dimers is a low-affinity reaction (KD = 10~-3- 10~-5 M, k_off = 1.8 s~-1,. k_on = 10~3-10~5 M-~1.s~-1) with relatively low unbinding force (35-55 pN at retrace velocities of 200-4,000 nm.s~-1). Higher order unbinding forces, that increase with inter- attion time, indicate association of cadherins into complexes with cumulative binding strength. These observations favor a model by which the inherently weak unit binding strength and affinity of cadherin trans-interaction requires clustering and cytoskeletal im- mobilization for amplification. Binding is regulated by low-affinity Ca~2+ binding sites (KD = 1.15 mM) with high cooperativity (Hill coefficient of 5.04). Local changes of free extracellular Ca2+ in the narrow intercellular space may be of physiological importance to facilitate rapid remodeling of intercellular adhesion and commu- nication.
机译：单分子原子力显微镜用于表征结构，结合强度（解结合力）。转染的中国仓鼠卵巢细胞分泌的经典钙粘着蛋白，血管内皮（VE）-钙粘着蛋白的结合动力学和结合动力学，是与人IgG Fc部分融合的顺式二聚全长外部结构域。在生理缓冲液中，VE-钙粘着蛋白二聚体的外部结构域是一个≈20nm长的棒状分子，在没有Ca〜2 +的情况下会塌陷并解离为单体（V形结构）。二聚体的反式相互作用是一种低亲和力反应（KD = 10〜-3- 10〜-5 M，k_off = 1.8 s〜-1 ,. k_on = 10〜3-10〜5 M-〜1.s〜 -1）具有相对较低的解粘力（在200-4,000 nm.s〜-1的回撤速度下为35-55 pN）。随着相互作用时间的增加，更高阶的解键力表明钙粘蛋白与具有累积结合强度的复合物缔合。这些观察结果支持一种模型，通过该模型固有的较弱的单元结合强度和钙粘蛋白反式相互作用的亲和力需要聚类和细胞骨架固定化来进行扩增。结合受到低亲和力的Ca〜2 +结合位点（KD = 1.15 mM）和高协同作用（希尔系数5.04）的调节。狭窄的细胞间隙中游离细胞外Ca2 +的局部变化可能对促进细胞间粘附和通讯的快速重塑具有重要的生理意义。

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