Baumgartner W, Hinterdorfer P, Ness W, Raab A, Vestweber D, Schindler H, Drenckhahn D
Institute of Anatomy, University of Würzburg, Koellikerstrasse 6, D-97070 Würzburg, Germany.
Proc Natl Acad Sci U S A. 2000 Apr 11;97(8):4005-10. doi: 10.1073/pnas.070052697.
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 approximately 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 (K(D) = 10(-3)-10(-5) M, k(off) = 1.8 s(-1), k(on) = 10(3)-10(5) M(-1) x s(-1)) with relatively low unbinding force (35-55 pN at retrace velocities of 200-4,000 nm x s(-1)). Higher order unbinding forces, that increase with interaction 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 immobilization for amplification. Binding is regulated by low-affinity Ca(2+) binding sites (K(D) = 1.15 mM) with high cooperativity (Hill coefficient of 5.04). Local changes of free extracellular Ca(2+) in the narrow intercellular space may be of physiological importance to facilitate rapid remodeling of intercellular adhesion and communication.
单分子原子力显微镜被用于表征由转染的中国仓鼠卵巢细胞分泌的一种经典钙黏蛋白——血管内皮（VE）-钙黏蛋白的结构、结合强度（解离力）和结合动力学。该钙黏蛋白作为与人类IgG的Fc部分融合的顺式二聚化全长胞外结构域。在生理缓冲液中，VE-钙黏蛋白二聚体的胞外结构域是一个约20纳米长的杆状分子，在没有Ca(2+)的情况下会折叠并解离成单体（V形结构）。二聚体的反式相互作用是一种低亲和力反应（K(D)=10(-3)-10(-5) M，k(off)=1.8 s(-1)，k(on)=10(3)-10(5) M(-1)×s(-1)），解离力相对较低（在200-4000纳米×s(-1)的回程速度下为35-55 pN）。随着相互作用时间增加的高阶解离力表明钙黏蛋白缔合成具有累积结合强度的复合物。这些观察结果支持一种模型，即钙黏蛋白反式相互作用固有的弱单位结合强度和亲和力需要聚集和细胞骨架固定来放大。结合由具有高协同性（希尔系数为5.04）的低亲和力Ca(2+)结合位点（K(D)=1.15 mM）调节。狭窄细胞间空间中游离细胞外Ca(2+)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生理重要性。
