Alattia J R, Ames J B, Porumb T, Tong K I, Heng Y M, Ottensmeyer P, Kay C M, Ikura M
Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Canada.
FEBS Lett. 1997 Nov 17;417(3):405-8. doi: 10.1016/s0014-5793(97)01333-1.
We report the Ca2+ binding characteristics of recombinant Ecad12, a construct spanning the first two repeats of epithelial cadherin, and demonstrate the links between Ca2+ binding and dimer formation. Sedimentation equilibrium and dynamic light scattering experiments show that weak dimerization of Ecad12 occurs in the presence of 10 mM Ca2+ (KdP = 0.17 mM), while no appreciable dimer formation was detected in the absence of Ca2+. Ca2+-induced dimerization was also observed in electron microscopy images of Ecad12. We conclude from Ca2+ titration experiments monitored by tryptophan fluorescence and flow dialysis that dimerization does not affect the equilibrium binding constant for Ca2+. However, the value of the Hill coefficient for Ca2+ binding increases from 1.5 to 2.4 as the protein concentration increases, showing that dimer formation largely contributes to the cooperativity in Ca2+ binding. Based on these observations and previous crystallographic studies, we propose that calcium acts more likely as a geometrical aligner ensuring the proper assembly of cadherin molecules, rather than a simple adhesive.
我们报告了重组Ecad12的Ca2+结合特性，该构建体跨越上皮钙黏蛋白的前两个重复序列，并证明了Ca2+结合与二聚体形成之间的联系。沉降平衡和动态光散射实验表明，在10 mM Ca2+存在下，Ecad12发生弱二聚化（KdP = 0.17 mM），而在没有Ca2+的情况下未检测到明显的二聚体形成。在Ecad12的电子显微镜图像中也观察到Ca2+诱导的二聚化。我们从色氨酸荧光监测的Ca2+滴定实验和流动透析得出结论，二聚化不会影响Ca2+的平衡结合常数。然而，随着蛋白质浓度的增加，Ca2+结合的希尔系数值从1.5增加到2.4，表明二聚体形成在很大程度上有助于Ca2+结合的协同性。基于这些观察结果和先前的晶体学研究，我们提出钙更可能作为一种几何排列剂，确保钙黏蛋白分子的正确组装，而不是一种简单的黏附剂。
