Choi Hee-Jung, Gross Julia C, Pokutta Sabine, Weis William I
Departments of Structural Biology and Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5126, USA.
J Biol Chem. 2009 Nov 13;284(46):31776-88. doi: 10.1074/jbc.M109.047928. Epub 2009 Sep 16.
Plakoglobin and beta-catenin are homologous armadillo repeat proteins found in adherens junctions, where they interact with the cytoplasmic domain of classical cadherins and with alpha-catenin. Plakoglobin, but normally not beta-catenin, is also a structural constituent of desmosomes, where it binds to the cytoplasmic domains of the desmosomal cadherins, desmogleins and desmocollins. Here, we report structural, biophysical, and biochemical studies aimed at understanding the molecular basis of selective exclusion of beta-catenin and alpha-catenin from desmosomes. The crystal structure of the plakoglobin armadillo domain bound to phosphorylated E-cadherin shows virtually identical interactions to those observed between beta-catenin and E-cadherin. Trypsin sensitivity experiments indicate that the plakoglobin arm domain by itself is more flexible than that of beta-catenin. Binding of plakoglobin and beta-catenin to the intracellular regions of E-cadherin, desmoglein1, and desmocollin1 was measured by isothermal titration calorimetry. Plakoglobin and beta-catenin bind strongly and with similar thermodynamic parameters to E-cadherin. In contrast, beta-catenin binds to desmoglein-1 more weakly than does plakoglobin. beta-Catenin and plakoglobin bind with similar weak affinities to desmocollin-1. Full affinity binding of desmoglein-1 requires sequences C-terminal to the region homologous to the catenin-binding domain of classical cadherins. Although pulldown assays suggest that the presence of N- and C-terminal beta-catenin "tails" that flank the armadillo repeat region reduces the affinity for desmosomal cadherins, calorimetric measurements show no significant effects of the tails on binding to the cadherins. Using purified proteins, we show that desmosomal cadherins and alpha-catenin compete directly for binding to plakoglobin, consistent with the absence of alpha-catenin in desmosomes.
桥粒斑珠蛋白和β-连环蛋白是在黏附连接中发现的同源犰狳重复蛋白，它们在那里与经典钙黏蛋白的细胞质结构域以及α-连环蛋白相互作用。桥粒斑珠蛋白，而非通常情况下的β-连环蛋白，也是桥粒的一种结构成分，在桥粒中它与桥粒钙黏蛋白、桥粒芯糖蛋白和桥粒胶蛋白的细胞质结构域结合。在此，我们报告了旨在理解β-连环蛋白和α-连环蛋白被选择性排除在桥粒之外的分子基础的结构、生物物理和生化研究。与磷酸化E-钙黏蛋白结合的桥粒斑珠蛋白犰狳结构域的晶体结构显示出与β-连环蛋白和E-钙黏蛋白之间观察到的几乎相同的相互作用。胰蛋白酶敏感性实验表明，桥粒斑珠蛋白的臂结构域本身比β-连环蛋白的臂结构域更具柔韧性。通过等温滴定量热法测量了桥粒斑珠蛋白和β-连环蛋白与E-钙黏蛋白、桥粒芯糖蛋白1和桥粒胶蛋白1细胞内区域的结合。桥粒斑珠蛋白和β-连环蛋白与E-钙黏蛋白强烈结合且具有相似的热力学参数。相比之下，β-连环蛋白与桥粒芯糖蛋白-1的结合比桥粒斑珠蛋白弱。β-连环蛋白和桥粒斑珠蛋白与桥粒胶蛋白-1的结合亲和力相似且较弱。桥粒芯糖蛋白-1的完全亲和力结合需要与经典钙黏蛋白连环蛋白结合结构域同源区域C端的序列。尽管下拉实验表明位于犰狳重复区域两侧的N端和C端β-连环蛋白“尾巴”的存在会降低对桥粒钙黏蛋白的亲和力，但量热测量结果显示这些尾巴对与钙黏蛋白结合没有显著影响。使用纯化的蛋白质，我们表明桥粒钙黏蛋白和α-连环蛋白直接竞争与桥粒斑珠蛋白的结合，这与桥粒中不存在α-连环蛋白一致。
