Ezrin mutants affecting dimerization and activation
Ezrin mutants affecting dimerization and activation
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DOI：
10.1021/bi0480382
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发表时间：
2005-03-15
期刊：
影响因子：
2.9
通讯作者：
Bretscher, A
中科院分区：
文献类型：
--
作者：
Chambers, DN;Bretscher, A
ERM（ezrin/radixin/moesin）蛋白在膜相关蛋白和肌动蛋白细胞骨架之间提供调节的连接。以前的工作表明，埃兹蛋白可以以休眠的单体状态存在，其中N-末端FERM结构域与C-ERMAD（羧基末端ERM缔合结构域）紧密缔合，掩蔽至少一些配体的结合位点，包括F-肌动蛋白和支架蛋白EBP 50。埃兹蛋白的激活需要解除分子内缔合，并且这被认为涉及苏氨酸567的磷酸化。因此，研究采用T567 D磷酸模拟物突变体来探索埃兹蛋白在体内活化的后果。Ezrin也以稳定的二聚体形式存在，其中两个亚基的方向未知，但可能涉及预测形成卷曲螺旋的中心α-螺旋区域。通过表征埃兹蛋白突变体，我们表明，救济的分子内协会在单体的结果在揭露配体结合，网站和显着的构象变化，T567 D突变有一个小的影响埃兹蛋白的生化激活，和预测的卷曲螺旋区不驱动二聚体的形成。这些结果提供了强有力的支持ezrin的构象激活模型，阐明了二聚体形成的基础，并揭示了一个突变体通常被认为是完全激活的不是。
ERM (ezrin/radixin/moesin) proteins provide a regulated linkage between membrane-associated proteins and the actin cytoskeleton. Previous work has shown that ezrin can exist in a dormant monomeric state in which the N-terminal FERM domain is tightly associated with the C-ERMAD (carboxyl-terminal ERM association domain), masking binding sites for at least some ligands, including F-actin and the scaffolding protein EBP50. Activation of ezrin requires relief of the intramolecular association, and this is believed to involve phosphorylation of threonine 567. Studies have therefore employed the T567D phosphomimetic mutant to explore the consequences of ezrin activation in vivo. Ezrin also exists as a stable dimer, in which the orientation of the two Subunits is unknown, but might involve the central alpha-helical region predicted to form a coiled-coil. By characterization of ezrin mutants, we show that relief of the intramolecular association in the monomer results in unmasking of ligand binding, sites and a significant conformational change, that the T567D mutation has a small effect on the biochemical activation of ezrin, and that the predicted coiled-coil region does not drive dimer formation. These results provide strong support for the conformational activation model of ezrin, elucidate the basis for dimer formation, and reveal that a mutant generally considered to be fully activated is not.