Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker
Activation of Moesin, a Protein That Links Actin Cytoskeleton to the Plasma Membrane, Occurs by Phosphatidylinositol 4,5-bisphosphate (PIP2) Binding Sequentially to Two Sites and Releasing an Autoinhibitory Linker
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DOI：
10.1074/jbc.m111.304881
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发表时间：
2012-05-11
影响因子：
4.8
通讯作者：
Shaw, Stephen
中科院分区：
文献类型：
--
作者：
Ben-Aissa, Khadija;Patino-Lopez, Genaro;Shaw, Stephen
许多细胞过程依赖于ERM（埃兹蛋白，膜突蛋白和根蛋白）蛋白介导质膜和肌动蛋白细胞骨架之间的调节连接。尽管ERM蛋白的构象激活是由膜PIP 2介导的，但所述两个PIP 2结合位点的已知性质不能解释激活。为了阐明可能的机制的结构基础，我们产生了信息膜突蛋白突变和测试三个属性：膜定位的表达膜突蛋白，膜突蛋白结合PIP 2，和PIP 2诱导释放膜突蛋白自身抑制。结果首次证明，在晶体结构上含有肌醇1，4，5-三磷酸的POCKET（“POCKET”Lys-63，Lys-278残基）介导所有三种功能。此外，第二个描述的PIP 2结合位点（“PATCH”，Lys-253/Lys-254，Lys-262/Lys-263）对于所有三种功能也是必需的。在天然自抑制的ERM蛋白中，POCKET是被酸性接头掩蔽的空腔，我们将其命名为“FLAP”。“对预测影响FLAP功能的三种突变膜突蛋白结构的分析表明，FLAP是一个功能性的自抑制区。此外，协同性和化学计量分析表明，PATCH和POCKET不同时结合PIP 2。基于我们的数据和支持已发表的数据，我们提出了一个模型的渐进激活的自抑制膜突蛋白由一个单一的PIP 2分子在膜上。PIP 2与PATCH的初始瞬时结合引发FLAP的释放，这使得相同的PIP 2分子能够过渡到新暴露的口袋中，在那里它稳定结合并完成构象激活。
Many cellular processes depend on ERM (ezrin, moesin, and radixin) proteins mediating regulated linkage between plasma membrane and actin cytoskeleton. Although conformational activation of the ERM protein is mediated by the membrane PIP2, the known properties of the two described PIP2-binding sites do not explain activation. To elucidate the structural basis of possible mechanisms, we generated informative moesin mutations and tested three attributes: membrane localization of the expressed moesin, moesin binding to PIP2, and PIP2-induced release of moesin autoinhibition. The results demonstrate for the first time that the POCKET containing inositol 1,4,5-trisphosphate on crystal structure (the "POCKET" Lys-63, Lys-278 residues) mediates all three functions. Furthermore the second described PIP2-binding site (the "PATCH," Lys253/Lys-254, Lys-262/Lys-263) is also essential for all three functions. In native autoinhibited ERM proteins, the POCKET is a cavity masked by an acidic linker, which we designate the "FLAP." Analysis of three mutant moesin constructs predicted to influence FLAP function demonstrated that the FLAP is a functional autoinhibitory region. Moreover, analysis of the cooperativity and stoichiometry demonstrate that the PATCH and POCKET do not bind PIP2 simultaneously. Based on our data and supporting published data, we propose a model of progressive activation of autoinhibited moesin by a single PIP2 molecule in the membrane. Initial transient binding of PIP2 to the PATCH initiates release of the FLAP, which enables transition of the same PIP2 molecule into the newly exposed POCKET where it binds stably and completes the conformational activation.