A Dynamic Model of Membrane-Bound Phospholipase Cβ2 Activation by Gβγ Subunits
A Dynamic Model of Membrane-Bound Phospholipase Cβ2 Activation by Gβγ Subunits
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DOI：
10.1124/mol.111.073403
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发表时间：
2011-09-01
影响因子：
3.6
通讯作者：
Weinstein, Harel
中科院分区：
文献类型：
--
作者：
Han, Daniel S.;Golebiewska, Urszula;Weinstein, Harel
磷脂酶 C (PLC) beta 2 是经过充分研究的酶家族成员，可催化膜脂磷脂酰肌醇 4,5-二磷酸 (PIP 2) 水解为第二信使，可被异源三聚体 G 蛋白的 G beta gamma 亚基激活，其激活方式取决于相关磷脂膜表面的存在和组成。 PLC beta 2 的 N 端 pleckstrin 同源 (PH) 结构域介导对 G beta gamma 的响应和膜结合，但这些相互作用如何耦合以产生活化的催化核心仍然未知。在这里，我们提出了一种基于截短的 PLC beta 2 分子模型的机制，其激活形式与 G beta gamma 复合，以及催化失活/膜结合形式，通过应用蛋白质-蛋白质对接算法和粗粒度分子动力学模拟获得。这些模型经过实验探索，并通过分子生物学和荧光测定相结合的结果证实了推论。分子模型的动态模拟及其与各种脂质双层的相互作用的结果确定了 PLC beta 2-PH 结构域对 G beta gamma 和脂质膜特异性的决定因素，并提出了先前报道的 G beta gamma 激活对相关膜组成的依赖性的机制。总之，这些发现解释了不同激活剂对 PH 和催化核心结构域相对于脂质膜的方向的影响。
Phospholipase C (PLC) beta 2, a well studied member of the family of enzymes that catalyze the hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP 2) into secondary messengers, can be activated by the G beta gamma subunits of heterotrimeric G-proteins in a manner that depends on the presence and composition of the associated phospholipid membrane surface. The N-terminal pleckstrin homology (PH) domain of PLC beta 2 mediates both the response to G beta gamma and membrane binding, but how these interactions are coupled to yield an activated catalytic core remains unknown. Here we propose a mechanism based on molecular models of truncated PLC beta 2 in its activated form complexed with G beta gamma and in the catalytically inactive/membrane-bound form, obtained with the application of protein-protein docking algorithms and coarse-grained molecular dynamics simulations. These models were probed experimentally, and the inferences were confirmed by results from a combination of molecular biology and fluorescence assays. Results from the dynamic simulations of the molecular models and their interactions with various lipid bilayers identify the determinants of PLC beta 2-PH domain specificity for G beta gamma and lipid membranes and suggest a mechanism for the previously reported dependence of G beta gamma activation on the associated membrane composition. Together, these findings explain the roles of the different activators in terms of their effect on the orientations of the PH and catalytic core domains relative to the lipid membranes.