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首页> 外文期刊>Journal of Molecular Biology >Membrane docking geometry and target lipid stoichiometry of membrane-bound PKCalpha C2 domain: a combined molecular dynamics and experimental study.
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Membrane docking geometry and target lipid stoichiometry of membrane-bound PKCalpha C2 domain: a combined molecular dynamics and experimental study.

机译：膜结合的PKCalpha C2域的膜对接几何和目标脂质化学计量：结合的分子动力学和实验研究。

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摘要

Protein kinase Calpha (PKCalpha) possesses a conserved C2 domain (PKCalpha C2 domain) that acts as a Ca(2+)-regulated membrane targeting element. Upon activation by Ca(2+), the PKCalpha C2 domain directs the kinase protein to the plasma membrane, thereby stimulating an array of cellular pathways. At sufficiently high Ca(2+) concentrations, binding of the C2 domain to the target lipid phosphatidylserine (PS) is sufficient to drive membrane association; however, at typical physiological Ca(2+) concentrations, binding to both PS and phosphoinositidyl-4,5-bisphosphate (PIP(2)) is required for specific plasma membrane targeting. Recent EPR studies have revealed the membrane docking geometries of the PKCalpha C2 domain docked to (i) PS alone and (ii) both PS and PIP(2) simultaneously. These two EPR docking geometries exhibit significantly different tilt angles relative to the plane of the membrane, presumably induced by the large size of the PIP(2) headgroup. The present study utilizes the two EPR docking geometries as starting points for molecular dynamics simulations that investigate atomic features of the protein-membrane interaction. The simulations yield approximately the same PIP(2)-triggered change in tilt angle observed by EPR. Moreover, the simulations predict a PIP(2):C2 stoichiometry approaching 2:1 at a high PIP(2) mole density. Direct binding measurements titrating the C2 domain with PIP(2) in lipid bilayers yield a 1:1 stoichiometry at moderate mole densities and a saturating 2:1 stoichiometry at high PIP(2) mole densities. Thus, the experiment confirms the target lipid stoichiometry predicted by EPR-guided molecular dynamics simulations. Potential biological implications of the observed docking geometries and PIP(2) stoichiometries are discussed.
机译：蛋白激酶Calpha（PKCalpha）拥有一个保守的C2域（PKCalpha C2域），其充当Ca（2+）调节的膜靶向元件。在由Ca（2+）激活后，PKCalpha C2域将激酶蛋白导向质膜，从而刺激一系列细胞途径。在足够高的Ca（2+）浓度下，C2域与目标脂质磷脂酰丝氨酸（PS）的结合足以驱动膜缔合。但是，在典型的生理Ca（2+）浓度下，特定质膜靶向要求同时结合PS和磷酸肌醇四基体4,5-双磷酸酯（PIP（2））。最近的EPR研究表明，PKCalpha C2域的膜对接几何结构分别对接（i）PS和（ii）PS和PIP（2）。这两个EPR对接的几何形状相对于膜的平面表现出明显不同的倾斜角，大概是由PIP（2）头基的大尺寸引起的。本研究利用两个EPR对接几何作为分子动力学模拟的起点，以研究蛋白质-膜相互作用的原子特征。该模拟产生了与EPR观察到的倾斜角大致相同的PIP（2）触发变化。此外，模拟预测在高PIP（2）摩尔密度下，PIP（2）：C2化学计量比接近2：1。用脂质双层中的PIP（2）滴定C2域的直接结合测量可在中等摩尔密度下产生1：1的化学计量，而在高PIP（2）摩尔密度下产生的饱和的2：1化学计量。因此，该实验证实了由EPR指导的分子动力学模拟所预测的目标脂质化学计量。讨论了对接的几何形状和PIP（2）的化学计量学的潜在生物学意义。

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