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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Phosphorylation-induced structural changes in smooth muscle myosin regulatory light chain
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Phosphorylation-induced structural changes in smooth muscle myosin regulatory light chain

机译：磷酸化诱导的平滑肌肌球蛋白调节轻链的结构变化

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摘要

We have performed complementary time-resolved fluorescence resonance energy transfer (TR-FRET) experiments and molecular dynamics (MD) simulations to elucidate structural changes in the phosphorylation domain (PD) of smooth muscle regulatory light chain (RLC) bound to myosin. PD is absent in crystal structures, leaving uncertainty about the mechanism of regulation. Donor-acceptor pairs of probes were attached to three site-directed di-Cys mutants of RLC, each having one Cys at position 129 in the C-terminal lobe and the other at position 2,3, or 7 in the N-terminal PD. Labeled RLC was reconstituted onto myosin subfragment 1 (S1). TR-FRET resolved two simultaneously populated structural states of RLC, closed and open, in both unphosphorylated and phosphorylated biochemical states. All three FRET pairs show that phosphorylation shifts the equilibrium toward the open state, increasing its mol fraction by ～20%. MD simulations agree with experiments in remarkable detail, confirming the coexistence of two structural states, with phosphorylation shifting the system toward the more dynamic open structural state. This agreement between experiment and simulation validates the additional structural details provided by MD simulations: In the closed state, PD is bent onto the surface of the C-terminal lobe, stabilized by interdomain salt bridges. In the open state, PD is more helical and straight, resides farther from the C-terminal lobe, and is stabilized by an intradomain salt bridge. The result is a vivid atomic-resolution visualization of the first step in the molecular mechanism by which phosphorylation activates smooth muscle.
机译：我们已经进行了互补的时间分辨荧光共振能量转移（TR-FRET）实验和分子动力学（MD）模拟，以阐明与肌球蛋白结合的平滑肌调节轻链（RLC）磷酸化结构域（PD）的结构变化。晶体结构中不存在PD，因此调节机理尚不确定。供体-受体对探针连接到RLC的三个定点di-Cys突变体，每个突变体在C末端叶的129位具有一个Cys，在N末端PD的2,3或7个具有另一个Cys 。将标记的RLC重构到肌球蛋白亚片段1（S1）上。 TR-FRET在未磷酸化和磷酸化的生化状态下解析了两个同时填充的RLC结构状态，即闭合和开放。这三对FRET均显示磷酸化使平衡向开态移动，使其摩尔分数增加了约20％。 MD模拟与实验非常吻合，证实了两种结构状态的共存，磷酸化使系统向更动态的开放结构状态转移。实验与模拟之间的这种一致性验证了MD模拟提供的其他结构细节：在闭合状态下，PD弯曲到C末端波瓣的表面上，并通过域间盐桥稳定。在开放状态下，PD更螺旋且笔直，距离C末端波瓣更远，并通过域内盐桥稳定。结果是分子机制第一步的生动原子分辨率可视化，通过该过程磷酸化激活平滑肌。

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