Espinoza-Fonseca L Michel, Alamo Lorenzo, Pinto Antonio, Thomas David D, Padrón Raúl
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Mol Biosyst. 2015 Aug;11(8):2167-79. doi: 10.1039/c5mb00162e.
Phosphorylation of myosin regulatory light chain (RLC) N-terminal extension (NTE) activates myosin in thick filaments. RLC phosphorylation plays a primary regulatory role in smooth muscles and a secondary (modulatory) role in striated muscles, which is regulated by Ca(2+)via TnC/TM on the thin filament. Tarantula striated muscle exhibits both regulatory systems: one switches on/off contraction through thin filament regulation, and another through PKC constitutively Ser35 phosphorylated swaying free heads in the thick filaments that produces quick force on twitches regulated from 0 to 50% and modulation is accomplished recruiting additional force-potentiating free and blocked heads via Ca(2+)4-CaM-MLCK Ser45 phosphorylation. We have used microsecond molecular dynamics (MD) simulations of tarantula RLC NTE to understand the structural basis for phosphorylation-based regulation in tarantula thick filament activation. Trajectory analysis revealed that an inter-domain salt bridge network (R39/E58,E61) facilitates the formation of a stable helix-coil-helix (HCH) motif formed by helices P and A in the unphosphorylated NTE of both myosin heads. Phosphorylation of the blocked head on Ser45 does not induce any substantial structural changes. However, phosphorylation of the free head on Ser35 disrupts this salt bridge network and induces a partial extension of helix P along RLC helix A. While not directly participating in the HCH folding, phosphorylation of Ser35 unlocks a compact structure and allows the NTE to spontaneously undergo coil-helix transitions. The modest structural change induced by the subsequent Ser45 diphosphorylation monophosphorylated Ser35 free head facilitates full helix P extension into a single structurally stable α-helix through a network of intra-domain salt bridges (pS35/R38,R39,R42). We conclude that tarantula thick filament activation is controlled by sequential Ser35-Ser45 phosphorylation via a conserved disorder-to-order transition.
肌球蛋白调节轻链（RLC）N端延伸区（NTE）的磷酸化激活了粗肌丝中的肌球蛋白。RLC磷酸化在平滑肌中起主要调节作用，在横纹肌中起次要（调节）作用，它通过细肌丝上的肌钙蛋白C/肌钙蛋白M（TnC/TM）由Ca(2+)调节。狼蛛横纹肌表现出两种调节系统：一种通过细肌丝调节开启/关闭收缩，另一种通过蛋白激酶C（PKC）组成性地使粗肌丝中摆动的自由头部的Ser35磷酸化，从而在0%至50%调节的抽搐中产生快速力，并且通过Ca(2+)4-钙调蛋白-肌球蛋白轻链激酶（CaM-MLCK）Ser45磷酸化募集额外的力增强自由头部和受阻头部来完成调节。我们使用狼蛛RLC NTE的微秒级分子动力学（MD）模拟来了解狼蛛粗肌丝激活中基于磷酸化调节的结构基础。轨迹分析表明，一个域间盐桥网络（R39/E58、E61）促进了由两个肌球蛋白头部未磷酸化NTE中的螺旋P和A形成的稳定螺旋-螺旋-螺旋（HCH）基序的形成。受阻头部Ser45的磷酸化不会引起任何实质性的结构变化。然而，自由头部Ser35的磷酸化破坏了这个盐桥网络，并诱导螺旋P沿着RLC螺旋A部分延伸。虽然Ser35的磷酸化不直接参与HCH折叠，但它解开了一个紧凑结构，使NTE能够自发地经历螺旋-螺旋转变。随后Ser45双磷酸化单磷酸化的Ser35自由头部引起的适度结构变化通过域内盐桥网络（pS35/R38、R39、R42）促进螺旋P完全延伸成单个结构稳定的α螺旋。我们得出结论，狼蛛粗肌丝激活是通过保守的无序到有序转变由Ser35-Ser45顺序磷酸化控制的。
