Centro de Biología Estructural, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020A, Venezuela.
J Mol Biol. 2011 Nov 18;414(1):44-61. doi: 10.1016/j.jmb.2011.09.017. Epub 2011 Sep 17.
Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). To elucidate the structural mechanism of activation, we have studied RLC phosphorylation in tarantula thick filaments, whose high-resolution structure is known. In the relaxed state, tarantula RLCs are ~50% non-phosphorylated and 50% mono-phosphorylated, while on activation, mono-phosphorylation increases, and some RLCs become bi-phosphorylated. Mass spectrometry shows that relaxed-state mono-phosphorylation occurs on Ser35, while Ca(2+)-activated phosphorylation is on Ser45, both located near the RLC N-terminus. The sequences around these serines suggest that they are the targets for protein kinase C and myosin light chain kinase (MLCK), respectively. The atomic model of the tarantula filament shows that the two myosin heads ("free" and "blocked") are in different environments, with only the free head serines readily accessible to kinases. Thus, protein kinase C Ser35 mono-phosphorylation in relaxed filaments would occur only on the free heads. Structural considerations suggest that these heads are less strongly bound to the filament backbone and may oscillate occasionally between attached and detached states ("swaying" heads). These heads would be available for immediate actin interaction upon Ca(2)(+) activation of the thin filaments. Once MLCK becomes activated, it phosphorylates free heads on Ser45. These heads become fully mobile, exposing blocked head Ser45 to MLCK. This would release the blocked heads, allowing their interaction with actin. On this model, twitch force would be produced by rapid interaction of swaying free heads with activated thin filaments, while prolonged exposure to Ca(2+) on tetanus would recruit new MLCK-activated heads, resulting in force potentiation.
许多肌肉的肌球蛋白丝通过其调节轻链（RLC）的磷酸化而被激活。为了阐明激活的结构机制，我们研究了已知高分辨率结构的狼蛛粗丝中的 RLC 磷酸化。在松弛状态下，狼蛛 RLC 约有 50%是非磷酸化的，50%是单磷酸化的，而在激活时，单磷酸化增加，并且一些 RLC 成为双磷酸化的。质谱分析表明，松弛状态下单磷酸化发生在 Ser35 上，而 Ca(2+)-激活的磷酸化发生在 Ser45 上，两者都位于 RLC N 端附近。这些丝氨酸周围的序列表明，它们分别是蛋白激酶 C 和肌球蛋白轻链激酶（MLCK）的靶标。狼蛛丝的原子模型表明，两个肌球蛋白头（“自由”和“受阻”）处于不同的环境中，只有自由头的丝氨酸容易被激酶接近。因此，在松弛丝中，蛋白激酶 C Ser35 单磷酸化只会发生在自由头上。结构考虑表明，这些头与丝的骨架结合较弱，并且可能偶尔在附着和脱离状态之间振荡（“摆动”头）。在 Ca(2+)激活薄丝后，这些头将可立即与肌动蛋白相互作用。一旦 MLCK 被激活，它就在 Ser45 上磷酸化自由头。这些头变得完全可移动，将受阻头的 Ser45 暴露于 MLCK。这将释放受阻头，允许它们与肌动蛋白相互作用。在该模型中，抽搐力将由快速摆动的自由头与激活的薄丝的快速相互作用产生，而在破伤风时持续暴露于 Ca(2+)将招募新的 MLCK 激活的头，从而增强力。
