A Molecular Model of Phosphorylation-Based Activation and Potentiation of Tarantula Muscle Thick Filaments
A Molecular Model of Phosphorylation-Based Activation and Potentiation of Tarantula Muscle Thick Filaments
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DOI：
10.1016/j.jmb.2011.09.017
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发表时间：
2011-11-18
影响因子：
5.6
通讯作者：
Padron，Raul
中科院分区：
文献类型：
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作者：
Brito，Reicy;Alamo，Lorenzo;Padron，Raul
来自许多肌肉的肌球蛋白丝被其调节轻链（rlc）的磷酸化激活。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RLC的磷酸化。在松弛状态下，狼蛛rlc为50%非磷酸化和50%单磷酸化，而在激活状态下，单磷酸化增加，一些rlc变为双磷酸化。质谱分析显示，松弛状态的单磷酸化发生在Ser35上，而Ca2+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）的靶点。狼蛛丝的原子模型表明，两个肌凝蛋白头部（“自由”和“受阻”）处于不同的环境中，只有自由的头部丝线容易被激酶接触。因此，松弛丝中的蛋白激酶C Ser35单磷酸化只发生在自由的头部。结构方面的考虑表明，这些头与细丝主干的结合较弱，可能偶尔在附着和分离状态之间振荡（“摇摆”头）。在Ca2+激活细丝时，这些头将可用于立即的肌动蛋白相互作用。一旦MLCK被激活，它会磷酸化Ser45上的游离头。这些头部变得完全移动，将阻塞的头部Ser45暴露给MLCK。这将释放阻塞的头部，允许它们与肌动蛋白相互作用。在该模型中，摆动的自由头与激活的细丝快速相互作用会产生抽动力，而长时间暴露于Ca2+会在破伤风上招募新的mlck激活的头，导致力增强。
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 Ca2+-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 Ca2+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 Ca2+on tetanus would recruit new MLCK-activated heads, resulting in force potentiation.