A hallmark feature of myosin-II is that it can spontaneously self-assemble into bipolar synthetic thick filaments (STFs) in low ionic strength buffers, thereby serving as a reconstituted in vitro model for muscle thick filament. While these STFs have been extensively used for structural characterization, their use for functional studies has been very limited. In this report, we show that the ultra-low ATP-consuming super-relaxed (SRX) state of myosin is electrostatically more stable in STFs as compared with shorter myosin sub-fragments that lack the distal tail required for thick filament assembly. However, this electrostatic stability of the SRX state is weakened by phosphorylation of myosin light chains or the hypertrophic cardiomyopathy-causing myosin R403Q mutation. We also show that ADP binding to myosin depopulates the SRX population in STFs made of wild-type (WT) myosin, but not in S1, HMM, or STFs made of mutant R403Q myosin. Collectively, these findings emphasize that a critical network of inter- and intra-molecular interactions that underlie the SRX state of myosin are mostly preserved in STFs, establishing it as a native-like tool to interrogate myosin regulation. Next, using STFs, we show that a clinical-stage small molecule inhibitor, mavacamten, is more effective in promoting the myosin SRX state in STFs than in S1 or HMM and that it is equally potent in STFs made of atrial-WT, ventricular-WT, and mutant-R403Q myosin. Also, we found that mavacamten-bound heads are not permanently protected in the SRX state but can be recruited in response to physiological perturbations, thus providing new insights into its inhibitory mechanism.

中文翻译：

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心肌合成厚丝中的保守相互作用在健康，疾病和马来酰胺治疗模型中不同地影响肌球蛋白的超松弛状态

肌球蛋白II的标志性特征是它可以在低离子强度的缓冲液中自发组装成双极合成粗丝（STF），从而成为肌肉粗丝的体外重建模型。尽管这些STF已被广泛用于结构表征，但它们在功能研究中的应用却非常有限。在此报告中，我们显示，与缺少缺乏粗丝组件所需的尾部末端的较短肌球蛋白亚片段相比，STF中肌球蛋白的超低ATP消耗超松弛（SRX）状态在静电上更稳定。但是，SRX状态的静电稳定性会因肌球蛋白轻链的磷酸化或肥大型心肌病引起的肌球蛋白R403Q突变而减弱。我们还显示，ADP与肌球蛋白的结合在由野生型（WT）肌球蛋白制成的STF中使SRX种群减少，但在由突变R403Q肌球蛋白制成的S1，HMM或STF中却没有。总的来说，这些发现强调指出，构成肌球蛋白SRX状态基础的分子间和分子间相互作用的关键网络大部分保留在STF中，从而将其确立为一种类似于肌球蛋白调节的天然工具。接下来，我们使用STFs显示，临床阶段的小分子抑制剂mavacamten在促进STFs中的肌球蛋白SRX状态方面比在S1或HMM中更有效，并且在由房颤，心室传导性和心房颤动制成的STFs中同样有效WT和突变型R403Q肌球蛋白。此外，我们发现，在SRX状态下，与马万寿菊结合的头部并没有受到永久保护，但是可以响应生理扰动而募集，