Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA.
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA.
J Biol Chem. 2021 Oct;297(4):101157. doi: 10.1016/j.jbc.2021.101157. Epub 2021 Sep 2.
The interacting-heads motif (IHM) is a structure of myosin that has been proposed to modulate cardiac output by occluding myosin molecules from undergoing the force-generating cycle. It is hypothesized to be the structural basis for the super-relaxed state (SRX), a low-ATPase kinetic state thought to be cardioprotective. The goal of the present study was to test this hypothesis by determining directly and quantitatively the fractions of myosin in the IHM and SRX under the same conditions in solution. To detect the structural IHM, we used time-resolved fluorescence resonance energy transfer to quantitate two distinct populations. One population was observed at a center distance of 2.0 nm, whereas the other was not detectable by fluorescence resonance energy transfer, implying a distance greater than 4 nm. We confirmed the IHM assignment to the 2.0-nm population by applying the same cross-linking protocol used previously to image the IHM by electron microscopy. Under the same conditions, we also measured the fraction of myosin in the SRX using stopped-flow kinetics. Our results show that the populations of SRX and IHM myosin were similar, unless treated with mavacamten, a drug that recently completed phase III clinical trials to treat hypertrophic cardiomyopathy and is proposed to act by stabilizing both the SRX and IHM. However, we found that mavacamten had a much greater effect on the SRX (55% increase) than on the IHM (4% increase). We conclude that the IHM structure is sufficient but not necessary to produce the SRX kinetic state.
相互作用的头部模体（IHM）是肌球蛋白的一种结构，它被提出通过阻止肌球蛋白分子经历力产生循环来调节心输出量。它被假设为超松弛状态（SRX）的结构基础，SRX 是一种低 ATP 酶动力学状态，被认为具有心脏保护作用。本研究的目的是通过在相同条件下直接和定量地确定溶液中 IHM 和 SRX 中的肌球蛋白分数来检验这一假设。为了检测结构 IHM，我们使用时间分辨荧光共振能量转移来定量两个不同的群体。一个群体在中心距离为 2.0nm 处观察到，而另一个群体则不能通过荧光共振能量转移检测到，这意味着距离大于 4nm。我们通过应用以前用于通过电子显微镜成像 IHM 的相同交联方案来确认 IHM 分配到 2.0nm 群体。在相同条件下，我们还使用停流动力学测量了 SRX 中的肌球蛋白分数。我们的结果表明，除非用 mavacamten 处理，否则 SRX 和 IHM 肌球蛋白的群体相似，mavacamten 是一种最近完成 III 期临床试验以治疗肥厚型心肌病的药物，被提议通过稳定 SRX 和 IHM 起作用。然而，我们发现 mavacamten 对 SRX 的影响（增加 55%）远大于对 IHM 的影响（增加 4%）。我们得出结论，IHM 结构足以产生 SRX 动力学状态，但不是必需的。
