Dilated cardiomyopathy mutation E525K in human beta-cardiac myosin stabilizes the interacting-heads motif and super-relaxed state of myosin.
Dilated cardiomyopathy mutation E525K in human beta-cardiac myosin stabilizes the interacting-heads motif and super-relaxed state of myosin.
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DOI：
10.7554/elife.77415
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发表时间：
2022-11-24
期刊：
影响因子：
7.7
通讯作者：
Yengo CM
中科院分区：
文献类型：
--
作者：
Rasicci DV;Tiwari P;Bodt SML;Desetty R;Sadler FR;Sivaramakrishnan S;Craig R;Yengo CM
心肌肌球蛋白的自动抑制、超放松（SRX）状态被认为对心脏收缩、放松和能量保存的调节至关重要。我们使用单ATP转换实验来证明，人β -心肌肌凝蛋白的扩张型心肌病（DCM）突变（E525K）增加了SRX状态下（ATP转换缓慢）肌凝蛋白头部的比例，特别是在生理离子强度条件下。我们还利用肌凝蛋白尾部c端GFP标签和结合在运动结构域活性位点的Cy3ATP之间的FRET来估计封闭的相互作用头部基序（IHM）中的头部比例；我们发现IHM和SRX状态之间有很强的相关性。阴性染色电镜和结构的二维平均显示，E525K突变增加了采用IHM的分子的比例。总之，我们的研究结果表明，E525K DCM突变可能通过稳定自抑制的SRX状态来降低肌肉的力量和功率。我们的研究也为心肌肌球蛋白中SRX生化状态与IHM结构状态之间的相关性提供了直接证据。此外，E525残基可能与关键的静电相互作用有关，这种静电相互作用调节了肌球蛋白的这种保守的、自我抑制的构象。
The auto-inhibited, super-relaxed (SRX) state of cardiac myosin is thought to be crucial for regulating contraction, relaxation, and energy conservation in the heart. We used single ATP turnover experiments to demonstrate that a dilated cardiomyopathy (DCM) mutation (E525K) in human beta-cardiac myosin increases the fraction of myosin heads in the SRX state (with slow ATP turnover), especially in physiological ionic strength conditions. We also utilized FRET between a C-terminal GFP tag on the myosin tail and Cy3ATP bound to the active site of the motor domain to estimate the fraction of heads in the closed, interacting-heads motif (IHM); we found a strong correlation between the IHM and SRX state. Negative stain electron microscopy and 2D class averaging of the construct demonstrated that the E525K mutation increased the fraction of molecules adopting the IHM. Overall, our results demonstrate that the E525K DCM mutation may reduce muscle force and power by stabilizing the auto-inhibited SRX state. Our studies also provide direct evidence for a correlation between the SRX biochemical state and the IHM structural state in cardiac muscle myosin. Furthermore, the E525 residue may be implicated in crucial electrostatic interactions that modulate this conserved, auto-inhibited conformation of myosin.