A Cardiomyopathy Mutation in the Myosin Essential Light Chain Alters Actomyosin Structure
A Cardiomyopathy Mutation in the Myosin Essential Light Chain Alters Actomyosin Structure
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DOI：
10.1016/j.bpj.2017.05.027
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发表时间：
2017-07-11
影响因子：
3.4
通讯作者：
Thomas, David D.
中科院分区：
文献类型：
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作者：
Guhathakurta, Piyali;Prochniewicz, Ewa;Thomas, David D.
我们已经使用了定点时间分辨荧光共振能量转移，以确定在人类心室肌球蛋白的基本轻链（hVELC）的病理突变的影响，肌动蛋白-肌球蛋白复合物的结构动力学。hVELC通过其N-末端延伸与肌动蛋白以及其C-末端叶与肌球蛋白重链的相互作用来调节肌动球蛋白的功能。hVELC中的几种突变与肥厚型心肌病（HCM）相关。这些突变的一些生化影响是已知的，但需要进一步了解它们对功能性肌动球蛋白结构动力学的影响。因此，我们将HCM突变E56 G引入到单半胱氨酸（C16）hVELC构建体中，并将其替换为牛心肌肌球蛋白亚片段1的VELC。使用供体荧光探针肌动蛋白（C374）和受体探针的C16的hVELC，我们进行了时间分辨荧光共振能量转移，直接检测功能过程中结合肌动球蛋白复合物内的结构变化。E56 G突变对ATP存在下肌动蛋白激活的ATP酶活性或肌动球蛋白亲和力没有显著影响，或者对ATP不存在下强结合S复合物的结构没有显著影响。然而，在饱和ATP的存在下，其中W（prepower-stroke）和S（postpowerstroke）的结构状态被观察到，突变体增加了S复合物的摩尔分数（增加占空比），而其余的W复合物的结构转移到S，表明结构重新分配到强有力的结合（力产生）复杂。我们认为这种效应是肌球蛋白中HCM突变引起的高收缩表型的原因。
We have used site-directed time-resolved fluorescence resonance energy transfer to determine the effect of a pathological mutation in the human ventricular essential light chain (hVELC) of myosin, on the structural dynamics of the actin-myosin complex. The hVELC modulates the function of actomyosin, through the interaction of its N-terminal extension with actin and its C-terminal lobe with the myosin heavy chain. Several mutations in hVELC are associated with hypertrophic cardiomyopathy (HCM). Some biochemical effects of these mutations are known, but further insight is needed about their effects on the structural dynamics of functioning actomyosin. Therefore, we introduced the HCM mutation E56G into a single-cysteine (C16) hVELC construct and substituted it for the VELC of bovine cardiac myosin subfragment 1. Using a donor fluorescent probe on actin (at C374) and an acceptor probe on C16 of hVELC, we performed time-resolved fluorescence resonance energy transfer, directly detecting structural changes within the bound actomyosin complex during function. The E56G mutation has no significant effect on actin-activated ATPase activity or actomyosin affinity in the presence of ATP, or on the structure of the strong-binding S complex in the absence of ATP. However, in the presence of saturating ATP, where both W (prepower-stroke) and S (postpowerstroke) structural states are observed, the mutant increases the mole fraction of the S complex (increasing the duty ratio), while shifting the structure of the remaining W complex toward that of S, indicating a structural redistribution toward the strongly bound (force-generating) complex. We propose that this effect is responsible for the hypercontractile phenotype induced by this HCM mutation in myosin.