PDF(Pubmed)
Abstract：
Inherited mutations in human beta-cardiac myosin (M2β) can lead to severe forms of heart failure. The E525K mutation in M2β is associated with dilated cardiomyopathy (DCM) and was found to stabilize the interacting heads motif (IHM) and autoinhibited super-relaxed (SRX) state in dimeric heavy meromyosin. However, in monomeric M2β subfragment 1 (S1) we found that E525K enhances (threefold) the maximum steady-state actin-activated ATPase activity (k cat) and decreases (eightfold) the actin concentration at which ATPase is one-half maximal (K ATPase). We also found a twofold to fourfold increase in the actin-activated power stroke and phosphate release rate constants at 30 μM actin, which overall enhanced the duty ratio threefold. Loaded motility assays revealed that the enhanced intrinsic motor activity translates to increased ensemble force in M2β S1. Glutamate 525, located near the actin binding region in the so-called activation loop, is highly conserved and predicted to form a salt bridge with another conserved residue (lysine 484) in the relay helix. Enhanced sampling molecular dynamics simulations predict that the charge reversal mutation disrupts the E525-K484 salt bridge, inducing conformations with a more flexible relay helix and a wide phosphate release tunnel. Our results highlight a highly conserved allosteric pathway associated with actin activation of the power stroke and phosphate release and suggest an important feature of the autoinhibited IHM is to prevent this region of myosin from interacting with actin. The ability of the E525K mutation to stabilize the IHM likely overrides the enhanced intrinsic motor properties, which may be key to triggering DCM pathogenesis.
摘要：
人β-心肌肌球蛋白(M2β)中的遗传突变可导致严重形式的心力衰竭。M2β中的E525K突变与扩张型心肌病（DCM）有关，并被发现可以稳定二聚重质肌球蛋白中相互作用的头基序（IHM）和自动抑制的超松弛（SRX）状态。然而,在单体M2β亚片段1（S1）中，我们发现E525K增强（三倍）最大稳态肌动蛋白激活的ATPase活性（kcat）并降低（八倍）ATPase为最大的一半时的肌动蛋白浓度（KATPase）。我们还发现，在30μM肌动蛋白时，肌动蛋白激活的动力冲程和磷酸盐释放速率常数增加了两倍至四倍，这总体上将占空比提高了三倍。负荷运动性测定显示，增强的内在运动活动转化为M2βS1中整体力的增加。谷氨酸525，位于所谓的激活环中的肌动蛋白结合区附近，高度保守，并预测与中继螺旋中的另一个保守残基（赖氨酸484）形成盐桥。增强的采样分子动力学模拟预测电荷反转突变会破坏E525-K484盐桥，诱导具有更灵活的中继螺旋和宽磷酸盐释放隧道的构象。我们的结果强调了与动力中风的肌动蛋白激活和磷酸盐释放相关的高度保守的变构途径，并表明自抑制的IHM的重要特征是防止肌球蛋白的该区域与肌动蛋白相互作用。E525K突变稳定IHM的能力可能会覆盖增强的内在电机特性，这可能是触发DCM发病机制的关键。
