Department of Cellular and Molecular Physiology, Pennsylvania State College of Medicine, Hershey, Pennsylvania, USA.
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Twin Cities, Minneapolis, Minnesota, USA.
J Biol Chem. 2020 Dec 18;295(51):17383-17397. doi: 10.1074/jbc.RA120.015632.
Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force-generating lever arm in myosin V. We directly compared the FRET results with single-molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating that it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), whereas our simulations suggest that the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre- and post-power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport.
肌球蛋白通过精确协调其机械和化学循环来产生力和运动，但这种协调的性质和时间仍存在争议。我们利用 FRET 方法研究了肌球蛋白 V 中产生力的杠杆臂结构变化的动力学。我们直接将 FRET 结果与通过光学捕获检查的单分子机械事件进行比较。我们在活性位点的保守开关 I 区域引入突变（S217A），以研究肌球蛋白如何将肌动蛋白和核苷酸结合区域的结构变化与力的产生联系起来。具体来说，S217A 增强了杠杆臂引发（恢复冲程）的最大速率，同时减缓了 ATP 水解，表明它将这两个步骤解耦。我们确定该突变极大地减慢了杠杆臂的肌动蛋白诱导旋转（功率冲程）和磷酸盐释放（≥10 倍），而我们的模拟表明，该突变不会改变这两个步骤的最大速率。时间分辨 FRET 显示，预功和后功构象的结构和这些构象的摩尔分数没有被突变改变。光学捕获结果表明，S217A 不会显著改变单位位移或减慢工作冲程速率常数，这与突变在功率冲程之前破坏肌动蛋白诱导的构象变化一致。我们提出，肌球蛋白的肌动蛋白和核苷酸结合区域之间的通讯确保在产生功率冲程之前形成适当的肌动蛋白结合界面和活性位点。这种耦合的可变性对于调节基于运动的功能（如肌肉收缩和细胞内运输）可能至关重要。
