Department of Cellular and Molecular Physiology, College of Medicine, Pennsylvania State University , Hershey, Pennsylvania 17033, United States.
Biochemistry. 2013 Jul 9;52(27):4710-22. doi: 10.1021/bi4004364. Epub 2013 Jun 25.
We investigated how magnesium (Mg) impacts key conformational changes during the ADP binding/release steps in myosin V and how these alterations impact the actomyosin mechanochemical cycle. The conformation of the nucleotide binding pocket was examined with our established FRET system in which myosin V labeled with FlAsH in the upper 50 kDa domain participates in energy transfer with mant labeled nucleotides. We examined the maximum actin-activated ATPase activity of MV FlAsH at a range of free Mg concentrations (0.1-9 mM) and found that the highest activity occurs at low Mg (0.1-0.3 mM), while there is a 50-60% reduction in activity at high Mg (3-9 mM). The motor activity examined with the in vitro motility assay followed a similar Mg-dependence, and the trend was similar with dimeric myosin V. Transient kinetic FRET studies of mantdADP binding/release from actomyosin V FlAsH demonstrate that the transition between the weak and strong actomyosin.ADP states is coupled to movement of the upper 50 kDa domain and is dependent on Mg with the strong state stabilized by Mg. We find that the kinetics of the upper 50 kDa conformational change monitored by FRET correlates well with the ATPase and motility results over a wide range of Mg concentrations. Our results suggest the conformation of the upper 50 kDa domain is highly dynamic in the Mg free actomyosin.ADP state, which is in agreement with ADP binding being entropy driven in the absence of Mg. Overall, our results demonstrate that Mg is a key factor in coupling the nucleotide- and actin-binding regions. In addition, Mg concentrations in the physiological range can alter the structural transition that limits ADP dissociation from actomyosin V, which explains the impact of Mg on actin-activated ATPase activity and in vitro motility.
我们研究了镁（Mg）如何影响肌球蛋白 V 中 ADP 结合/释放步骤中的关键构象变化，以及这些变化如何影响肌球蛋白-肌动蛋白机械化学循环。核苷酸结合口袋的构象通过我们建立的 FRET 系统进行检查，其中标记在上部 50 kDa 结构域的 FlAsH 的肌球蛋白 V 与 mant 标记的核苷酸参与能量转移。我们在一系列游离 Mg 浓度（0.1-9 mM）下检查 MV FlAsH 的最大肌动蛋白激活 ATP 酶活性，发现最高活性发生在低 Mg（0.1-0.3 mM）下，而在高 Mg（3-9 mM）下活性降低 50-60%。用体外运动分析检查的运动活性遵循类似的 Mg 依赖性，并且这种趋势与二聚肌球蛋白 V 相似。瞬态动力学 FRET 研究表明，从肌球蛋白 V FlAsH 上结合/释放 mantdADP 的弱和强肌球蛋白-ADP 状态之间的转变与上 50 kDa 结构域的运动有关，并且依赖于 Mg，强状态由 Mg 稳定。我们发现，通过 FRET 监测的上 50 kDa 构象变化的动力学与 ATP 酶和运动结果在广泛的 Mg 浓度范围内很好地相关。我们的结果表明，在上 50 kDa 无 Mg 肌球蛋白-ADP 状态下，该结构域的构象高度动态，这与 ADP 结合在没有 Mg 时是熵驱动的一致。总体而言，我们的结果表明 Mg 是连接核苷酸和肌动蛋白结合区域的关键因素。此外，生理范围内的 Mg 浓度可以改变限制 ADP 从肌球蛋白 V 解离的结构转变，这解释了 Mg 对肌动蛋白激活 ATP 酶活性和体外运动的影响。
