Kad Neil M, Trybus Kathleen M, Warshaw David M
Department of Molecular Physiology & Biophysics, University of Vermont, Burlington, Vermont 05405, USA.
J Biol Chem. 2008 Jun 20;283(25):17477-84. doi: 10.1074/jbc.M800539200. Epub 2008 Apr 27.
Myosin V is a processive actin-based motor protein that takes multiple 36-nm steps to deliver intracellular cargo to its destination. In the laser trap, applied load slows myosin V heavy meromyosin stepping and increases the probability of backsteps. In the presence of 40 mm phosphate (P(i)), both forward and backward steps become less load-dependent. From these data, we infer that P(i) release commits myosin V to undergo a highly load-dependent transition from a state in which ADP is bound to both heads and its lead head trapped in a pre-powerstroke conformation. Increasing the residence time in this state by applying load increases the probability of backstepping or detachment. The kinetics of detachment indicate that myosin V can detach from actin at two distinct points in the cycle, one of which is turned off by the presence of P(i). We propose a branched kinetic model to explain these data. Our model includes P(i) release prior to the most load-dependent step in the cycle, implying that P(i) release and load both act as checkpoints that control the flux through two parallel pathways.
肌球蛋白V是一种基于肌动蛋白的持续性运动蛋白，它通过多个36纳米的步幅将细胞内货物运送到目的地。在激光阱中，施加的负载会减缓肌球蛋白V重酶解肌球蛋白的步幅，并增加后退步的概率。在存在40 mM磷酸盐（P(i)）的情况下，向前和向后的步幅对负载的依赖性都降低。从这些数据中，我们推断P(i)的释放使肌球蛋白V经历一个高度依赖负载的转变，从一种两个头部都结合ADP且其领头头部被困在预动力冲程构象的状态转变。通过施加负载增加在该状态下的停留时间会增加后退步或脱离的概率。脱离动力学表明肌球蛋白V可以在循环中的两个不同点从肌动蛋白上脱离，其中一个点在P(i)存在时被关闭。我们提出一个分支动力学模型来解释这些数据。我们的模型包括在循环中最依赖负载的步骤之前释放P(i)，这意味着P(i)的释放和负载都充当控制通过两条平行途径通量的检查点。
