FRET and Optical Trapping Measurements Reveal Relationship between Phosphate Release and the Power Stroke in Myosin V

Abstract

Myosin V is able to processively walk along actin filaments by coordinating its mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. Our previous FRET measurements of the power stroke in myosin V containing a single IQ domain (MV 1IQ) revealed a fast rotation of the lever arm prior to the phosphate release step and a slower rotation of the lever that occurs during the transition between actomyosin.ADP states. Here we introduce a mutation in the active site (S217A) that is proposed to slow entry of phosphate into the exit tunnel. The mutation causes both fast lever arm rotation and phosphate release to occur with the same rate constant (slowing the maximum rate 10-fold), suggesting entry of phosphate into the exit tunnel gates the lever arm swing. In single molecule laser trapping studies we found the step size of S217A was similar to WT (4nm). Furthermore, raising the Pi concentration in the laser trap assay, which should increase the population of the actomyosin.ADP.Pi state, also did not affect the step size of S217A MV 1IQ. Interestingly, the attached time increased dramatically in the presence of 15 mM Pi. We also found that Pi can inhibit actin gliding in the vitro motility assay in both S217A and WT MV 1IQ in a dose-dependent manner, which is consistent with the optical trapping studies, while Pi did not impact steady-state ATPase activity. Our results suggest a model in which attachment to actin and the power stroke (lever arm swing) occur rapidly before phosphate is released in myosin V. In addition, Pi rebinding to the actomyosin.ADP state may be capable of reversing the power stroke without altering the actin binding interface.