Direct Visualization of Walking Myosin V Molecules by High-Speed AFM

Abstract

Facilitated by the processivity of myosin V, numerous single-molecule studies have demonstrated that it moves hand over hand with a ∼36 nm advance for every ATP hydrolysis. However, these results are obtained using optical microscopy and therefore the molecule itself is invisible in the observations. The motor has been visualized using electron microscopy but the obtained images are static. Thus, the detailed structural dynamic of walking myosin V and its functional mechanism are not yet fully described. Here, we directly visualized walking M5-HMM (tail-truncated myosin V) using high-speed AFM with nanometer and sub-second spatiotemporal resolution. The molecular movies provided not only corroborative visual evidence for established and speculated behaviors but also revealed unappreciated ones. First, it was cleared that the forward movement is driven not by bending but by rotation of the leading head. The leading head often showed ‘foot-stomping-like brief detachment and rebinding followed by forward step. In ADP, the coiled-coil tail of the two-headed bound molecule occasionally unwound, which was followed by the leading lever-arm swing, indicating the presence of intramolecular tension responsible for the powerstroke. From these observations, it is concluded that tension for forward movement can be produced without transitioning through a weak-binding ADP-Pi-bound state. Moreover, in low concentrations of ADP, the leading head showed conformational switching between the straight and sharp bend conformations, from which the rate of ADP dissociation from the leading head was estimated to be 0.1/s. This is straightforward evidence that the ATPase cycle is essentially stalled at the leading head. Thus, dynamic visualization of functioning M5-HMM by high-speed AFM lead to a comprehensive understanding of how it operates to function.