Direct observation of the mechanochemical coupling in myosin Va during processive movement

Abstract

Myosin Va is a two-headed molecular motor which transports cargo inside cells by moving along actin filaments. The trailing head detaches and swings 72 nm forward to bind to a new leading position. During the processive movement at least one of the heads remain bound to actin. In this report Sellers and colleagues visualized the movement of a fluorescently labelled myosin Va molecule while simultaneously observing the binding and dissociation of a fluorescent ATP analogue. Each head swing is tightly coupled to the binding and dissociation of the nucleotide and the lead head retains nucleotide, presumably the diphosphate form, as long as the trail head is attached indicating gating between the two heads. This is the first direct demonstration of nucleotide binding to and movement of myosin V motors during stepping. Myosin Va is a two-headed molecular motor that transports cargo inside cells by moving along actin filaments. The trailing head detaches and swings 72 nm forward to bind to a new leading position. During the processive movement at least one of the heads remain bound to actin. This report visualizes the movement of a fluorescently-labelled myosin Va molecule while simultaneously observing the binding and dissociation of a fluorescent ATP analogue. This is the first direct demonstration of nucleotide binding to and movement of myosin V motors during stepping. Myosin Va transports intracellular cargoes along actin filaments in cells1. This processive, two-headed motor takes multiple 36-nm steps in which the two heads swing forward alternately towards the barbed end of actin driven by ATP hydrolysis2. The ability of myosin Va to move processively is a function of its long lever arm, the high duty ratio of its kinetic cycle and the gating of the kinetics between the two heads such that ADP release from the lead head is greatly retarded3,4,5,6,7,8,9,10. Mechanical studies at the multiple- and the single-molecule level suggest that there is tight coupling (that is, one ATP is hydrolysed per power stroke), but this has not been directly demonstrated4,5,11. We therefore investigated the coordination between the ATPase mechanism of the two heads of myosin Va and directly visualized the binding and dissociation of single fluorescently labelled nucleotide molecules, while simultaneously observing the stepping motion of the fluorescently labelled myosin Va as it moved along an actin filament. Here we show that preferential ADP dissociation from the trail head of mouse myosin Va is followed by ATP binding and a synchronous 36-nm step. Even at low ATP concentrations, the myosin Va molecule retained at least one nucleotide (ADP in the lead head position) when moving. Thus, we directly demonstrate tight coupling between myosin Va movement and the binding and dissociation of nucleotide by simultaneously imaging with near nanometre precision.