Direct observation of the mechanochemical coupling in myosin Va during processive movement.
Myosin Va transports intracellular cargoes along actin filaments in cells. 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 hydrolysis. 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 retarded. 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 demonstrated. 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.
Sakamoto
Takeshi
T
Laboratory of Molecular Physiology, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892, USA.
Webb
Martin R
MR
Forgacs
Eva
E
White
Howard D
HD
Sellers
James R
JR
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D003374:Coumarins / Q000378:metabolism; D005456:Fluorescent Dyes; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D009068:Movement; D018995:Myosin Heavy Chains / Q000378:metabolism* / Q000648:ultrastructure; D015879:Myosin Subfragments / Q000378:metabolism / Q000648:ultrastructure; D024701:Myosin Type V / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding
C532242:3'-(7-diethylaminocoumarin-3-carbonylamino)-3'-deoxyadenosine-5'-triphosphate; D003374:Coumarins; D005456:Fluorescent Dyes; C513082:Myo5a protein, mouse; D015879:Myosin Subfragments; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D024701:Myosin Type V; D018995:Myosin Heavy Chains