A Branched Kinetic Scheme Describes the Mechanochemical Coupling of Myosin Va Processivity in Response to Substrate (original) (raw)

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

Nature, 2008

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 2 . 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 single molecule level suggest that there is tight coupling (i.e. one ATP is hydrolyzed per power stroke), but this has not been directly demonstrated 4,5,11 . 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-V as it moves along an actin filament. Here we show that preferential ADP dissociation from the trail head of myosin Va is followed by ATP binding and a synchronous 36-nm step. Even at low ATP concentrations, the myosin-V molecule retains at least one nucleotide (ADP in the lead head position) while moving. Thus we directly demonstrate tight coupling between myosin Va movement and the binding and dissociation of nucleotide by simultaneously imaging with nanometer precision.

Myosin-V stepping kinetics: A molecular model for processivity

Proceedings of The National Academy of Sciences, 2000

Myosin-V is a molecular motor that moves processively along its actin track. We have used a feedback-enhanced optical trap to examine the stepping kinetics of this movement. By analyzing the distribution of time periods separating discrete Ϸ36-nm mechanical steps, we characterize the number and duration of rate-limiting biochemical transitions preceding each such step. These data show that myosin-V is a tightly coupled motor whose cycle time is limited by ADP release. On the basis of these results, we propose a model for myosin-V processivity.

Coupling of two non-processive myosin 5c dimers enables processive stepping along actin filaments

Scientific reports, 2014

Myosin 5c (Myo5c) is a low duty ratio, non-processive motor unable to move continuously along actin filaments though it is believed to participate in secretory vesicle trafficking in vertebrate cells. Here, we measured the ATPase kinetics of Myo5c dimers and tested the possibility that the coupling of two Myo5c molecules enables processive movement. Steady-state ATPase activity and ADP dissociation kinetics demonstrated that a dimer of Myo5c-HMM (double-headed heavy meromyosin 5c) has a 6-fold lower Km for actin filaments than Myo5c-S1 (single-headed myosin 5c subfragment-1), indicating that the two heads of Myo5c-HMM increase F-actin-binding affinity. Nanometer-precision tracking analyses showed that two Myo5c-HMM dimers linked with each other via a DNA scaffold and moved processively along actin filaments. Moreover, the distance between the Myo5c molecules on the DNA scaffold is an important factor for the processive movement. Individual Myo5c molecules in two-dimer complexes move...

Role of the lever arm in the processive stepping of myosin V

Proceedings of the National Academy of Sciences, 2002

Myosin V is a two-headed molecular motor that binds six light chains per heavy chain, which creates unusually long lever arms. This motor moves processively along its actin track in discrete 36-nm steps. Our model is that one head of the two-headed myosin V tightly binds to actin and swings its long lever arm through a large angle, providing a stroke. We created single-headed constructs with different-size lever arms and show that stroke size is proportional to lever arm length. In a two-headed molecule, the stroke provides the directional bias, after which the unbound head diffuses to find its binding site, 36 nm forward. Our two-headed construct with all six light chains per head reconstitutes the 36-nm processive step seen in tissue-purified myosin V. Two-headed myosin V molecules with only four light chains per head are still processive, but their step size is reduced to 24 nm. A further reduction in the length of the lever arms to one light chain per head results in a motor that is unable to walk processively. This motor produces single small Ϸ6-nm strokes, and ATPase and pyrene actin quench measurements show that only one of the heads of this dimer rapidly binds to actin for a given binding event. These data show that for myosin V with its normal proximal tail domain, both heads and a long lever arm are required for large, processive steps.

Robust processivity of myosin V under off-axis loads

Nature Chemical Biology, 2010

The dimeric motor myosin V transports organelles along actin filament tracks over long distances in cells. Myosin V is a smart 'walker' that is able to swiftly adjust to variable 'road conditions' to continue its processive movement across dense cellular environments. Coordination between the two heads via intramolecular load modulates biochemical kinetics and ensures highly efficient unidirectional motion. However, little is known about how load-induced regulation of the processive stepping occurs in vivo, where myosin V experiences significant off-axis loads applied in various directions. To reveal how myosin V remains processive in cells, we measured the effect of the off-axis loads, applied to individual actomyosin V bonds in a range of angles, on the coordination between the two heads and myosin V processive stepping. We found that myosin V remains highly processive under diagonal loads owing to asymmetrical ADP affinities and that the native 6IQ lever optimizes the subunit coordination, which indicates that myosin V is designed to be an intracellular transporter. Myosin V transports cargos toward the barbed end of actin filaments in cells 1,2 , taking multiple ~36-nm steps by alternately swinging forward two lever arms in a 'hand-over-hand' fashion 3-7. Single-molecule experiments reveal that one ATP molecule is consumed for each step, which confirms the tight coupling between mechanical and enzymatic events 8. Combined with the unidirectionality of the processive movement, this mechanism implies that the ATPase cycles in two catalytic domains are precisely coordinated. Directional loads modulate ATPase kinetics in myosin V 9-11 , which suggests that two subunits communicate via the intramolecular load, which is generated during binding to actin with both heads, to achieve the coordinated mechanical performance.

Chemomechanical Coupling and Motor Cycles of Myosin V

Biophysical Journal, 2011

The molecular motor myosin V has been studied extensively both in bulk and single molecule experiments. Based on the chemical states of the motor, we construct a systematic network theory that includes experimental observations about the stepping behavior of myosin V. We utilize constraints arising from nonequilibrium thermodynamics to determine motor parameters and demonstrate that the motor behavior is governed by three chemomechanical motor cycles. The competition between these cycles can be understood via the influence of external load forces onto the chemical transition rates for the binding of adenosine triphosphate and adenosine diphosphate. In addition, we also investigate the functional dependence of the mechanical stepping rates on these forces. For substall forces, the dominant pathway of the motor network is profoundly different from the one for superstall forces, which leads to a stepping behavior that is in agreement with the experimental observations. Our theory provides a unified description of the experimental data as obtained for myosin V in single motor experiments.

Mechanochemical coupling of two substeps in a single myosin V motor

Nature Structural & Molecular Biology, 2004

Myosin V belongs to the myosin superfamily of actin-based molecular motors and is involved in the intracellular transport of organelles 1-4 . Myosin V consists of two identical heavy chains, each composed of an N-terminal motor domain ('head'), a domain comprising six IQ motifs that bind light chains ('neck'), a coiled coil dimerization domain and a globular cargo-binding tail domain 1,3 . Myosin V is a processive motor that 'walks' along an actin filament toward the barbed end over a long distance without dissociating from the filament 5,6 . Electron microscopy of actomyosin V in the presence of low ATP concentrations shows both motor domains of myosin V bound to the actin filament at sites spaced 36 nm apart, which corresponds to the half pitch of the filament long-pitch helix 7 . Experiments using optical tweezers identified processive 36-nm steps of a bead, on which single myosin V molecules were adsorbed 6,8 . Moreover, it was shown that myosin V walks as a left-handed spiral motor along an actin filament, because the average step size is slightly shorter than the half pitch of the long-pitch actin helix 9 . The hand-over-hand walking model has received strong support from two recent experiments that (i) observed the orientation of the neck domain of myosin V by monitoring the polarization of a single fluorophore covalently attached to a light chain 10 and (ii) measured the stepwise displacement of a single fluorophore labeled at one of six light chains of myosin V 11 .