Direct real-time detection of the actin-activated power stroke within the myosin catalytic domain

Abstract

We have used transient kinetics, nanosecond time-resolved fluorescence resonance energy transfer (FRET), and kinetics simulations to resolve a structural transition in the Dictyostelium myosin II relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in myosin, coupling biochemical changes in the ATPase site with the force-transducing rotation of the myosin light-chain domain. Previous research in the absence of actin showed that ATP binding to myosin induces a dynamic equilibrium between a bent prepower stroke state of the relay helix and a straight postpower stroke state, which dominates in the absence of ATP or when ADP is bound. We now ask whether actin binding reverses this transition and if so, how this reversal is coordinated with actin-activated phosphate release. We labeled a Cys-lite Dictyostelium myosin II motor domain with donor and acceptor probes at two engineered Cys residues designed to detect relay helix bending. We then performed transient timeresolved FRET following stopped-flow mixing of actin with labeled myosin, preincubated with ATP. We determined the kinetics of actin-activated phosphate release, using fluorescent phosphatebinding protein. The results show that actin binding to the myosin.ADP.P complex straightens the relay helix before phosphate dissociation. This actin-activated relay helix straightening is reversible, but phosphate irreversibly dissociates from the postpower stroke state, preventing reversal of the power stroke. Thus, relay helix straightening gates phosphate dissociation, whereas phosphate dissociation provides the thermodynamic driving force underlying force production.muscle | time-resolved FRET | structural kinetics T he myosin ATPase family uses the free energy of ATP hydrolysis to power a cycle of changes in myosin structure. These changes perform mechanical work, essential for motility in all eukaryotic cells. The efficient production of work by myosins is controlled by nucleotide and actin binding in coordination with a reversible rotation of the myosin light-chain domain, hypothesized to act as a force transducing "lever arm" (1-3). The myosin relay helix is crucial for this coordination (1, 4, 5).The structure of the relay helix is coupled to nucleotide binding and to the orientation of the light-chain domain. The relay helix is straight when myosin is crystallized with bound nonhydrolyzed ATP, prehydrolysis ATP analogs, or ADP or in the absence of nucleotide (abbreviated apo) and bent when crystallized with bound posthydrolysis ATP analogs (Fig. 1A, nucleotide free, IFMV, and ADP.Vanadate bound, 1VOM, crystal structures shown) (3, 6-9). There is no crystal structure of myosin bound to actin, but the relay helix is straight in a myosin-V crystal structure that is hypothesized to exhibit features of an actin-bound state (10) and also in a 0.8-nm resolution cryo-electron microscopy model of actin-bound myosin (11). In each of these studies, the orientation of the light-chain domain mirrors the structural s...

High-resolution helix orientation in actin-bound myosin determined with a bifunctional spin label

“…S1 and Table S1). There was significant variation among the three constructs, but the observed kinetics fall within the range previously observed for Dicty myosin S1dC (28,29), and labeling affected neither V max nor K ATPase by more than a factor of 2. Thus, labeling does not alter the fundamental mechanism of myosin's enzymatic activity.…”

“…A solvent-exposed location on the C-terminal end of myosin's relay helix was chosen for initial study, because crystal structures and spectroscopic studies of isolated myosin have shown that the orientation of this helical segment is sensitive to nucleotide binding (25)(26)(27)(28). MTSSL was reacted monofunctionally at position 492 ( Fig.…”

Biological Nanomotors with a Revolution, Linear, or Rotation Motion Mechanism

“…There is a good agreement on the details of movement of the lever arm during the recovery stroke. A FRET study reported that the reverse movement of the relay helix from a straight to a kinked conformation is associated with the reversal of the power stroke or the recovery stroke (208). Other studies also agree that the straight-to-kinked transition of the relay helix occurs after ATP binding and before hydrolysis (194,207).…”

“…The relay helix near the lower 50-kDa domain is a 4.7-nm-long ␣ helix that has been well documented to be an essential feature of the force-generating region of myosin (194,207,208). It connects the nucleotide binding site to the lever arm region and goes from a kinked to a straight conformation during formation of the pre-power stroke state (194,207,208). The HO helix and the relay helix are connected via the switch II loop.…”

“…The lever arm movement during the power and recovery stroke stages of the catalytic cycle has been probed in a number of studies, including intrinsic tryptophan fluorescence (209,210) polarization (211,212), electron paramagnetic resonance (213), and more recently FRET (208) studies. There is still controversy about the timing of the power stroke in relationship to the product release steps of the ATPase cycle.…”