ABSTRACT: Fundamental to cellular processes are directional movements driven by molecular motors. A common theme for these and other molecular machines driven by ATP is that controlled release of hydrolysis products is essential for using the chemical energy efficiently. Mechanochemical transduction by myosin motors on actin is coupled to unknown structural changes that result in the sequential release of inorganic phosphate (Pi) and MgADP. We present here a myosin structure possessing an actin-binding interface and a tunnel (back door) that creates an escape route for Pi with a minimal rotation of the myosin lever arm that drives movements. We propose that this state represents the beginning of the powerstroke on actin and that Pi translocation from the nucleotide pocket triggered by actin binding initiates myosin force generation. This elucidates how actin initiates force generation and movement and may represent a strategy common to many molecular machines.
Project description:Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly bound, whereas myosin is strongly bound to actin. We have generated an 8-Å cryoEM reconstruction of this state for myosin V and used molecular dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The ADP-bound structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This state is accomplished by a previously unseen conformation of the ?-sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5° rotation of the myosin lever arm, coupled to a ?-sheet rearrangement. Thus, the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release that is essential for processive myosins, such as myosin V.
Project description:Elevated levels of phosphate (Pi) reduce isometric force, providing support for the notion that the release of Pi from myosin is closely associated with the generation of muscular force. Pi is thought to rebind to actomyosin in an ADP-bound state and reverse the force-generating steps, including the rotation of the lever arm (i.e., the powerstroke). Despite extensive study, this mechanism remains controversial, in part because it fails to explain the effects of Pi on isometric ATPase and unloaded shortening velocity. To gain new insight into this process, we determined the effect of Pi on the force-generating capacity of a small ensemble of myosin (?12 myosin heads) using a three-bead laser trap assay. In the absence of Pi, myosin pulled the actin filament out of the laser trap an average distance of 54 ± 4 nm, translating into an average peak force of 1.2 pN. By contrast, in the presence of 30 mM Pi, myosin generated only enough force to displace the actin filament by 13 ± 1 nm, generating just 0.2 pN of force. The elevated Pi also caused a >65% reduction in binding-event lifetime, suggesting that Pi induces premature detachment from a strongly bound state. Definitive evidence of a Pi-induced powerstroke reversal was not observed, therefore we determined if a branched kinetic model in which Pi induces detachment from a strongly bound, postpowerstroke state could explain these observations. The model was able to accurately reproduce not only the data presented here, but also the effects of Pi on both isometric ATPase in muscle fibers and actin filament velocity in a motility assay. The ability of the model to capture the findings presented here as well as previous findings suggests that Pi-induced inhibition of force may proceed along a kinetic pathway different from that of force generation.
Project description:Understanding the basis for the action of myosin motors and related molecular machines requires a quantitative energy-based description of the overall functional cycle. Previous theoretical attempts to do so have provided interesting insights on parts of the cycle but could not generate a structure-based free energy landscape for the complete cycle of myosin. In particular, a nonphenomenological structure/energy-based understanding of the unidirectional motion is still missing. Here we use a coarse-grained model of myosin V and generate a structure-based free energy surface of the largest conformational change, namely the transition from the post- to prepowerstroke movement. We also couple the observed energetics of ligand binding/hydrolysis and product release to that of the conformational surface and reproduce the energetics of the complete mechanochemical cycle. It is found that the release in electrostatic free energy upon changing the conformation of the lever arm and the convertor domain from its post- to prepowerstroke state provides the necessary energy to bias the system towards the unidirectional movement of myosin V on the actin filament. The free energy change of 11 kcal is also in the range of ?2-3 pN, which is consistent with the experimentally observed stalling force required to stop the motor completely on its track. The conformational-chemical coupling generating a successful powerstroke cycle is believed to be conserved among most members of the myosin family, thus highlighting the importance of the previously unknown role of electrostatics free energy in guiding the functional cycle in other actin-based myosin motors.
Project description:The release of phosphate (Pi) is an important element in actomyosin function and has been shown to be accelerated by the binding of myosin to actin. To provide information about the structural elements important for Pi release, possible escape pathways from various isolated myosin II structures have been determined by molecular dynamics simulations designed for studying such slow processes. The residues forming the pathways were identified and their role was evaluated by mutant simulations. Pi release is slow in the pre-powerstroke structure, an important element in preventing the powerstroke prior to actin binding, and is much more rapid for Pi modeled into the post-rigor and rigor-like structures. The previously proposed backdoor route is dominant in the pre-powerstroke and post-rigor states, whereas a different path is most important in the rigor-like state. This finding suggests a mechanism for the actin-activated acceleration of Pi release.
Project description:A structure-based model of myosin motor is built in the same spirit of our early work for kinesin-1 and Ncd towards physical understanding of its mechanochemical cycle. We find a structural adaptation of the motor head domain in post-powerstroke state that signals faster ADP release from it compared to the same from the motor head in the pre-powerstroke state. For dimeric myosin, an additional forward strain on the trailing head, originating from the postponed powerstroke state of the leading head in the waiting state of myosin, further increases the rate of ADP release. This coordination between the two heads is the essence of the processivity of the cycle. Our model provides a structural description of the powerstroke step of the cycle as an allosteric transition of the converter domain in response to the Pi release. Additionally, the variation in structural elements peripheral to catalytic motor domain is the deciding factor behind diverse directionalities of myosin motors (myosin V & VI). Finally, we observe that there are general rules for functional molecular motors across the different families. Allosteric structural adaptation of the catalytic motor head in different nucleotide states is crucial for mechanochemistry. Strain-mediated coordination between motor heads is essential for processivity and the variation of peripheral structural elements is essential for their diverse functionalities.
Project description:Omecamtiv mecarbil (OM), a putative heart failure therapeutic, increases cardiac contractility. We hypothesize that it does this by changing the structural kinetics of the myosin powerstroke. We tested this directly by performing transient time-resolved FRET on a ventricular cardiac myosin biosensor. Our results demonstrate that OM stabilizes myosin's prepowerstroke structural state, supporting previous measurements showing that the drug shifts the equilibrium constant for myosin-catalyzed ATP hydrolysis toward the posthydrolysis biochemical state. OM slowed the actin-induced powerstroke, despite a twofold increase in the rate constant for actin-activated phosphate release, the biochemical step in myosin's ATPase cycle associated with force generation and the conversion of chemical energy into mechanical work. We conclude that OM alters the energetics of cardiac myosin's mechanical cycle, causing the powerstroke to occur after myosin weakly binds to actin and releases phosphate. We discuss the physiological implications for these changes.
Project description:Kinesins and myosins hydrolyze ATP, producing force that drives spindle assembly, vesicle transport and muscle contraction. How do motors do this? Here we discuss mechanisms of motor force transduction, based on their mechanochemical cycles and conformational changes observed in crystal structures. Distortion or twisting of the central ?-sheet - proposed to trigger actin-induced Pi and ADP release by myosin, and microtubule-induced ADP release by kinesins - is shown in a movie depicting the transition between myosin ATP-like and nucleotide-free states. Structural changes in the switch I region form a tube that governs ATP hydrolysis and Pi release by the motors, explaining the essential role of switch I in hydrolysis. Comparison of the motor power strokes reveals that each stroke begins with the force-amplifying structure oriented opposite to the direction of rotation or swing. Motors undergo changes in their mechanochemical cycles in response to small-molecule inhibitors, several of which bind to kinesins by induced fit, trapping the motors in a state that resembles a force-producing conformation. An unusual motor activator specifically increases mechanical output by cardiac myosin, potentially providing valuable information about its mechanism of function. Further study is essential to understand motor mechanochemical coupling and energy transduction, and could lead to new therapies to treat human disease.
Project description:We used small-angle X-ray solution scattering (SAXS) technique to investigate the nucleotide-mediated conformational changes of the head domains [subfragment 1 (S1)] of myosin V and VI processive motors that govern their directional preference for motility on actin. Recombinant myosin V-S1 with two IQ motifs (MV-S1IQ2) and myosin VI-S1 (MVI-S1) were engineered from Sf9 cells using a baculovirus expression system. The radii of gyration (R(g)) of nucleotide-free MV-S1IQ2 and MVI-S1 were 48.6 and 48.8 A, respectively. In the presence of ATP, the R(g) value of MV-S1IQ2 decreased to 46.7 A, while that of MVI-S1 increased to 51.7 A, and the maximum chord length of the molecule decreased by ca 9% for MV-S1IQ2 and increased by ca 6% for MVI-S1. These opposite directional changes were consistent with those occurring in S1s with ADP and Vi or AlF(4)(-2) bound (i.e., in states mimicking the ADP/Pi-bound state of ATP hydrolysis). Binding of AMPPNP induced R(g) changes of both constructs similar to those in the presence of ATP, suggesting that the timing of the structural changes for their motion on actin is upon binding of ATP (the pre-hydrolysis state) during the ATPase cycle. Binding of ADP to MV-S1IQ2 and MVI-S1 caused their R(g) values to drop below those in the nucleotide-free state. Thus, upon the release of Pi, the reverse conformational change could occur, coupling to drive the directional motion on actin. The amount of R(g) change upon the release of Pi was ca 6.4 times greater in MVI-S1 than in MV-S1IQ2, relating to the production of the large stroke of the MVI motor during its translocation on actin. Atomic structural models for these S1s based upon the ab initio shape reconstruction from X-ray scattering data were constructed, showing that MVI-S1 has the light-chain-binding domain positioned in the opposite direction to MV-S1IQ2 in both the pre- and post-powerstroke transition. The angular change between the light chain-binding domains of MV-S1IQ2 in the pre- to post-powerstroke transition was approximately 50 degrees, comparable to that of MII-S1. On the other hand, that of MVI-S1 was approximately 100 degrees or approximately 130 degrees much less than the currently postulated changes to allow the maximal stroke size of myosin VI-S1 but still significantly larger than those of other myosins reported so far. The results suggest that some additional alterations or elements are required for MVI-S1 to take maximal working strokes along the actin filament.
Project description:Dynamics of the actomyosin cytoskeleton regulate cellular processes such as secretion, cell division, cell motility, and shape change. Actomyosin dynamics are themselves regulated by proteins that control actin filament polymerization and depolymerization, and myosin motor contractility. Previous theoretical work has focused on translational movement of actin filaments but has not considered the role of filament rotation. Since filament rotational movements are likely sources of forces that direct cell shape change and movement we explicitly model the dynamics of actin filament rotation as myosin II motors traverse filament pairs, drawing them into alignment. Using Monte Carlo simulations we find an optimal motor velocity for alignment of actin filaments. In addition, when we introduce polymerization and depolymerization of actin filaments, we find that alignment is reduced and the filament arrays exist in a stable, asynchronous state. Further analysis with continuum models allows us to investigate factors contributing to the stability of filament arrays and their ability to generate force. Interestingly, we find that two different morphologies of F-actin arrays generate the same amount of force. We also identify a phase transition to alignment which occurs when either polymerization rates are reduced or motor velocities are optimized. We have extended our analysis to include a maximum allowed stretch of the myosin motors, and a non-uniform length for filaments leading to little change in the qualitative results. Through the integration of simulations and continuum analysis, we are able to approach the problem of understanding rotational alignment of actin filaments by myosin II motors.
Project description:Myosin VI challenges the prevailing theory of how myosin motors move on actin: the lever arm hypothesis. While the reverse directionality and large powerstroke of myosin VI can be attributed to unusual properties of a subdomain of the motor (converter with a unique insert), these adaptations cannot account for the large step size on actin. Either the lever arm hypothesis needs modification, or myosin VI has some unique form of extension of its lever arm. We determined the structure of the region immediately distal to the lever arm of the motor and show that it is a three-helix bundle. Based on C-terminal truncations that display the normal range of step sizes on actin, CD, fluorescence studies, and a partial deletion of the bundle, we demonstrate that this bundle unfolds upon dimerization of two myosin VI monomers. This unconventional mechanism generates an extension of the lever arm of myosin VI.