Direct measurements of the coordination of lever arm swing and the catalytic cycle in myosin V

Myosins use a conserved structural mechanism to convert the energy from ATP hydrolysis into a large swing of the force-generating lever arm. The precise timing of the lever arm movement with respect to the steps in the actomyosin ATPase cycle has not been determined. We have developed a FRET system in myosin V that uses three donor–acceptor pairs to examine the kinetics of lever arm swing during the recovery and power stroke phases of the ATPase cycle. During the recovery stroke the lever arm swing is tightly coupled to priming the active site for ATP hydrolysis. The lever arm swing during the power stroke occurs in two steps, a fast step that occurs before phosphate release and a slow step that occurs before ADP release. Time-resolved FRET demonstrates a 20-Å change in distance between the pre- and postpower stroke states and shows that the lever arm is more dynamic in the postpower stroke state. Our results suggest myosin binding to actin in the ADP.Pi complex triggers a rapid power stroke that gates the release of phosphate, whereas a second slower power stroke may be important for mediating strain sensitivity.Myosins are molecular machines that use the energy from ATP hydrolysis to generate force and motion through a cyclic interaction with actin filaments. Actomyosin-based force generation is used to drive muscle contraction, organelle transport, cytokinesis, membrane tension generation, and numerous biological tasks (1). Most myosins display a conserved structural fold and ATPase mechanism, suggesting the mechanism of energy transduction is similar in the myosin superfamily. A long α-helix which extends from the motor core binds a variable number of light chains and is referred to as the “lever arm” (2). A relative sliding motion of myosin (thick) and actin (thin) filaments in muscle forms the basis of the cross-bridge hypothesis which provides a more general view of the mechanism of muscle contraction (3). The swinging lever arm hypothesis provides a more molecular basis of muscle contraction. In this hypothesis, the lever arm swing is associated with the actin-activated product release steps, in turn leading to force generation by the attached cross-bridge (4). However, the precise timing of the lever arm swing and product release steps has remained a central question since early studies of actomyosin (5).Scheme I represents a simplified actomyosin ATPase cycle that can be used to describe the kinetics of key steps in the catalytic cycle (5, 6). The weak actin-binding states of myosin are indicated in bold, and the actin-bound biochemical transitions are indicated by equilibrium constants with a prime. ATP binding to myosin occurs in two steps, an initial collision complex (K′₁) followed by a structural change that is associated with a weak actin-binding conformation (open actin-binding cleft) and high affinity for ATP (closed nucleotide-binding pocket) (K′₂). The movement of the lever arm into the prepower stroke state is thought to occur during one of the ATP-binding steps and before ATP hydrolysis. The ATP hydrolysis step (K₃) occurs while myosin remains in a weak actin-binding conformation. When myosin binds to actin with the hydrolyzed products in the active site, there is a dramatic acceleration of the product release steps, first phosphate (Pi) (K′₄) and then ADP (K′₅). It is during the actin-activated product release steps that the lever shifts from a pre- to a postpower stroke state and force generation occurs. In addition, myosin shifts from a weak to a strong actin-binding conformation as a result of actin-induced closure of the actin-binding cleft. Thus, to determine the structural mechanism of actomyosin-based force generation, it is crucial to design a method of measuring the position of the lever arm during the formation of the prepower stroke state (recovery stroke) as well as during the transition from the pre- to postpower stroke states (power stroke).Open in a separate windowScheme 1.The actomyosin ATPase cycle.In the current study we engineered myosin V (MV), a motor that is well characterized both kinetically and structurally, to contain three site-specific donor–acceptor pairs that allowed us to measure the lever arm swing directly by FRET. We provide direct evidence that the lever arm swings into the prepower stroke state (recovery stroke) when the active site is primed for ATP hydrolysis. The force-generating swing (the power stroke) occurs in two steps, with the first, fast step occurring before phosphate release. The slower power stroke step occurs before ADP release which is hypothesized to be a strain-sensitive step in the catalytic cycle of MV. Our work provides crucial insights into the structural details of lever arm swing in relation to the different steps in the catalytic cycle of myosin motors.     

Structural mechanism of the recovery stroke in the myosin molecular motor

The power stroke pulling myosin along actin filaments during muscle contraction is achieved by a large rotation ( approximately 60 degrees ) of the myosin lever arm after ATP hydrolysis. Upon binding the next ATP, myosin dissociates from actin, but its ATPase site is still partially open and catalytically off. Myosin must then close and activate its ATPase site while returning the lever arm for the next power stroke. A mechanism for this coupling between the ATPase site and the distant lever arm is determined here by generating a continuous series of optimized intermediates between the crystallographic end-states of the recovery stroke. This yields a detailed structural model for communication between the catalytic and the force-generating regions that is consistent with experimental observations. The coupling is achieved by an amplifying cascade of conformational changes along the relay helix lying between the ATPase and the domain carrying the lever arm.     

Characterization of the pre-force-generation state in the actomyosin cross-bridge cycle

Myosin is an actin-based motor protein that generates force by cycling between actin-attached (strong binding: ADP or rigor) and actin-detached (weak binding: ATP or ADP.P(i)) states during its ATPase cycle. However, it remains unclear what specific conformational changes in the actin binding site take place on binding to actin, and how these structural changes lead to product release and the production of force and motion. We studied the dynamics of the actin binding region of myosin V by using fluorescence resonance energy transfer (FRET) to monitor conformational changes in the upper-50-kDa domain of the actin binding cleft in the weak and strong actin binding states. Steady-state and lifetime data monitoring the FRET signal suggest that the cleft is in a more open conformation in the weak actin binding states. Transient kinetic experiments suggest that a rapid conformational change occurs, which is consistent with cleft closure before actin-activated phosphate release. Our results have identified a pre-force-generation actomyosin ADP.P(i) state, and suggest force generation may occur from a state not yet seen by crystallography in which the actin binding cleft and the nucleotide binding pocket are closed. Computational modeling uncovers dramatic changes in the rigidity of the upper-50-kDa domain in different nucleotide states, which suggests that the intrinsic flexibility of this domain allows myosin motors to accomplish simultaneous tight nucleotide binding (closed nucleotide binding pocket) and high-affinity actin binding (closed actin binding cleft).     

The myosin step size: measurement of the unit displacement per ATP hydrolyzed in an in vitro assay.

Chemomechanical coupling in muscle contraction may be due to "swinging crossbridges," such that a change in the angle at which the myosin head binds to the actin filament is tightly coupled to release of products of ATP hydrolysis. This model would limit the step size, the unit displacement of actin produced by a single ATP hydrolysis, to less than twice the chord length of the myosin head. Recent measurements have found the step size to be significantly larger than this geometric limit, bringing into question any direct correspondence between the crossbridge and ATP-hydrolysis cycles. We have measured the rate of ATP hydrolysis due to actin sliding movement in an in vitro motility assay consisting of purified actin and purified myosin. We have calculated an apparent myosin step size well within the geometric limit set by the size of the myosin head. These data are consistent with tight coupling between myosin crossbridge movement and ATP hydrolysis.     

Kinetics and Energetics of The Crossbridge Cycle

Myosin heads interacting with actin filaments, a process fueled by MgATP and regulated by calcium, powers the pump-like action of the human heart. Hydrolysis of MgATP, the competition between MgATP, its products of hydrolysis, and actin for binding to myosin, and the sequence of shifting affinities in that competition, constitute the central mechanism of muscular contraction. The force, work, and power produced during the cardiac cycle stems from an isomerization of the myosin head that is closely associated with strong binding of myosin to actin and release of phosphate. While fluctuations of intracellular [Ca²⁺] bound to troponin and related shifts in tropomyosin on the thin filaments regulate the number of crossbridges on a beat-to-beat basis, the oscillatory work produced is augmented by a delayed force response to stretch that develops during diastole. This stretch-activated myogenic response is facilitated by specialized myofilament structures, including actin-binding portions of the myosin essential light chain and myosin binding protein C, which are thought to guide and orient the myosin head or enhance thin filament activation. Phosphorylation of the myosin regulatory light chain, myosin binding protein C, and troponin T also assist in this regard. Animal models show isoform shifts in myosin and other myofibrillar proteins have major effects on power output, but isoform shifts in human myocardium are modest at best and are therefore likely to play only a minor role in modulating crossbridge kinetics compared to disease-related post-translational modifications of the contractile proteins and to changes in their chemical environment.     

Determination of the myosin step size from mechanical and kinetic data.

During muscle contraction, work is generated when a myosin cross-bridge attaches to an actin filament and exerts a force on it through some power-stroke distance, h. At the end of this power stroke, attached myosin heads are carried into regions where they exert a negative force on the actin filament (the drag stroke) and where they are released rapidly from actin by ATP binding. Although the length of the power stroke remains controversial, average distance traversed in the drag-stroke region can be determined when one knows both rate of cross-bridge dissociation and filament-sliding velocity. At maximum contraction velocity, the average force exerted in the drag stroke must balance that exerted in the power stroke. We discuss here a simple model of cross-bridge interaction that allows one to calculate the force exerted in the drag stroke and to relate this to the power-stroke distance h traversed by cross-bridges in the positive-force region. Both the rate at which myosin can be dissociated from actin and the velocity at which an actin filament can be translated have been measured for a series of myosin isozymes and for different substrates, producing a wide range of values for each. Nonetheless, we show here that the rate of myosin dissociation from actin correlates well with the velocity of filament sliding, providing support for the simple model presented and suggesting that the power stroke is approximately 10 nm in length.     

Mapping the actin filament with myosin

Structural studies have shown that the heads of the myosin motor molecule bind preferentially to "target zones" of favorably oriented sites on the helices of the actin filament. We present direct evidence for target zones from the interactions of a single myosin head with an actin filament held between two optically trapped beads. With compliant traps, thermal motions of the filament allow the fixed myosin-S1 to interact with at least two zones, observed as a bi-modal distribution of filament displacements due to myosin binding, whose spacing is near the 36-nm helix repeat distance. The number of binding events and the "apparent working stroke" (mean displacement with myosin bound), vary periodically as the filament is moved past the fixed myosin by displacing the traps; observed periods are close to 36 nm and the apparent stroke varies from 0-10 nm. We also observe a strong modulation at the 5.5-nm actin monomer repeat confirming that myosin interacts with a single strand and that the actin is not free to rotate. Each interaction can be assigned to an actin monomer and each active zone on the helix is made up of three actin monomers.     

Structural mechanism of the ATP-induced dissociation of rigor myosin from actin

Myosin is a true nanomachine, which produces mechanical force from ATP hydrolysis by cyclically interacting with actin filaments in a four-step cycle. The principle underlying each step is that structural changes in separate regions of the protein must be mechanically coupled. The step in which myosin dissociates from tightly bound actin (the rigor state) is triggered by the 30 Å distant binding of ATP. Large conformational differences between the crystal structures make it difficult to perceive the coupling mechanism. Energetically accessible transition pathways computed at atomic detail reveal a simple coupling mechanism for the reciprocal binding of ATP and actin.     

Direct real-time detection of the structural and biochemical events in the myosin power stroke

A principal goal of molecular biophysics is to show how protein structural transitions explain physiology. We have developed a strategic tool, transient time-resolved FRET [(TR)²FRET], for this purpose and use it here to measure directly, with millisecond resolution, the structural and biochemical kinetics of muscle myosin and to determine directly how myosin’s power stroke is coupled to the thermodynamic drive for force generation, actin-activated phosphate release, and the weak-to-strong actin-binding transition. We find that actin initiates the power stroke before phosphate dissociation and not after, as many models propose. This result supports a model for muscle contraction in which power output and efficiency are tuned by the distribution of myosin structural states. This technology should have wide application to other systems in which questions about the temporal coupling of allosteric structural and biochemical transitions remain unanswered.Myosin family proteins use ATP hydrolysis to generate force and movement required for normal physiology. They drive muscle contraction, help control cell division and cellular motility, move organelles through the cytoplasm, and are important elements of the cellular mechanical-sensing machinery (1, 2). The key to understanding how myosin and related enzymes function in cells, and how to modulate their activity to treat disease, is to determine how the protein’s structural dynamics and biochemical kinetics are coupled. Although high-resolution crystal structures provide best-guess snapshots of protein structure over a range of biochemical states, determining the physiological relevance of these snapshots remains one of the central challenges of structural biophysics.How myosin generates force remains debated despite more than 50 y of intense research (1–3). The most popular current model (2, 4) proposes that after ATP hydrolysis, myosin interacts weakly with actin and this interaction initiates an ordered series of structural and biochemical transitions that culminate in the dissociation of hydrolyzed phosphate, followed by the isomerization of the actin-binding interface to a state that binds actin with nanomolar affinity and then the rotation of the myosin light-chain domain (LCD) toward the plus end of the actin filament. This rotation converts the thermodynamic energy of phosphate release and actin binding into mechanical energy that performs work. A number of results question this model, however, including spectroscopic data showing that a structural transition in the myosin relay helix, hypothesized to be coupled to LCD rotation, precedes P_i release (5) and force development precedes P_i release in muscle fibers (6).Determining how these events take place in solution and in cells is an important question, because (i) differences in the mechanics of different myosins likely reflect differences in how the biochemical and structural transitions described above are coordinated (4); (ii) disease-causing mutations in the myosin heavy chain should alter this coordination (7, 8); (iii) recent studies show that modulating mechanochemical coupling in myosin is a viable therapeutic approach to treat myosin-associated diseases (9, 10); and (iv) myosin remains a model system for mechanochemical coupling in general, so what we learn about the myosin power stroke will inform work on related enzymes (3). Understanding the coordination of actin binding, phosphate release, and LCD rotation will address each of these goals. However, direct detection of the kinetics of both LCD rotation and phosphate release, the key to understanding mechanochemical coupling in myosin, has not been achieved (4).We have developed a spectroscopic approach that allows us to detect angstrom-scale structural changes in protein samples using time-resolved FRET (TR-FRET) measured on the submillisecond time scale. We call this approach transient time-resolved FRET [(TR)²FRET] (5, 11) and use it to determine the structural kinetic mechanism of the myosin power stroke. The results show that actin induces LCD rotation before phosphate dissociation, suggesting a more nuanced interpretation of recent high-resolution crystal structures of myosins complexed with ADP and phosphate (4). They also prompt a general model for tuning myosin force generation where the nonequilibrium distribution of pre- and postpower stroke LCD orientations controls power output and muscle efficiency.     

Conformational change of the actomyosin complex drives the multiple stepping movement

Actin-myosin (actomyosin) generates mechanical force by consuming ATP molecules. We apply the energy landscape perspective to address a controversial issue as to whether the myosin head moves with multiple steps after a single ATP hydrolysis or only a single mechanical event of the lever-arm swinging follows a single ATP hydrolysis. Here we propose a theoretical model in which the refolding of the partially unfolded actomyosin complex and the movement of the myosin head along the actin filament are coupled. A single ATP hydrolysis is followed by the formation of a high free-energy partially unfolded actomyosin complex, which then gradually refolds with a concomitant multiple stepping movement on the way to the lowest free-energy rigor state. The model quantitatively explains the single-molecular observation of the multiple stepping movement and is consistent with structural observations of the disorder in the actomyosin-binding process. The model also explains the observed variety in dwell time before each step, which is not accounted for by previous models, such as the lever-arm or ratchet models.     

Three-dimensional image reconstruction of dephosphorylated smooth muscle heavy meromyosin reveals asymmetry in the interaction between myosin heads and placement of subfragment 2

Regulation of the actin-activated ATPase of smooth muscle myosin II is known to involve an interaction between the two heads that is controlled by phosphorylation of the regulatory light chain. However, the three-dimensional structure of this inactivated form has been unknown. We have used a lipid monolayer to obtain two-dimensional crystalline arrays of the unphosphorylated inactive form of smooth muscle heavy meromyosin suitable for structural studies by electron cryomicroscopy of unstained, frozen-hydrated specimens. The three-dimensional structure reveals an asymmetric interaction between the two myosin heads. The ATPase activity of one head is sterically "blocked" because part of its actin-binding interface is positioned onto the converter domain of the second head. ATPase activity of the second head, which can bind actin, appears to be inhibited through stabilization of converter domain movements needed to release phosphate and achieve strong actin binding. When the subfragment 2 domain of heavy meromyosin is oriented as it would be in an actomyosin filament lattice, the position of the heads is very different from that needed to bind actin, suggesting an additional contribution to ATPase inhibition in situ.     

Crystallographic findings on the internally uncoupled and near-rigor states of myosin: further insights into the mechanics of the motor

Here we report a 2.3-A crystal structure of scallop myosin S1 complexed with ADP.BeF(x), as well as three additional structures (at 2.8-3.8 A resolution) for this S1 complexed with ATP analogs, some of which are cross-linked by para-phenyl dimaleimide, a short intramolecular cross-linker. In all cases, the complexes are characterized by an unwound SH1 helix first seen in an unusual 2.5-A scallop myosin-MgADP structure and described as corresponding to a previously unrecognized actin-detached internally uncoupled state. The unwinding of the SH1 helix effectively uncouples the converter/lever arm module from the motor and allows cross-linking by para-phenyl dimaleimide, which has been shown to occur only in weak actin-binding states of the molecule. Mutations near the metastable SH1 helix that disable the motor can be accounted for by viewing this structural element as a clutch controlling the transmission of torque to the lever arm. We have also determined a 3.2-A nucleotide-free structure of scallop myosin S1, which suggests that in the near-rigor state there are two conformations in the switch I loop, depending on whether nucleotide is present. Analysis of the subdomain motions in the weak actin-binding states revealed by x-ray crystallography, together with recent electron microscopic results, clarify the mechanical roles of the parts of the motor in the course of the contractile cycle and suggest how strong binding to actin triggers both the power stroke and product release.     

Toward understanding actin activation of myosin ATPase: the role of myosin surface loops

To understand the complicated interplay when a traveling myosin head reaches interaction distance with two actins in a filament we looked to three myosin loops that early on exert their influences from the "outside" of the myosin. On these we conduct, functionally test, and interpret strategically chosen mutations at sites thought from crystallography to be a patch for binding the "first" of the two actins. One loop bears a hydrophobic triplet of residues, one is the so-called "loop 2," and the third is the "cardiomyopathy" loop. So far as we know, the myosin sites that first respond are the two lysine-rich loops that produce an ionic strength-dependent weak-binding complex with actin. Subsequently, the three loops of interest bind the first actin simultaneously, and all three assist in closing the cleft in the 50-kDa domain of the myosin, a closure that results in transition from weak to strong binding and precedes rapid Pi release and motility. Mutational analysis shows that each such loop contact is distinctive in the route by which it communicates with its specific target elsewhere in myosin. The strongest contact with actin, for example, is that of the triplet-bearing loop. On the other hand, that of loop 2 (dependent on drawing close two myosin lysines and two actin aspartates) is probably responsible for opening switch I and uncovering the gamma-phosphate moiety of bound ATP. Taking into account these findings, we begin to arrange in order many molecular events in muscle function.     

Reconciling the working strokes of a single head of skeletal muscle myosin estimated from laser-trap experiments and crystal structures

Myosin generates force by a rotation of its lever arm. Crystal structures of myosin II indicate an unloaded working stroke of 10-12 nm, a range confirmed by recent x-ray interference experiments. However, when an actin filament, held between two weakly, optically trapped beads is made to interact with a single head of skeletal myosin, the bead displacements have often been reported as having a mean value of 5-6 nm, a value that is commonly interpreted as the working stroke. In general, the observed displacement is not expected to be equal to the working stroke because the kinetics of the stroke is necessarily strain-dependent: this effect biases the frequency of binding events to different actin sites so that displacements smaller than the working stroke are preferentially selected. Our analysis is tailored to current trap experiments, in which the time resolution is insufficient to detect pre-rigor states. If the preceding transitions are in equilibrium, the mean displacement is zero, contrary to observations in the presence of ATP. However, under ATP-cycling conditions, we find that the mean displacement is deflated to 0.3-0.7 of the true working stroke, depending on the equilibrium constant of the stroke and the rate at which the first myosin product state can detach from actin. The primary working stroke of processive myosin motors as measured by optical trapping is similarly uncertain.     

The working stroke upon myosin-nucleotide complexes binding to actin

For many years, it has been known that myosin binds to actin tightly, but it had not been possible to devise a muscle fiber experiment to determine whether this binding energy is directly coupled to the working stroke of the actomyosin crossbridge cycle. Addressing the question at the single-molecule level with optical tweezers allows the problem to be resolved. We have compared the working stroke on the binding of four myosin complexes (myosin, myosin-ADP, myosin-pyrophosphate, and myosin-adenyl-5'yl imidodiphosphate) with that observed while hydrolyzing ATP. None of the four was observed to give a working stroke significantly different from zero. A working stroke (5.4 nm) was observed only with ATP, which indicates that the other states bind to actin in a rigor-like conformation and that myosin products (M.ADP.Pi), the state that binds to actin during ATPase activity, binds in a different, prestroke conformation. We conclude that myosin, while dissociated from actin, must be able to take up at least two mechanical conformations and show that our results are consistent with these conformations corresponding to the two states characterized at high resolution, which are commonly referred to in terms of having open and closed nucleotide binding pockets.     

Two independent mechanical events in the interaction cycle of skeletal muscle myosin with actin

During skeletal muscle contraction, regular arrays of actin and myosin filaments slide past each other driven by the cyclic ATP-dependent interaction of the motor protein myosin II (the cross-bridge) with actin. The rate of the cross-bridge cycle and its load-dependence, defining shortening velocity and energy consumption at the molecular level, vary widely among different isoforms of myosin II. However, the underlying mechanisms remain poorly understood. We have addressed this question by applying a single-molecule approach to rapidly ( approximately 300 mus) and precisely ( approximately 0.1 nm) detect acto-myosin interactions of two myosin isoforms having large differences in shortening velocity. We show that skeletal myosin propels actin filaments, performing its conformational change (working stroke) in two steps. The first step ( approximately 3.4-5.2 nm) occurs immediately after myosin binding and is followed by a smaller step ( approximately 1.0-1.3 nm), which occurs much faster in the fast myosin isoform than in the slow one, independently of ATP concentration. On the other hand, the rate of the second phase of the working stroke, from development of the latter step to dissociation of the acto-myosin complex, is very similar in the two isoforms and depends linearly on ATP concentration. The finding of a second mechanical event in the working stroke of skeletal muscle myosin provides the molecular basis for a simple model of actomyosin interaction. This model can account for the variation, in different fiber types, of the rate of the cross-bridge cycle and provides a common scheme for the chemo-mechanical transduction within the myosin family.     

Catalytic consequences of oligomeric organization: kinetic evidence for "tethered" acto-heavy meromyosin at low ATP concentrations.

The influence of the supramolecular organization of myosin on its ATPase activity was investigated at a range of ATP concentrations, using as a model system subfragment 1 (S1) and heavy meromyosin (HMM), which are respectively monomeric and dimeric proteolytic fragments of myosin. At low ATP levels in the presence of a molar excess of actin, dimeric HMM showed an increased rate of ATP hydrolysis relative to that for monomeric S1. This increased ATPase for HMM was inhibited by high concentrations of ATP, which reduced the acto-HMM ATPase rate to the lower level of acto-S1. This observation is consistent with the rapid ATP hydrolysis of acto-HMM at low ATP being due to rapid product release from a "tethered" acto-HMM species, which has product bound to one head group while the other head group remains bound to actin. At high concentrations of ATP, ATP binds to both head groups, resulting in net dissociation of HMM from actin. This model is supported by 18O exchange data. Acto-HMM hydrolyzed ATP with extensive exchange of water oxygens into Pi at high ATP levels, but not at low ATP levels. Acto-S1 exhibited extensive exchange at both high and low ATP levels. This result is consistent with rapid product release from a tethered acto-HMM intermediate at low ATP.     

On the mechanism of actomyosin ATPase from fast muscle.

The labeled inorganic phosphate formed by enzymatic hydrolysis of [gamma-18O]ATP in normal water was derivatized to trimethyl phosphate and analyzed for the proportions of [18O3]Pi, [18O2]Pi, [18O1]Pi, and [18O0]Pi. The proportions observed were correlated with the kinetics of intermediate exchange by using a kinetic relationship in which it is assumed that binding of ATP and subsequent release of products are irreversible. Actomyosin and acto-heavy meromyosin catalyze intermediate exchange at a mean rate that is more than 1 order of magnitude slower than that predicted by rapid kinetic studies or implied by the essentially complete intermediate exchange observed with myosin alone. The reason for the slow apparent exchange is that there are two ATPase activities with different exchange properties. The effect of varying heavy meromyosin concentrations at a constant actin concentration shows that the two activities are interrelated and suggests further that one is due to ATP hydrolysis by the undissociated actomyosin crossbridge.     

Functional significance of myofilament protein oxidation.

During muscle contraction, a molecular interaction takes placebetween the myofilament proteins actin and myosin, which istriggered by a rise in intracellular calcium and is driven bythe energy from ATP hydrolysis. The tropomyosin–troponincomplex inhibits the actin–myosin interaction at low intracellular-freecalcium. This inhibition is released when intracellular-freecalcium increases and calcium binding to troponin C takes placeresulting in a conformational change of the troponin–tropomyosincomplex. Movement     

Cardiac myosin binding protein-C modulates actomyosin binding and kinetics in the in vitro motility assay

The modulatory role of whole cardiac myosin binding protein-C (cMyBP-C) on myosin force and motion generation was assessed in an in vitro motility assay. The presence of cMyBP-C at an approximate molar ratio of cMyBP-C to whole myosin of 1:2, resulted in a 25% reduction in thin filament velocity (P < 0.002) with no effect on relative isometric force under maximally activated conditions (pCa 5). Cardiac MyBP-C was capable of inhibiting actin filament velocity in a concentration-dependent manner using either whole myosin, HMM or S1, indicating that the cMyBP-C does not have to bind to myosin LMM or S2 subdomains to exert its effect. The reduction in velocity by cMyBP-C was independent of changes in ionic strength or excess inorganic phosphate. Co-sedimentation experiments demonstrated S1 binding to actin is reduced as a function of cMyBP-C concentration in the presence of ATP. In contrast, S1 avidly bound to actin in the absence of ATP and limited cMyBP-C binding, indicating that cMyBP-C and S1 compete for actin binding in an ATP-dependent fashion. However, based on the relationship between thin filament velocity and filament length, the cMyBP-C induced reduction in velocity was independent of the number of cross-bridges interacting with the thin filament. In conclusion, the effects of cMyBP-C on velocity and force at both maximal and submaximal activation demonstrate that cMyBP-C does not solely act as a tether between the myosin S2 and LMM subdomains but likely affects both the kinetics and recruitment of myosin cross-bridges through its direct interaction with actin and/or myosin head.