X-ray interference studies of crossbridge action in muscle contraction: evidence from muscles during steady shortening

During normal muscle shortening, the myosin heads must undergo many cycles of interaction with the actin filaments sliding past them. It is important to determine what range of configurations is found under these circumstances, and, in terms of the tilting lever arm model, what range of orientations the lever arms undergo. We have studied this using the X-ray interference technique described in the previous article, focusing mainly on the changes in the first order meridional reflection (M3) as compared to isometric. The change in ratio of the heights of the interference peaks indicates how far the mean lever arm angle has moved towards the end of the working stroke; the total intensity change depends on the angle change, on the number of heads now attached at any one time, and on the dispersion of lever arm angles. The latter provides a measure of the distance over which myosin heads remain attached to actin as they go through their working strokes. Surprisingly, the mean position of the attached heads moves only about 1 nm inwards (towards the center of the A-band) at low velocity shortening (around 0.9 T0): their dispersion changes very little. This shows that they must be detaching very early in the working stroke. However, at loads around 0.5 T0, the mean lever arm angle is about half way towards the end of the working stroke, and the dispersion of lever arm angles (with a uniform dispersion) is such as to distribute the heads throughout the whole of the working stroke. At higher velocities of shortening (at 0.3 T0), the mean position shifts further towards the end of the stroke, and the dispersion increases further. The details of the measurements, together with other data on muscle indicate that the force-generating mechanism within the myosin heads must have some unexpected properties.     

Myosin S2 Origins Track Evolution of Strong Binding on Actin by Azimuthal Rolling of Motor Domain

Myosin crystal structures have given rise to the swinging lever arm hypothesis, which predicts a large axial tilt of the lever arm domain during the actin-attached working stroke. Previous work imaging the working stroke in actively contracting, fast-frozen Lethocerus muscle confirmed the axial tilt; but strongly bound myosin heads also showed an unexpected azimuthal slew of the lever arm around the thin filament axis, which was not predicted from known crystal structures. We hypothesized that an azimuthal reorientation of the myosin motor domain on actin during the weak-binding to strong-binding transition could explain the lever arm slew provided that myosin’s α-helical coiled-coil subfragment 2 (S2) domain emerged from the thick filament backbone at a particular location. However, previous studies did not adequately resolve the S2 domain. Here we used electron tomography of rigor muscle swollen by low ionic strength to pull S2 clear of the thick filament backbone, thereby revealing the azimuth of its point of origin. The results show that the azimuth of S2 origins of those rigor myosin heads, bound to the actin target zone of actively contracting muscle, originate from a restricted region of the thick filament. This requires an azimuthal reorientation of the motor domain on actin during the weak to strong transition.     

X-ray interference studies of crossbridge action in muscle contraction: evidence from quick releases

We have used a high-resolution small angle X-ray scattering system, together with a high-performance CCD camera, on the BioCAT beamline at the APS synchrotron radiation facility at the Argonne National Laboratory, to study X-ray interference effects in the meridional reflections generated by the arrays of myosin crossbridges in contracting muscle. These give information about axial movements of the myosin heads during contraction with sub-nanometer resolution. Using whole intact muscle preparations (frog sartorius) we have been able to record the detailed behavior of M3 (the first order meridional reflection from the myosin crossbridges, at 14.56 nm) at each of a number of quick releases of increasing magnitude, on the same specimen, and at the same time make similar measurements on higher order myosin meridional reflections, particularly M6. The latter provides information about the dispersion of lever arm angles of the actin-attached myosin heads. The observations show that in isometric contraction the lever arm angles are dispersed through +/- 20-25 degrees on either side of a mean orientation that is about 60 degrees away from their orientation at the end of the working stroke: and that they move towards that orientation in synchronized fashion, with constant dispersion, during quick releases. The relationship between the shift in the interference fringes (which measures the shift of the myosin heads scattering mass towards the center of the sarcomere, and the changes in the total intensity of the reflections, which measures the changes in the axial profile of the heads, is consistent with the tilting lever arm mechanism of muscle contraction. Significant fixed contributions to the meridional reflections come from unattached myosin heads and from backbone components of the myosin filaments, and the interaction of these with the contributions from actin-attached myosin heads determines the behavior of these reflections.     

Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber

Muscle contraction results from an attachment–detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.     

Cardiomyopathy mutations impact the actin-activated power stroke of human cardiac myosin

Cardiac muscle contraction is driven by the molecular motor myosin, which uses the energy from ATP hydrolysis to generate a power stroke when interacting with actin filaments, although it is unclear how this mechanism is impaired by mutations in myosin that can lead to heart failure. We have applied a fluorescence resonance energy transfer (FRET) strategy to investigate structural changes in the lever arm domain of human β-cardiac myosin subfragment 1 (M2β-S1). We exchanged the human ventricular regulatory light chain labeled at a single cysteine (V105C) with Alexa 488 onto M2β-S1, which served as a donor for Cy3ATP bound to the active site. We monitored the FRET signal during the actin-activated product release steps using transient kinetic measurements. We propose that the fast phase measured with our FRET probes represents the macroscopic rate constant associated with actin-activated rotation of the lever arm during the power stroke in M2β-S1. Our results demonstrated M2β-S1 has a slower actin-activated power stroke compared with fast skeletal muscle myosin and myosin V. Measurements at different temperatures comparing the rate constants of the actin-activated power stroke and phosphate release are consistent with a model in which the power stroke occurs before phosphate release and the two steps are tightly coupled. We suggest that the actin-activated power stroke is highly reversible but followed by a highly irreversible phosphate release step in the absence of load and free phosphate. We demonstrated that hypertrophic cardiomyopathy (R723G)- and dilated cardiomyopathy (F764L)-associated mutations both reduced actin activation of the power stroke in M2β-S1. We also demonstrate that both mutations alter in vitro actin gliding in the presence and absence of load. Thus, examining the structural kinetics of the power stroke in M2β-S1 has revealed critical mutation-associated defects in the myosin ATPase pathway, suggesting these measurements will be extremely important for establishing structure-based mechanisms of contractile dysfunction.     

Atomic structure of scallop myosin subfragment S1 complexed with MgADP: a novel conformation of the myosin head.

The crystal structure of a proteolytic subfragment from scallop striated muscle myosin, complexed with MgADP, has been solved at 2.5 A resolution and reveals an unusual conformation of the myosin head. The converter and the lever arm are in very different positions from those in either the pre-power stroke or near-rigor state structures; moreover, in contrast to these structures, the SH1 helix is seen to be unwound. Here we compare the overall organization of the myosin head in these three states and show how the conformation of three flexible "joints" produces rearrangements of the four major subdomains in the myosin head with different bound nucleotides. We believe that this novel structure represents one of the prehydrolysis ("ATP") states of the contractile cycle in which the myosin heads stay detached from actin.     

Direct modeling of x-ray diffraction pattern from skeletal muscle in rigor

Available high-resolution structures of F-actin, myosin subfragment 1 (S1), and their complex, actin-S1, were used to calculate a 2D x-ray diffraction pattern from skeletal muscle in rigor. Actin sites occupied by myosin heads were chosen using a "principle of minimal elastic distortion energy" so that the 3D actin labeling pattern in the A-band of a sarcomere was determined by a single parameter. Computer calculations demonstrate that the total off-meridional intensity of a layer line does not depend on disorder of the filament lattice. The intensity of the first actin layer A1 line is independent of tilting of the "lever arm" region of the myosin heads. Myosin-based modulation of actin labeling pattern leads not only to the appearance of the myosin and "beating" actin-myosin layer lines in rigor diffraction patterns, but also to changes in the intensities of some actin layer lines compared to random labeling. Results of the modeling were compared to experimental data obtained from small bundles of rabbit muscle fibers. A good fit of the data was obtained without recourse to global parameter search. The approach developed here provides a background for quantitative interpretation of the x-ray diffraction data from contracting muscle and understanding structural changes underlying muscle contraction.     

Probing Muscle Myosin Motor Action: X-Ray (M3 and M6) Interference Measurements Report Motor Domain Not Lever Arm Movement

The key question in understanding how force and movement are produced in muscle concerns the nature of the cyclic interaction of myosin molecules with actin filaments. The lever arm of the globular head of each myosin molecule is thought in some way to swing axially on the actin-attached motor domain, thus propelling the actin filament past the myosin filament. Recent X-ray diffraction studies of vertebrate muscle, especially those involving the analysis of interference effects between myosin head arrays in the two halves of the thick filaments, have been claimed to prove that the lever arm moves at the same time as the sliding of actin and myosin filaments in response to muscle length or force steps. It was suggested that the sliding of myosin and actin filaments, the level of force produced and the lever arm angle are all directly coupled and that other models of lever arm movement will not fit the X-ray data. Here, we show that, in addition to interference across the A-band, which must be occurring, the observed meridional M3 and M6 X-ray intensity changes can all be explained very well by the changing diffraction effects during filament sliding caused by heads stereospecifically attached to actin moving axially relative to a population of detached or non-stereospecifically attached heads that remain fixed in position relative to the myosin filament backbone. Crucially, and contrary to previous interpretations, the X-ray interference results provide little direct information about the position of the myosin head lever arm; they are, in fact, reporting relative motor domain movements. The implications of the new interpretation are briefly assessed.     

Frequency-dependent distortion of meridional intensity changes during sinusoidal length oscillations of activated skeletal muscle

Bundles of intact, tetanized skeletal muscle fibers from Rana temporaria were subjected to sinusoidal length oscillations in the frequency domain 100 Hz to 3 kHz while measuring force and sarcomere length. Simultaneously, intensity of the third-order x-ray reflection of the axial myosin unit cell (I(M3)) was measured using synchrotron radiation. At oscillation frequencies <1 kHz, I(M3) was distorted during the shortening phase of the sinusoid (i.e., where bundle length was less than rest length). Otherwise, during the stretch phase of oscillations at all frequencies, during the shortening phase of oscillations above 1 kHz, and for bundles in the rigor state, I(M3) was approximately sinusoidal in form. Mean I(M3) during oscillations was reduced by 20% compared to the isometric value, suggesting a possible change in S1 disposition during oscillations. However, the amplitude of length change required to produce distortion (estimated from the phase angle at which distortion was first evident) corresponded to that of a step release sufficient to reach the maximum I(M3), indicating a mean S1 disposition during oscillations close to that during an isometric tetanus. The mechanical properties of the bundle during oscillations were also consistent with an unaltered S1 disposition during oscillations.     

Recent X-ray diffraction studies of muscle contraction and their implications

Recent studies on the interference fringes in the myosin meridional reflections provide a new source of structural information on cross-bridge movement during mechanical transients and steady shortening. Many observations can be interpreted satisfactorily by the tilting lever-arm model, with some assumptions, including the presence of fixed repeating structures contributing to the M3 and higher-order meridional reflections. In isometric contraction, the lever arms are oriented near the start of the working stroke, with a dispersion of ca+/-20-25 degrees . Upon a rapid release of 10-12 nm, they move to the end of the stroke, with a well-known T2 delay of 1-2 ms. This delay must represent additional processes, which have to occur even in tension-generating heads, or activation of attached heads, which initially do not develop force. Surprisingly, in muscles shortening at moderate loads (0.5-0.6 P0), the mean position of the heads moves only 2-3 nm closer to the M-line than in the isometric case, reminiscent of the Piazzesi-Lombardi model.     

The "roll and lock" mechanism of force generation in muscle

Muscle force results from the interaction of the globular heads of myosin-II with actin filaments. We studied the structure-function relationship in the myosin motor in contracting muscle fibers by using temperature jumps or length steps combined with time-resolved, low-angle X-ray diffraction. Both perturbations induced simultaneous changes in the active muscle force and in the extent of labeling of the actin helix by stereo-specifically bound myosin heads at a constant total number of attached heads. The generally accepted hypothesis assumes that muscle force is generated solely by tilting of the lever arm, or the light chain domain of the myosin head, about its catalytic domain firmly bound to actin. Data obtained suggest an additional force-generating step: the "roll and lock" transition of catalytic domains of non-stereo-specifically attached heads to a stereo-specifically bound state. A model based on this scheme is described to quantitatively explain the data.     

Five Alternative Myosin Converter Domains Influence Muscle Power,Stretch Activation,and Kinetics

Muscles have evolved to power a wide variety of movements. A protein component critical to varying power generation is the myosin isoform present in the muscle. However, how functional variation in muscle arises from myosin structure is not well understood. We studied the influence of the converter, a myosin structural region at the junction of the lever arm and catalytic domain, using Drosophila because its single myosin heavy chain gene expresses five alternative converter versions (11a–e). We created five transgenic fly lines, each forced to express one of the converter versions in their indirect flight muscle (IFM) fibers. Electron microscopy showed that the converter exchanges did not alter muscle ultrastructure. The four lines expressing converter versions (11b–e) other than the native IFM 11a converter displayed decreased flight ability. IFM fibers expressing converters normally found in the adult stage muscles generated up to 2.8-fold more power and displayed up to 2.2-fold faster muscle kinetics than fibers with converters found in the embryonic and larval stage muscles. Small changes to stretch-activated force generation only played a minor role in altering power output of IFM. Muscle apparent rate constants, derived from sinusoidal analysis of the chimeric converter fibers, showed a strong positive correlation between optimal muscle oscillation frequency and myosin attachment kinetics to actin, and an inverse correlation with detachment related cross-bridge kinetics. This suggests the myosin converter alters at least two rate constants of the cross-bridge cycle with changes to attachment and power stroke related kinetics having the most influence on setting muscle oscillatory power kinetics.     

Mutating the Converter-Relay Interface of Drosophila Myosin Perturbs ATPase Activity, Actin Motility, Myofibril Stability and Flight Ability

We used an integrative approach to probe the significance of the interaction between the relay loop and converter domain of the myosin molecular motor from Drosophila melanogaster indirect flight muscle. During the myosin mechanochemical cycle, ATP-induced twisting of the relay loop is hypothesized to reposition the converter, resulting in cocking of the contiguous lever arm into the pre-power stroke configuration. The subsequent movement of the lever arm through its power stroke generates muscle contraction by causing myosin heads to pull on actin filaments. We generated a transgenic line expressing myosin with a mutation in the converter domain (R759E) at a site of relay loop interaction. Molecular modeling suggests that the interface between the relay loop and converter domain of R759E myosin would be significantly disrupted during the mechanochemical cycle. The mutation depressed calcium as well as basal and actin-activated MgATPase (V_max) by ∼ 60% compared to wild-type myosin, but there is no change in apparent actin affinity (K_m). While ATP or AMP-PNP (adenylyl-imidodiphosphate) binding to wild-type myosin subfragment-1 enhanced tryptophan fluorescence by ∼ 15% or ∼ 8%, respectively, enhancement does not occur in the mutant. This suggests that the mutation reduces lever arm movement. The mutation decreases in vitro motility of actin filaments by ∼ 35%. Mutant pupal indirect flight muscles display normal myofibril assembly, myofibril shape, and double-hexagonal arrangement of thick and thin filaments. Two-day-old fibers have occasional “cracking” of the crystal-like array of myofilaments. Fibers from 1-week-old adults show more severe cracking and frayed myofibrils with some disruption of the myofilament lattice. Flight ability is reduced in 2-day-old flies compared to wild-type controls, with no upward mobility but some horizontal flight. In 1-week-old adults, flight capability is lost. Thus, altered myosin function permits myofibril assembly, but results in a progressive disruption of the myofilament lattice and flight ability. We conclude that R759 in the myosin converter domain is essential for normal ATPase activity, in vitro motility and locomotion. Our results provide the first mutational evidence that intramolecular signaling between the relay loop and converter domain is critical for myosin function both in vitro and in muscle.     

Conformation of myosin interdomain interactions during contraction: deductions from muscle fibers using polarized fluorescence

Myosin cross-bridge subfragment 1 (S1) is the ATP catalyzing motor protein in muscle. It consists of three domains that catalyze ATP and bind actin (catalytic), conduct energy transduction (converter), and transport the load (lever arm). Force development during contraction is thought to result from rotary lever arm movement with the cross-bridge attached to actin. To elucidate cross-bridge structure during force development, two crystal structures of S1 were extrapolated to working "in solution" or oriented "in tissue" forms, using structure-sensitive optical spectroscopic signals from two extrinsic probes. The probes were located at two interfaces containing the catalytic, converter, and lever arm domains of S1. Observed signals included circular dichroism (CD) and absorption originating from S1 in solution in the presence and absence of actin and fluorescence polarization from cross-bridges in muscle fibers. Theoretical signals were calculated from S1 crystal structure models perturbed with lever arm movement from swiveling at three conserved glycines, 699, 703, and 710 (chicken skeletal myosin numbering). Best agreement between the computed and observed signals gave structures showing that actin binding to S1 causes movement of the lever arm. A three-state model of S1 conformation during contraction consists of three actin-bound cross-bridge states observed from muscle fibers in isometric contraction, in the presence of MgADP, and in rigor. Structures best representing these states show that most of the lever arm rotation occurs between isometric contraction and the MgADP states, i.e., during phosphate release. Smaller but significant lever arm rotation occurs with ADP dissociation. Structural changes within the S1 interfaces studied are discussed in the accompanying paper [Burghardt et al. (2001) Biochemistry 40, 4834-4843].     

Chemical Decoupling of ATPase Activation and Force Production from the Contractile Cycle in Myosin by Steric Hindrance of Lever-Arm Movement

The myosin motor protein generates force in muscle by hydrolyzing Adenosine 5′-triphosphate (ATP) while interacting transiently with actin. Structural evidence suggests the myosin globular head (subfragment 1 or S1) is articulated with semi-rigid catalytic and lever-arm domains joined by a flexible converter domain. According to the prevailing hypothesis for energy transduction, ATP binding and hydrolysis in the catalytic domain drives the relative movement of the lever arm. Actin binding and reversal of the lever-arm movement (power stroke) applies force to actin. These domains interface at the reactive lysine, Lys84, where trinitrophenylation (TNP-Lys84-S1) was observed in this work to block actin activation of myosin ATPase and in vitro sliding of actin over myosin. TNP-Lys84-S1's properties and interactions with actin were examined to determine how trinitrophenylation causes these effects. Weak and strong actin binding, the rate of mantADP release from actomyosin, and actomyosin dissociation by ATP were equivalent in TNP-Lys84-S1 and native S1. Molecular dynamics calculations indicate that lever-arm movement inhibition during ATP hydrolysis and the power stroke is caused by steric clashes between TNP and the converter or lever-arm domains. Together these findings suggest that TNP uncouples actin activation of myosin ATPase and the power stroke from other steps in the contraction cycle by inhibiting the converter and lever-arm domain movements.     

X-ray diffraction studies of the contractile mechanism in single muscle fibres

The molecular mechanism of muscle contraction was investigated in intact muscle fibres by X-ray diffraction. Changes in the intensities of the axial X-ray reflections produced by imposing rapid changes in fibre length establish the average conformation of the myosin heads during active isometric contraction, and show that the heads tilt during the elastic response to a change in fibre length and during the elementary force generating process: the working stroke. X-ray interference between the two arrays of myosin heads in each filament allows the axial motions of the heads following a sudden drop in force from the isometric level to be measured in situ with unprecedented precision. At low load, the average working stroke is 12 nm, which is consistent with crystallographic studies. The working stroke is smaller and slower at a higher load. The compliance of the actin and myosin filaments was also determined from the change in the axial spacings of the X-ray reflections following a force step, and shown to be responsible for most of the sarcomere compliance. The mechanical properties of the sarcomere depend on both the motor actions of the myosin heads and the compliance of the myosin and actin filaments.     

Cooperativity of myosin molecules through strain-dependent chemistry

There is mounting evidence that the myosin head domain contains a lever arm which amplifies small structural changes that occur at the nucleotide-binding site. The mechanical work associated with movement of the lever affects the rates at which the products of ATP hydrolysis are released. During muscle contraction, this strain-dependent chemistry leads to cooperativity of the myosin molecules within a thick filament. Two aspects of cooperative action are discussed, in the context of a simple stochastic model. (i) A modest motion of the lever arm on ADP release can serve to regulate the fraction of myosin bound to the thin filament, in order to recruit more heads at higher loads. (ii) If the lever swings through a large angle when phosphate is released, the chemical cycles of the myosin molecules can be synchronized at high loads. This leads to stepwise sliding of the filaments and suggests that the isometric condition is not a steady state.     

The conformation of myosin head domains in rigor muscle determined by X-ray interference

In the absence of adenosine triphosphate, the head domains of myosin cross-bridges in muscle bind to actin filaments in a rigor conformation that is expected to mimic that following the working stroke during active contraction. We used x-ray interference between the two head arrays in opposite halves of each myosin filament to determine the rigor head conformation in single fibers from frog skeletal muscle. During isometric contraction (force T(0)), the interference effect splits the M3 x-ray reflection from the axial repeat of the heads into two peaks with relative intensity (higher angle/lower angle peak) 0.76. In demembranated fibers in rigor at low force (<0.05 T(0)), the relative intensity was 4.0, showing that the center of mass of the heads had moved 4.5 nm closer to the midpoint of the myosin filament. When rigor fibers were stretched, increasing the force to 0.55 T(0), the heads' center of mass moved back by 1.1-1.6 nm. These motions can be explained by tilting of the light chain domain of the head so that the mean angle between the Cys(707)-Lys(843) vector and the filament axis increases by approximately 36 degrees between isometric contraction and low-force rigor, and decreases by 7-10 degrees when the rigor fiber is stretched to 0.55 T(0).     

CaATP as a substrate to investigate the myosin lever arm hypothesis of force generation

In an effort to test the lever arm model of force generation, the effects of replacing magnesium with calcium as the ATP-chelated divalent cation were determined for several myosin and actomyosin reactions. The isometric force produced by glycerinated muscle fibers when CaATP is the substrate is 20% of the value obtained with MgATP. For myosin subfragment 1 (S1), the degree of lever arm rotation, determined using transient electric birefringence to measure rates of rotational Brownian motion in solution, is not significantly changed when calcium replaces magnesium in an S1-ADP-vanadate complex. Actin activates S1 CaATPase activity, although less than it does MgATPase activity. The increase in actin affinity when S1. CaADP. P(i) is converted to S1. CaADP is somewhat greater than it is for the magnesium case. The ionic strength dependence of actin binding indicates that the change in apparent electrostatic charge at the acto-S1 interface for the S1. CaADP. P(i) to S1. CaADP step is similar to the change when magnesium is bound. In general, CaATP is an inferior substrate compared to MgATP, but all the data are consistent with force production by a lever arm mechanism for both substrates. Possible reasons for the reduced magnitude of force when CaATP is the substrate are discussed.     

Force generation by recombinant myosin heads trapped between two functionalized surfaces

Fluorescence resonance energy transfer measurements have revealed that the lever-arm domain of myosin swings when it hydrolyzes Mg-ATP. It is generally accepted that this swing of the lever arm of myosin is the molecular basis of force generation. On the other hand, the possibility that the force might be generated at the interface between actin and myosin cannot be ignored. However, there is a third possibility, namely, that myosin itself generates force without actin. Thus, using recombinant subfragment 1 molecules of Dictyostelium myosin II that were trapped between two functionalized surfaces of a surface-force apparatus, we determined whether myosin itself could actually generate force. Here, we report that, despite the absence of actin, myosin heads themselves have a capacity to generate a force (at least ~0.2 pN/molecule) that is coupled to the structural changes. Although the role of actin should not be neglected because muscle physiologically shortens as a result of the interaction between actin and myosin, in this work the focus is on the question of whether the catalytic domain of myosin has the capacity to generate force.