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.     

Different degrees of lever arm rotation control myosin step size

Myosins are actin-based motors that are generally believed to move by amplifying small structural changes in the core motor domain via a lever arm rotation of the light chain binding domain. However, the lack of a quantitative agreement between observed step sizes and the length of the proposed lever arms from different myosins challenges this view. We analyzed the step size of rat myosin 1d (Myo1d) and surprisingly found that this myosin takes unexpectedly large steps in comparison to other myosins. Engineering the length of the light chain binding domain of rat Myo1d resulted in a linear increase of step size in relation to the putative lever arm length, indicative of a lever arm rotation of the light chain binding domain. The extrapolated pivoting point resided in the same region of the rat Myo1d head domain as in conventional myosins. Therefore, rat Myo1d achieves its larger working stroke by a large calculated approximately 90 degrees rotation of the light chain binding domain. These results demonstrate that differences in myosin step sizes are not only controlled by lever arm length, but also by substantial differences in the degree of lever arm rotation.     

Ultra-fastChara myosin: A test case for the swinging lever arm model for force production by myosin

Recent breakthroughs and technological improvements are rapidly generating evidence supporting the “swinging lever arm model” for force production by myosin. Unlike previous models, this model posits that the globular domain of the myosin motor binds to actin with a constant orientation during force generation. Movement of the neck domain of the motor is hypothesized to occur relative to the globular domain much like a lever arm. This intramolecular conformational change drives the movement of the bound actin. The swinging lever arm model is supported by or consistent with a large number of experimental data obtained with skeletal muscle or slime mold myosins, all of which move actin filaments at rates between 1 and 10 μm/sin vitro. Recently myosin was purified, fromChara internodal cells.In vitro the purifiedChara myosin moves actin filaments at rates one order of magnitude faster than the “fast” skeletal muscle myosin. While this ultra fast movement is not necessarily inconsistent with the swinging lever arm model, one or more specific facets of the motor must be altered in theChara motor in order to accommodate such rapid movement. These characteristics are experimentally testable, thus the ultra fast movement byChara myosin represents a powerful and compelling test of the swinging lever arm model.     

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.     

Trinitrophenylated reactive lysine residue in myosin detects lever arm movement during the consecutive steps of ATP hydrolysis.

Trinitrophenylation of the reactive lysine (Lys84) in skeletal myosin subfragment 1 (S1) introduces a chiral probe (TNP) into an interface of the catalytic and lever arm domains of S1 [Muhlrad (1977) Biochim. Biophys. Acta 493, 154-166]. Characteristics of the TNP absorption and circular dichroism (CD) spectra in TNP-modified S1 (TNP-Lys84-S1), and the Lys84 trinitrophenylation rate in native S1, indicate a one-to-one correspondence between ATPase transients and trapped phosphate analogues. Phosphate analogue-induced structures of TNP-Lys84-S1 were modeled using the crystallographic coordinates of S1 [Rayment et al. (1993) Science 261, 50-58] with swivels at Gly699 and Gly710 to approximate conformational changes during ATPase. The CD and absorption spectral characteristics of the model structures were compared to those observed for analogue-induced structures. The model calculations, first tested on a trinitrophenylated hexapeptide with known structure, were applied to TNP-Lys84-S1. They showed that ATP binding initiates swiveling at Gly699 and that swiveling at both Gly710 and Gly699 accompanied ATP splitting just prior to product release. The computed lever arm trajectory during ATPase suggests (i) a plausible mechanism for the nucleotide-induced inhibition of Lys84 trinitrophenylation, and (ii) trinitrophenylation-induced changes in S1 Mg2+- and K+-EDTA ATPase are from collision of the lever arm with TNP at Lys84. TNP is a site-specific structural perturbant of S1 and a chiral reporter group for the effect of Lys84 modification on dynamic S1 structure. As such, TNP-Lys84-S1 is equivalent to a genetically engineered mutant with intrinsic sensitivity to structure local to the modified residue.     

Selective perturbation of the myosin recovery stroke by point mutations at the base of the lever arm affects ATP hydrolysis and phosphate release

After ATP binding the myosin head undergoes a large structural rearrangement called the recovery stroke. This transition brings catalytic residues into place to enable ATP hydrolysis, and at the same time it causes a swing of the myosin lever arm into a primed state, which is a prerequisite for the power stroke. By introducing point mutations into a subdomain interface at the base of the myosin lever arm at positions Lys(84) and Arg(704), we caused modulatory changes in the equilibrium constant of the recovery stroke, which we could accurately resolve using the fluorescence signal of single tryptophan Dictyostelium myosin II constructs. Our results shed light on a novel role of the recovery stroke: fine-tuning of this reversible equilibrium influences the functional properties of myosin through controlling the effective rates of ATP hydrolysis and phosphate release.     

Mutations in the relay loop region result in dominant-negative inhibition of myosin II function in Dictyostelium

Dominant-negative inhibition is a powerful genetic tool for the characterization of gene function in vivo, based on the specific impairment of a gene product by the coexpression of a mutant version of the same gene product. We describe the detailed characterization of two myosin constructs containing either point mutations F487A or F506G in the relay region. Dictyostelium cells transformed with F487A or F506G myosin are unable to undergo processes that require myosin II function, including fruiting-body formation, normal cytokinesis and growth in suspension. Our results show that the dominant-negative inhibition of myosin function is caused by disruption of the communication between active site and lever arm, which blocks motor activity completely, and perturbation of the communication between active site and actin-binding site, leading to an ~100-fold increase in the mutants' affinity for actin in the presence of ATP.     

Detection of the swings of the lever arm of a myosin motor by fluorescence resonance energy transfer of green and blue fluorescent proteins

The "lever-arm" model of a myosin motor predicts that the lever-arm domain in the myosin head tilts and swings against the catalytic domain during ATP hydrolysis, resulting in force generation. To investigate if this "swing" of the lever arm really occurs during the hydrolysis of ATP, we employed fluorescence resonance energy transfer (FRET) between two fluorescent proteins [green (GFP) and blue (BFP)] fused to the N and C termini of the Dictyostelium myosin-motor domain. FRET measurements showed that the C-terminal BFP in the fusion protein first swings against the N-terminal GFP at the isomerization step of the ATP hydrolysis cycle and then swings back at the phosphate-release step. Because the C-terminal BFP mimics the motion of the lever arm, the result indicates that the lever arm swings at the specific steps of the ATP hydrolysis cycle, i.e., at the isomerization and phosphate-release steps. The latter swing may correspond to the power stroke of myosin, while the former may be related to the recovery stroke.     

Myosin VI steps via a hand-over-hand mechanism with its lever arm undergoing fluctuations when attached to actin

Myosin VI is a reverse direction myosin motor that, as a dimer, moves processively on actin with an average center-of-mass movement of approximately 30 nm for each step. We labeled myosin VI with a single fluorophore on either its motor domain or on the distal of two calmodulins (CaMs) located on its putative lever arm. Using a technique called FIONA (fluorescence imaging with one nanometer accuracy), step size was observed with a standard deviation of <1.5 nm, with 0.5-s temporal resolution, and observation times of minutes. Irrespective of probe position, the average step size of a labeled head was approximately 60 nm, strongly supporting a hand-over-hand model of motility and ruling out models in which the unique myosin VI insert comes apart. However, the CaM probe displayed large spatial fluctuations (presence of ATP but not ADP or no nucleotide) around the mean position, whereas the motor domain probe did not. This supports a model of myosin VI motility in which the lever arm is either mechanically uncoupled from the motor domain or is undergoing reversible isomerization for part of its motile cycle on actin.     

The regulatory domain of the myosin head behaves as a rigid lever.

The regulatory domain of the myosin head is believed to serve as a lever arm that amplifies force generated in the catalytic domain and transmits this strain to the thick filament. The lever arm itself either can be passive or may have a more active role storing some of the energy created by hydrolysis of ATP. A structural correlate which might distinguish between these two possibilities (a passive or an active role) is the stiffness of the domain in question. To this effect we have examined the motion of the proximal (ELC) and distal (RLC) subdomains of the regulatory domain in reconstituted myosin filaments. Each subdomain was labeled with a spin label at a unique cysteine residue, Cys-136 of ELC or Cys-154 of mutant RLC, and its mobility was determined using saturation transfer electron paramagnetic resonance spectroscopy. The mobility of the two domains was similar; the effective correlation time (tau(eff)) for ELC was 17 micros and that for RLC was 22 micros. Additionally, following a 2-fold change of the global dynamics of the myosin head, effected by decreasing the interactions with the filament surface (or the other myosin head), the coupling of the intradomain dynamics remained unchanged. These data suggest that the regulatory domain of the myosin head acts as a single mechanically rigid body, consistent with the regulatory domain serving as a passive lever.     

Secretory vesicle transport velocity in living cells depends on the myosin-V lever arm length

Myosins are molecular motors that exert force against actin filaments. One widely conserved myosin class, the myosin-Vs, recruits organelles to polarized sites in animal and fungal cells. However, it has been unclear whether myosin-Vs actively transport organelles, and whether the recently challenged lever arm model developed for muscle myosin applies to myosin-Vs. Here we demonstrate in living, intact yeast that secretory vesicles move rapidly toward their site of exocytosis. The maximal speed varies linearly over a wide range of lever arm lengths genetically engineered into the myosin-V heavy chain encoded by the MYO2 gene. Thus, secretory vesicle polarization is achieved through active transport by a myosin-V, and the motor mechanism is consistent with the lever arm model.     

Experimental and modeling investigation of the mechanism of synaptic vesicles recycling

Under the condition of microelectrode recording and fluorescence microscopy with dye FM 1-43 the research of exo-/endocytosis of synaptic vesicles in motor nerve terminals (NT) of frog cutaneous pectoris and white mice diaphragm muscles during high frequency stimulation (20 imp/s) was carried out. A mathematical modeling allowed us to conclude that the obtained experimental data can be explained in the following framework. Three pools of synaptic vesicles are involved in neurotransmitter release in the frog motor NT. Recovery of these pools is provided by endocytosis of two types: fast endocytosis with limited capacity and slow endocytosis. Fast-reconstructing vesicles refill the mobilization pool and slow endocytosis recovers the reserve pool. Our modeling investigation has revealed in frog NT independent recruiting of reserve and mobilization pools to the neurotransmitter secretion, i.e. this pools work concurrently. Experimental data, obtained on mice preparations, are well described with the framework of two-pools model including single type of endocytosis (fast endocytosis).     

Myosin V movement: lessons from molecular dynamics studies of IQ peptides in the lever arm

Myosin V moves along actin filaments by an arm-over-arm motion, known as the lever mechanism. Each of its arms is composed of six consecutive IQ peptides that bind light chain proteins, such as calmodulin or calmodulin-like proteins. We have employed a multistage approach in order to investigate the mechanochemical structural basis of the movement of myosin V from the budding yeast Saccharomyces cerevisiae. For that purpose, we previously carried out molecular dynamics simulations of the Mlc1p-IQ2 and the Mlc1p-IQ4 protein-peptide complexes, and the present study deals with the structures of the IQ peptides when stripped from the Mlc1p protein. We have found that the crystalline structure of the IQ2 peptide retains a stable rodlike configuration in solution, whereas that of the IQ4 peptide grossly deviates from its X-ray conformation exhibiting an intrinsic tendency to curve and bend. The refolding process of the IQ4 peptide is initially driven by electrostatic interactions followed by nonpolar stabilization. Its bending appears to be affected by the ionic strength, when ionic strength higher than approximately 300 mM suppresses it from flexing. Considering that a poly-IQ sequence is the lever arm of myosin V, we suggest that the arm may harbor a joint, localized within the IQ4 sequence, enabling the elasticity of the neck of myosin V. Given that a poly-IQ sequence is present at the entire class of myosin V and the possibility that the yeast's myosin V molecule can exist either as a nonprocessive monomer or as a processive dimer depending on conditions (Krementsova, E. B., Hodges, A. R., Lu, H., and Trybus, K. M. (2006) J. Biol. Chem. 281, 6079-6086), our observations may account for a general structural feature for the myosins' arm embedded flexibility.     

Rotation of the lever arm of Myosin in contracting skeletal muscle fiber measured by two-photon anisotropy

The rotation of the lever arm of myosin cross-bridges is believed to be responsible for muscle contraction. To resolve details of this rotation, it is necessary to observe a single cross-bridge. It is still impossible to do so in muscle fiber, but it is possible to investigate a small population of cross-bridges by simultaneously activating myosin in a femtoliter volume by rapid release of caged ATP. In earlier work, in which the number of observed cross-bridges was limited to approximately 600 by confocal microscopy, we were able to measure the rates of cross-bridge detachment and rebinding. However, we were unable to resolve the power stroke. We speculated that the reason for this was that the number of observed cross-bridges was too large. In an attempt to decrease this number, we used two-photon microscopy which permitted observation of approximately 1/2 as many cross-bridges as before with the same signal/noise ratio. With the two-photon excitation, the number of cross-bridges was small enough to resolve the beginning of the power stroke. The results indicated that the power stroke begins approximately 170 ms after the rigor cross-bridge first binds ATP.     

Kinetics of structural changes in the relay loop and SH3 domain of myosin

The intrinsic fluorescence of smooth muscle myosin signals conformational changes associated with different catalytic states of the ATPase cycle. To elucidate this relationship, we have examined the pre-steady-state kinetics of nucleotide binding, hydrolysis, and product release in motor domain-essential light chain mutants containing a single endogenous tryptophan, either residue 512 in the rigid relay loop or residue 29 adjacent to the SH3 domain. The intrinsic fluorescence of W512 is sensitive to both nucleotide binding and hydrolysis, and appears to report structural changes at the active site, presumably through a direct connection with switch II. The intrinsic fluorescence of W29 is sensitive to nucleotide binding but not hydrolysis, and does not appear to be tightly linked with structural changes occurring at the active site. We propose that the SH3 domain may be sensitive to conformational changes in the lever arm through contacts with the essential light chain.     

Evidence that the N-terminal region of A1-light chain of myosin interacts directly with the C-terminal region of actin. A proton magnetic resonance study

Earlier 1H-NMR experiments on the myosin subfragment-1 (S1) light chain isoenzymes from rabbit fast muscle, containing either the A1 or the A2 alkali light chains [S1(A1) or S1(A2)], have shown that the 41-residue N-terminal extension of A1, rich in proline, alanine and lysine residues, is freely mobile in solution but that this mobility is constrained in the acto-S1(A1) complex [Prince et al. (1981) Eur. J. Biochem. 121, 213-219]. It is now established that this N-terminal region of the A1-light chain interacts directly with the C-terminal region of actin in the acto-S1(A1) complex. This was shown by covalently labelling the Cys-374 residue of actin with a spin-label and observing the enhanced relaxation this paramagnetic centre induced in the 1H-NMR spectrum of S1(A1). In particular, the signal arising from the -N+(CH3)3 protons of alpha-N-trimethylalanine (Me3Ala) were monitored as this residue is uniquely sited at the N-terminus of the A1 light chain [Henry et al. (1982) FEBS Lett. 144, 11-15]. Experiments using complexes of actin with either the N-terminal 37-residue peptide of A1, S1(A1) or heavy meromyosin indicate that the N-terminal region of A1 is binding in a similar manner to actin in each case, with the N-terminal Me3Ala residue within 1.5 nm of the spin label introduced to Cys-374 of actin. A similar strategy was adopted to show that the Me3Ala residue can also be found close (less than 1.5 nm) to the fast-reacting SH1 thiol group on the S1 heavy chain. These data, together with published work, have been used to suggest a possible organisation for the polypeptide chains in the myosin head.     

Kinetic mechanism of human myosin IIIA

Myosin IIIA is specifically expressed in photoreceptors and cochlea and is important for the phototransduction and hearing processes. In addition, myosin IIIA contains a unique N-terminal kinase domain and C-terminal tail actin-binding motif. We examined the kinetic properties of baculovirus expressed human myosin IIIA containing the kinase, motor, and two IQ domains. The maximum actin-activated ATPase rate is relatively slow (k(cat) = 0.77 +/- 0.08 s(-1)), and high actin concentrations are required to fully activate the ATPase rate (K(ATPase) = 34 +/- 11 microm). However, actin co-sedimentation assays suggest that myosin III has a relatively high steady-state affinity for actin in the presence of ATP (K(actin) approximately 7 microm). The rate of ATP binding to the motor domain is quite slow both in the presence and absence of actin (K(1)k(+2) = 0.020 and 0.001 microm(-1).s(-1), respectively). The rate of actin-activated phosphate release is more than 100-fold faster (85 s(-1)) than the k(cat), whereas ADP release in the presence of actin follows a two-step mechanism (7.0 and 0.6 s(-1)). Thus, our data suggest a transition between two actomyosin-ADP states is the rate-limiting step in the actomyosin III ATPase cycle. Our data also suggest the myosin III motor spends a large fraction of its cycle in an actomyosin ADP state that has an intermediate affinity for actin (K(d) approximately 5 microm). The long lived actomyosin-ADP state may be important for the ability of myosin III to function as a cellular transporter and actin cross-linker in the actin bundles of sensory cells.     

The myosin relay helix to converter interface remains intact throughout the actomyosin ATPase cycle

Crystal structures of the myosin motor domain in the presence of different nucleotides show the lever arm domain in two basic angular states, postulated to represent prestroke and poststroke states, respectively (Rayment, I. (1996) J. Biol. Chem. 271, 15850-15853; Dominguez, R., Freyzon, Y., Trybus, K. M., and Cohen, C. (1998) Cell 94, 559-571). Contact is maintained between two domains, the relay and the converter, in both of these angular states. Therefore it has been proposed by Dominguez et al. (cited above) that this contact is critical for mechanically driving the angular change of the lever arm domain. However, structural information is lacking on whether this contact is maintained throughout the actin-activated myosin ATPase cycle. To test the functional importance of this interdomain contact, we introduced cysteines into the sequence of a "cysteine-light" myosin motor at position 499 on the lower cleft and position 738 on the converter domain (Shih, W. M., Gryczynski, Z., Lakowicz, J. L., and Spudich, J. A. (2000) Cell 102, 683-694). Disulfide cross-linking could be induced. The cross-link had minimal effects on actin binding, ATP-induced actin release, and actin-activated ATPase. These results demonstrate that the relay/converter interface remains intact in the actin strongly bound state of myosin and throughout the entire actin-activated myosin ATPase cycle.