Insight into the mechanism of fast movement of myosin from Chara corallina

Chara myosin, two-headed plant myosin belonging to class XI, slides F-actin at maximally 60 microm s(-1). To elucidate the mechanism of this fast sliding, we extensively investigated its mechanochemical properties. The maximum actin activated ATPase activity, Vmax, was 21.3 s(-1) head(-1) in a solution, but when myosin was immobilized on the surface, its activity was 57.6 s(-1) head(-1) at 2 mg ml(-1) of F-actin. The sliding velocity and the actin activated ATPase activity were greatly inhibited by ADP, suggesting that ADP dissociation was the rate limiting step. With the extensive assay of motility by varying the surface density, the duty ratio of Chara myosin was found to be 0.49-0.44 from velocity measurements and 0.34 from the landing rate analysis. At the surface density of 10 molecules microm(-2), Chara myosin exhibited pivot movement under physiological conditions. Based on the results obtained, we will discuss the sliding mechanism of Chara myosin according to the working stroke model in terms of its physiological aspects. aspects.     

Kinetic mechanism of the fastest motor protein, Chara myosin

Chara corallina class XI myosin is by far the fastest molecular motor. To investigate the molecular mechanism of this fast movement, we performed a kinetic analysis of a recombinant motor domain of Chara myosin. We estimated the time spent in the strongly bound state with actin by measuring rate constants of ADP dissociation from actin.motor domain complex and ATP-induced dissociation of the motor domain from actin. The rate constant of ADP dissociation from acto-motor domain was >2800 s(-1), and the rate constant of ATP-induced dissociation of the motor domain from actin at physiological ATP concentration was 2200 s(-1). From these data, the time spent in the strongly bound state with actin was estimated to be <0.82 ms. This value is the shortest among known values for various myosins and yields the duty ratio of <0.3 with a V(max) value of the actin-activated ATPase activity of 390 s(-1). The addition of the long neck domain of myosin Va to the Chara motor domain largely increased the velocity of the motility without increasing the ATP hydrolysis cycle rate, consistent with the swinging lever model. In addition, this study reveals some striking kinetic features of Chara myosin that are suited for the fast movement: a dramatic acceleration of ADP release by actin (1000-fold) and extremely fast ATP binding rate.     

Kinetic and spectroscopic evidence for three actomyosin:ADP states in smooth muscle

Smooth muscle myosin II undergoes an additional movement of the regulatory domain with ADP release that is not seen with fast skeletal muscle myosin II. In this study, we have examined the interactions of smooth muscle myosin subfragment 1 with ADP to see if this additional movement corresponds to an identifiable state change. These studies indicate that for this myosin:ADP, both the catalytic site and the actin-binding site can each assume one of two conformations. Relatively loose coupling between these two binding sites leads to three discrete actin-associated ADP states. Following an initial, weakly bound state, binding of myosin:ADP to actin shifts the equilibrium toward a mixture of two states that each bind actin strongly but differ in the conformation of their catalytic sites. By contrast, fast myosins, including Dictyostelium myosin II, have reciprocal coupling between the actin- and ADP-binding sites, so that either actin or nucleotide, but not both, can be tightly bound. This uncoupling, which generates a second strongly bound actomyosin ADP state in smooth muscle, would prolong the fraction of the ATPase cycle time that this actomyosin spends in a force-generating conformation and may be central to explaining the physiologic differences between this and other myosins.     

Cooperative actions between myosin heads bring effective functions

A recent study with single molecule measurements has reported that muscle myosin, a molecular motor, stochastically generates multiple steps along an actin filament associated with the hydrolysis of a single ATP molecule [Kitamura, K., Tokunaga, M., Esaki, S., Iwane, A.H., Yanagida, T., 2005. Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro. Biophysics 1, 1-19]. We have built a model reproducing such a stochastic movement of a myosin molecule incorporated with ATPase reaction cycles and demonstrated that the thermal fluctuation was a key for the function of myosin molecules [Esaki, S., Ishii, Y., Yanagida, T., 2003. Model describing the biased Brownian movement of myosin. Proc. Jpn. Acad. 79 (Ser B), 9-14]. The size of the displacement generated during the hydrolysis of single ATP molecules was limited within a half pitch of an actin filament when a single myosin molecules work separately. However, in muscle the size of the displacement has been reported to be greater than 60 nm [Yanagida, T., Arata, T., Oosawa, F., 1985. Sliding distance of actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle. Nature 316, 366-369; Higuchi et al., 1991]. The difference suggests cooperative action between myosin heads in muscle. Here we extended the model built for an isolated myosin head to a system in which myosin heads are aligned in muscle arrangement to understand the cooperativity between heads. The simulation showed that the rotation of the actin filament [Takezawa, Y., Sugimoto, Y., Wakabayashi, K., 1998. Extensibility of the actin and myosin filaments in various states of skeletal muscles as studied by X-ray diffraction. Adv. Exp. Med. Biol. 453, 309-317; Wakabayashi, K., Ueno, Y., Takezawa, Y., Sugimoto, Y., 2001. Muscle contraction mechanism: use of X-ray synchrotron radiation. Nat. Enc. Life Sci. 1-11] associated with the release of ATPase products and binding of ATP as well as interaction between myosin heads allowed the myosin filament to move greater than a half pitch of the actin filament while a single ATP molecule is hydrolyzed. Our model demonstrated that the movement is loosely coupled to the ATPase cycle as observed in muscle.     

Movement of smooth muscle tropomyosin by myosin heads.

It has been proposed that during the activation of muscle contraction the initial binding of myosin heads to the actin thin filament contributes to switching on the thin filament and that this might involve the movement of actin-bound tropomyosin. The movement of smooth muscle tropomyosin on actin was investigated in this work by measuring the change in distance between specific residues on tropomyosin and actin by fluorescence resonance energy transfer (FRET) as a function of myosin head binding to actin. An energy transfer acceptor was attached to Cys374 of actin and a donor to the tropomyosin heterodimer at either Cys36 of the beta-chain or Cys190 of the alpha-chain. FRET changed for the donor at both positions of tropomyosin upon addition of skeletal or smooth muscle myosin heads, indicating a movement of the whole tropomyosin molecule. The changes in FRET were hyperbolic and saturated at about one head per seven actin subunits, indicating that each head cooperatively affects several tropomyosin molecules, presumably via tropomyosin's end-to-end interaction. ATP, which dissociates myosin from actin, completely reversed the changes in FRET induced by heads, whereas in the presence of ADP the effect of heads was the same as in its absence. The results indicate that myosin with and without ADP, intermediates in the myosin ATPase hydrolytic pathway, are effective regulators of tropomyosin position, which might play a role in the regulation of smooth muscle contraction.     

Inhibition of the relative movement of actin and myosin by caldesmon and calponin.

Contractile activity of myosin II in smooth muscle and non-muscle cells requires phosphorylation of myosin by myosin light chain kinase. In addition, these cells have the potential for regulation at the thin filament level by caldesmon and calponin, both of which bind calmodulin. We have investigated this regulation using in vitro motility assays. Caldesmon completely inhibited the movement of actin filaments by either phosphorylated smooth muscle myosin or rabbit skeletal muscle heavy meromyosin. The amount of caldesmon required for inhibition was decreased when tropomyosin is present. Similarly, calponin binding to actin resulted in inhibition of actin filament movement by both smooth muscle myosin and skeletal muscle heavy meromyosin. Tropomyosin had no effect on the amount of calponin needed for inhibition. High concentrations of calmodulin (10 microM) in the presence of calcium completely reversed the inhibition. The nature of the inhibition by the two proteins was markedly different. Increasing caldesmon concentrations resulted in graded inhibition of the movement of actin filaments until complete inhibition of movement was obtained. Calponin inhibited actin sliding in a more "all or none" fashion. As the calponin concentration was increased the number of actin filaments moving was markedly decreased, but the velocity of movement remained near control values.     

Kinetic analysis of Drosophila muscle myosin isoforms suggests a novel mode of mechanochemical coupling

The molecular mechanism of myosin function was addressed by measuring transient kinetic parameters of naturally occurring and chimeric Drosophila muscle myosin isoforms. We assessed the native embryonic isoform, the native indirect flight muscle isoform, and two chimeric isoforms containing converter domains exchanged between the indirect flight muscle and embryonic isoforms. Myosin was purified from the indirect flight muscles of transgenic flies, and S1 was produced by alpha-chymotryptic digestion. Previous studies in vertebrate and scallop myosins have shown a correlation between actin filament velocity in motility assays and cross-bridge detachment rate, specifically the rate of ADP release. In contrast, our study showed no correlation between the detachment rate and actin filament velocity in Drosophila myosin isoforms and further that the converter domain does not significantly influence the biochemical kinetics governing the detachment of myosin from actin. We suggest that evolutionary pressure on a single muscle myosin gene may maintain a fast detachment rate in all isoforms. As a result, the attachment rate and completion of the power stroke or the equilibrium between actin.myosin.ADP states may define actin filament velocity for these myosin isoforms.     

Effect of phosphorylation on the binding of smooth muscle heavy meromyosin X ADP to actin

Relaxation of both smooth and skeletal muscles appears to be caused primarily by inhibition of the step associated with Pi release in the actomyosin ATPase cycle, rather than by a block in the binding of the myosin X ATP and myosin X ADP X Pi complexes to actin. In skeletal muscle, troponin-tropomyosin not only causes marked inhibition of Pi release, but it also markedly inhibits the binding of myosin subfragment-1 X ADP to actin, raising the possibility that the two phenomena are coupled in some way. In the present study we determined whether phosphorylation of smooth muscle heavy meromyosin (HMM) also affects both the binding of HMM X ADP to actin and the Pi release step. This was done by having phosphorylated and unphosphorylated HMM X ADP compete for sites on F-actin. At mu = 30 mM, phosphorylation increased the affinity of the HMM molecule for actin about 12-fold and at mu = 170 mM, there was less than a 3-fold increase in the affinity of HMM. If phosphorylation affects the binding of each head of HMM to the same extent, then phosphorylation caused about a 4- and 2-fold increase in the affinity of each head of HMM for actin at mu = 30 and 170 mM, respectively. In contrast, at both ionic strengths, phosphorylation caused more than 100-fold actin activation of the ATPase activity of smooth muscle HMM. Therefore, the marked activation of Pi release in the acto X HMM ATPase cycle upon phosphorylation of HMM is not accompanied by a comparable increase in the affinity of HMM X ADP for actin. We have also found that phosphorylation increases by only 4-fold the rate of Pi release from HMM alone. These results suggest that in smooth muscle, phosphorylation accelerates the step associated with the release of Pi both in the forward and the reverse direction without correspondingly affecting the binding of myosin X ADP to actin.     

Magnesium regulates ADP dissociation from myosin V

Processivity in myosin V is mediated through the mechanical strain that results when both heads bind strongly to an actin filament, and this strain regulates the timing of ADP release. However, what is not known is which steps that lead to ADP release are affected by this mechanical strain. Answering this question will require determining which of the several potential pathways myosin V takes in the process of ADP release and how actin influences the kinetics of these pathways. We have addressed this issue by examining how magnesium regulates the kinetics of ADP release from myosin V and actomyosin V. Our data support a model in which actin accelerates the release of ADP from myosin V by reducing the magnesium affinity of a myosin V-MgADP intermediate. This is likely a consequence of the structural changes that actin induces in myosin to release phosphate. This effect on magnesium affinity provides a plausible explanation for how mechanical strain can alter this actin-induced acceleration. For actomyosin V, magnesium release follows phosphate release and precedes ADP release. Increasing magnesium concentration to within the physiological range would thus slow both the ATPase activity and the velocity of movement of this motor.     

Vertebrate myosin VIIb is a high duty ratio motor adapted for generating and maintaining tension

Kinetic adaptation of muscle and non-muscle myosins plays a central role in defining the unique cellular functions of these molecular motor enzymes. The unconventional vertebrate class VII myosin, myosin VIIb, is highly expressed in polarized cells and localizes to highly ordered actin filament bundles such as those found in the microvilli of the intestinal brush border and kidney. We have cloned mouse myosin VIIb from a cDNA library, expressed and purified the catalytic motor domain, and characterized its actin-activated ATPase cycle using quantitative equilibrium and kinetic methods. The myosin VIIb steady-state ATPase activity is slow (approximately 1 s(-1)), activated by very low actin filament concentrations (K(ATPase) approximately 0.7 microm), and limited by ADP release from actomyosin. The slow ADP dissociation rate constant generates a long lifetime of the strong binding actomyosin.ADP states. ADP and actin binding is uncoupled, which enables myosin VIIb to remain strongly bound to actin and ADP at very low actin concentrations. In the presence of 2 mm ATP and 2 microm actin, the duty ratio of myosin VIIb is approximately 0.8. The enzymatic properties of actomyosin VIIb are suited for generating and maintaining tension and favor a role for myosin VIIb in anchoring membrane surface receptors to the actin cytoskeleton. Given the high conservation of vertebrate class VII myosins, deafness phenotypes arising from disruption of normal myosin VIIa function are likely to reflect a loss of tension in the stereocilia of inner ear hair cells.     

Some motile properties of fast characean myosin

We improved a motility assay system by using an affinity-purified antibody against the C-terminal globular domain of characean myosin. This improvement allowed us to study the sensitivity to ionic strength or the processivity of characean myosin. The sliding velocity of actin filaments on a characean myosin-coated surface was unaffected by ionic strength. This property is unlike that of skeletal or smooth muscle myosin and suggests that the binding manner of characean myosin to actin is different from that in other muscle myosins. The sliding velocity decreased when the MgADP concentration was raised. The extent of inhibition by MgADP on the motile activity of characean myosin was almost the same as in skeletal muscle or cardiac myosin. The number of sliding filaments on the characean myosin-coated surface decreased drastically with a decrease in the motor density. The motor density required to produce a successful movement of actin filament was about 200 molecules/microm(2). These results suggest that the characean myosin is not a processive motor protein.     

Mechanoenzymatic characterization of human myosin Vb

There are three isoforms of class V myosin in mammals. While myosin Va has been studied well, little is known about the function of other myosin V isoforms (Vb and Vc) at a molecular level. Here we report the mechanoenzymatic function of human myosin Vb (HuM5B) for the first time. Electron microscopic observation showed that HuM5B has a double-headed structure with a long neck like myosin Va. V(max) and K(actin) of the actin-activated ATPase activity of HuM5B were 9.7 +/- 0.4 s(-)(1) and 8.5 +/- 0.1 microM, respectively. K(actin) and K(ATP) of the actin-activated ATPase activity were significantly higher than those of myosin Va. ADP markedly inhibited the ATPase activity. The rate of release of ADP from acto-HuM5B was 12.2 +/- 0.5 s(-)(1), which was comparable to the V(max) of the actin-activated ATPase activity. These results suggest that ADP release is the rate-limiting step for the actin-activated ATPase cycle; thus, HuM5B is a high duty ratio myosin. Consistently, the actin gliding velocity (0.22 +/- 0.03 microm/s) remained constant at a low motor density. The actin filament landing assay revealed that a single HuM5B molecule is sufficient to move the actin filament continuously, indicating that HuM5b is a processive motor.     

Motor function and regulation of myosin X.

Myosin X is a member of the diverse myosin superfamily that is ubiquitously expressed in various mammalian tissues. Although its association with actin in cells has been shown, little is known about its biochemical and mechanoenzymatic function at the molecular level. We expressed bovine myosin X containing the entire head, neck, and coiled-coil domain and purified bovine myosin X in Sf9 cells. The Mg(2+)-ATPase activity of myosin X was significantly activated by actin with low K(ATP). The actin-activated ATPase activity was reduced at Ca(2+) concentrations above pCa 5 in which 1 mol of calmodulin light chain dissociates from the heavy chain. Myosin X translocates F-actin filaments with the velocity of 0.3 microm/s with the direction toward the barbed end. The actin translocating activity was inhibited at concentrations of Ca(2+) at pCa 6 in which no calmodulin dissociation takes place, suggesting that the calmodulin dissociation is not required for the inhibition of the motility. Unlike class V myosin, which shows a high affinity for F-actin in the presence of ATP, the K(actin) of the myosin X ATPase was much higher than that of myosin V. Consistently nearly all actin dissociated from myosin X in the presence of ATP. ADP did not significantly inhibit the actin-activated ATPase activity of myosin X, suggesting that the ADP release step is not rate-limiting. These results suggest that myosin X is a nonprocessive motor. Consistently myosin X failed to support the actin translocation at low density in an in vitro motility assay where myosin V, a processive motor, supports the actin filament movement.     

The biochemical kinetics underlying actin movement generated by one and many skeletal muscle myosin molecules

To better understand how skeletal muscle myosin molecules move actin filaments, we determine the motion-generating biochemistry of a single myosin molecule and study how it scales with the motion-generating biochemistry of an ensemble of myosin molecules. First, by measuring the effects of various ligands (ATP, ADP, and P(i)) on event lifetimes, tau(on), in a laser trap, we determine the biochemical kinetics underlying the stepwise movement of an actin filament generated by a single myosin molecule. Next, by measuring the effects of these same ligands on actin velocities, V, in an in vitro motility assay, we determine the biochemistry underlying the continuous movement of an actin filament generated by an ensemble of myosin molecules. The observed effects of P(i) on single molecule mechanochemistry indicate that motion generation by a single myosin molecule is closely associated with actin-induced P(i) dissociation. We obtain additional evidence for this relationship by measuring changes in single molecule mechanochemistry caused by a smooth muscle HMM mutation that results in a reduced P(i)-release rate. In contrast, we observe that motion generation by an ensemble of myosin molecules is limited by ATP-induced actin dissociation (i.e., V varies as 1/tau(on)) at low [ATP], but deviates from this relationship at high [ATP]. The single-molecule data uniquely provide a direct measure of the fundamental mechanochemistry of the actomyosin ATPase reaction under a minimal load and serve as a clear basis for a model of ensemble motility in which actin-attached myosin molecules impose a load.     

Slip sliding away: load-dependence of velocity generated by skeletal muscle myosin molecules in the laser trap

Skeletal muscle's ability to shorten and lengthen against a load is a fundamental property, presumably reflecting the inherent load-dependence of the myosin molecular motor. Here we report the velocity of a single actin filament translocated by a mini-ensemble of skeletal myosin approximately 8 heads under constant loads up to 15 pN in a laser trap assay. Actin filament velocity decreased with increasing load hyberbolically, with unloaded velocity and stall force differing by a factor of 2 with [ATP] (30 vs. 100 muM). Analysis of actin filament movement revealed that forward motion was punctuated with rapid backward 60-nm slips, with the slip frequency increasing with resistive load. At stall force, myosin-generated forward movement was balanced by backward slips, whereas at loads greater than stall, myosin could no longer sustain forward motion, resulting in negative velocities as in eccentric contractions of whole muscle. Thus, the force-velocity relationship of muscle reflects both the inherent load-dependence of the actomyosin interaction and the balance between forward and reverse motion observed at the molecular level.     

Steady-state force–velocity relation of ATP-dependent sliding between slime mold myosin, arranged on paramyosin filaments, and algal cell actin cables

To investigate characteristics of ATP-dependent sliding of a non-muscle cell myosin, obtained from a cellular slime mold Dictyostelium discoideum, on actin filament, we prepared hybrid thick filaments, in which Dictyostelium myosin was regularly arranged around paramyosin filaments obtained from a molluscan smooth muscle. A single to a few hybrid filaments were attached to a polystyrene bead (diameter, 4.5 μm; specific gravity, 1.5), and the filaments were made to slide on actin filament arrays (actin cables) in the internodal cell of an alga Chara corallina, mounted on the rotor of a centrifuge microscope. The filament-attached bead was observed to move with a constant velocity under a constant external load for many seconds. The steady-state force–velocity relation of Dictyostelium myosin sliding on actin cables was hyperbolic in shape except for large loads ≤0.7–0.8 P₀, being qualitatively similar to that of skeletal muscle fibres, despite a considerable variation in the number of myosin molecules interacting with actin cables. Comparison of the P–V curves between Dictyostelium myosin and muscle myosins sliding on actin cables suggests that the time of attachment to actin in a single attachment–detachment cycle is much longer in Dictyostelium myosin than in muscle myosins.     

Recombinant motor domain constructs of Chara corallina myosin display fast motility and high ATPase activity

The mechanism and structural features that are responsible for the fast motility of Chara corallina myosin (CCM) have not been elucidated, so far. The low yields of native CCM that can be purified to homogeneity were the major reason for this. Here, we describe the expression of recombinant CCM motor domains, which support the fast movement of actin filaments in an in vitro motility assay. A CCM motor domain without light chain binding site moved actin filaments at a velocity of 8.8 microm/s at 30 degrees C and a CCM motor domain with an artificial lever arm consisting of two alpha-actinin repeats moved actin filaments at 16.2 microm/s. Both constructs displayed high actin-activated ATPase activities ( approximately 500 Pi/s/head), which is indicative of a very fast hydrolysis step. Our results provide an excellent system to dissect the specific structural and functional features that distinguish the myosin responsible for fast cytoplasmic streaming.     

Caldesmon freezes the structure of actin filaments during the actomyosin ATPase cycle

Hybrid contractile apparatus was reconstituted in skeletal muscle ghost fibers by incorporation of skeletal muscle myosin subfragment 1 (S1), smooth muscle tropomyosin and caldesmon. The spatial orientation of FITC-phalloidin-labeled actin and IAEDANS-labeled S1 during sequential steps of the acto-S1 ATPase cycle was studied by measurement of polarized fluorescence in the absence or presence of nucleotides conditioning the binding affinity of both proteins. In the fibers devoid of caldesmon addition of nucleotides evoked unidirectional synchronous changes in the orientation of the fluorescent probes attached to F-actin or S1. The results support the suggestion on the multistep rotation of the cross-bridge (myosin head and actin monomers) during the ATPase cycle. The maximal cross-bridge rotation by 7 degrees relative to the fiber axis and the increase in its rigidity by 30% were observed at transition between A**.M**.ADP.Pi (weak binding) and A--.M--.ADP (strong binding) states. When caldesmon was present in the fibers (OFF-state of the thin filament) the unidirectional changes in the orientation of actin monomers and S1 were uncoupled. The tilting of the myosin head and of the actin monomer decreased by 29% and 90%, respectively. It is suggested that in the "closed" position caldesmon "freezes" the actin filament structure and induces the transition of the intermediate state of actomyosin towards the weak-binding states, thereby inhibiting the ATPase activity of the actomyosin.     

Using optical tweezers to relate the chemical and mechanical cross-bridge cycles

In most current models of muscle contraction there are two translational steps, the working stroke, whereby an attached myosin cross-bridge moves relative to the actin filament, and the repriming step, in which the cross-bridge returns to its original orientation. The development of single molecule methods has allowed a more detailed investigation of the relationship of these mechanical steps to the underlying biochemistry. In the normal adenosine triphosphate cycle, myosin.adenosine diphosphate.phosphate (M.ADP.Pi) binds to actin and moves it by ca. 5 nm on average before the formation of the end product, the rigor actomyosin state. All the other product-like intermediate states tested were found to give no net movement indicating that M.ADP.Pi alone binds in a pre-force state.Myosin states with bound, unhydrolysed nucleoside triphosphates also give no net movement, indicating that these must also bind in a post-force conformation and that the repriming, post- to pre-transition during the forward cycle must take place while the myosin is dissociated from actin. These observations fit in well with the structural model in which the working stroke is aligned to the opening of the switch 2 element of the ATPase site.     

The stiffness of skeletal muscle in isometric contraction and rigor: the fraction of myosin heads bound to actin.

Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of myosin cross-bridges in the region of overlap between the myosin filament and the actin filament remains constant, and only the length of the nonoverlapped region of the actin filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the myosin filament compliance is 1.3 nm microm(-1) T0(-1). With these values for filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor.