Myosin V from head to tail.

Myosin V (myoV), a processive cargo transporter, has arguably been the most well-studied unconventional myosin of the past decade. Considerable structural information is available for the motor domain, the IQ motifs with bound calmodulin or light chains, and the cargo-binding globular tail, all of which have been crystallized. The repertoire of adapter proteins that link myoV to a particular cargo is becoming better understood, enabling cellular transport processes to be dissected. MyoV is processive, meaning that it takes many steps on actin filaments without dissociating. Its extended lever arm results in long 36-nm steps, making it ideal for single molecule studies of processive movement. In addition, electron microscopy revealed the structure of the inactive, folded conformation of myoV when it is not transporting cargo. This review provides a background on myoV, and highlights recent discoveries that show why myoV will continue to be an active focus of investigation.

Myosin V exhibits a high duty cycle and large unitary displacement.

Myosin V is a double-headed unconventional myosin that has been implicated in organelle transport. To perform this role, myosin V may have a high duty cycle. To test this hypothesis and understand the properties of this molecule at the molecular level, we used the laser trap and in vitro motility assay to characterize the mechanics of heavy meromyosin-like fragments of myosin V (M5(HMM)) expressed in the Baculovirus system. The relationship between actin filament velocity and the number of interacting M5(HMM) molecules indicates a duty cycle of > or =50%. This high duty cycle would allow actin filament translocation and thus organelle transport by a few M5(HMM) molecules. Single molecule displacement data showed predominantly single step events of 20 nm and an occasional second step to 37 nm. The 20-nm unitary step represents the myosin V working stroke and is independent of the mode of M5(HMM) attachment to the motility surface or light chain content. The large M5(HMM) working stroke is consistent with the myosin V neck acting as a mechanical lever. The second step is characterized by an increased displacement variance, suggesting a model for how the two heads of myosin V function in processive motion.

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.

ADP binding induces an asymmetry between the heads of unphosphorylated myosin.

Light chain phosphorylation is the key event that regulates smooth and non-muscle myosin II ATPase activity. Here we show that both heads of smooth muscle heavy meromyosin (HMM) bind tightly to actin in the absence of nucleotide, irrespective of the state of light chain phosphorylation. In striking contrast, only one of the two heads of unphosphorylated HMM binds to actin in the presence of ADP, and the heads have different affinities for ADP. This asymmetry suggests that phosphorylation alters the mechanical coupling between the heads of HMM. A model that incorporates strain between the two heads is proposed to explain the data, which have implications for how one head of a motor protein can gate the response of the other.

Coiled-coil unwinding at the smooth muscle myosin head-rod junction is required for optimal mechanical performance.

Myosin II has two heads that are joined together by an alpha-helical coiled-coil rod, which can separate in the region adjacent to the head-rod junction (Trybus, K. M. 1994. J. Biol. Chem. 269:20819-20822). To test whether this flexibility at the head-rod junction is important for the mechanical performance of myosin, we used the optical trap to measure the unitary displacements of heavy meromyosin constructs in which a stable coiled-coil sequence derived from the leucine zipper was introduced into the myosin rod. The zipper was positioned either immediately after the heads (0-hep zip) or following 15 heptads of native sequence (15-hep zip). The unitary displacement (d) decreased from d = 9.7 +/- 0.6 nm for wild-type heavy meromyosin (WT HMM) to d = 0.1 +/- 0.3 nm for the 0-hep zip construct (mean +/- SE). Native values were restored in the 15-hep zip construct (d = 7.5 +/- 0.7 nm). We conclude that flexibility at the myosin head-rod junction, which is provided by an unstable coiled-coil region, is essential for optimal mechanical performance.

Two conserved lysines at the 50/20-kDa junction of myosin are necessary for triggering actin activation.

Actin stimulates myosin's activity by inducing structural alterations that correlate with the transition from a weakly to a strongly bound state, during which time inorganic phosphate (P(i)) is released from myosin's active site. The surface loop at the 50/20-kDa junction of myosin (loop 2) is part of the actin interface. Here we demonstrate that elimination of two highly conserved lysines at the C-terminal end of loop 2 specifically blocks the ability of heavy meromyosin to undergo a weak to strong binding transition with actin in the presence of ATP. Removal of these lysines has no effect on strong binding in the absence of nucleotide, on the rate of ADP binding or release, or on the basal ATPase activity. We further show that the 16 amino acids of loop 2 preceding the lysine-rich region are not essential for actin activation, although they do modulate myosin's affinity for actin in the presence of ATP. We conclude that interaction of the conserved lysines with acidic residues in subdomain 1 of actin either triggers a structural change or stabilizes a conformation that is necessary for actin-activated release of P(i) and completion of the ATPase cycle.

Evidence for cleft closure in actomyosin upon ADP release.

Structural insights into the interaction of smooth muscle myosin with actin have been provided by computer-based fitting of crystal structures into three-dimensional reconstructions obtained by electron cryomicroscopy, and by mapping of structural and dynamic changes in the actomyosin complex. The actomyosin structures determined in the presence and absence of MgADP differ significantly from each other, and from all crystallographic structures of unbound myosin. Coupled to a complex movement ( approximately 34 A) of the light chain binding domain upon MgADP release, we observed a approximately 9 degrees rotation of the myosin motor domain relative to the actin filament, and a closure of the cleft that divides the actin binding region of the myosin head. Cleft closure is achieved by a movement of the upper 50 kDa region, while parts of the lower 50 kDa region are stabilized through strong interactions with actin. This model supports a mechanism in which binding of MgATP at the active site opens the cleft and disrupts the interface, thereby releasing myosin from actin.

Regulation of asymmetric smooth muscle myosin II molecules.

The emerging view of smooth/nonmuscle myosin regulation suggests that the attainment of the completely inhibited state requires numerous weak interactions between components of the two heads and the myosin rod. To further examine the nature of the structural requirements for regulation, we engineered smooth muscle heavy meromyosin molecules that contained one complete head and truncations of the second head. These truncations eliminated the motor domain but retained two, one, or no light chains. All constructs contained 37 heptads of rod sequence. None of the truncated constructs displayed complete regulation of both ATPase and motility, reinforcing the idea that interactions between motor domains are necessary for complete regulation. Surprisingly, the rate of ADP release was slowed by regulatory light chain dephosphorylation of the truncated construct that contained all four light chains and one motor domain. These data suggest that there is a second step (ADP release) in the smooth muscle myosin-actin-activated ATPase cycle that is modulated by regulatory light chain phosphorylation. This may be part of the mechanism underlying "latch" in smooth muscle.

The light chain binding domain of expressed smooth muscle heavy meromyosin acts as a mechanical lever.

Structural data led to the proposal that the molecular motor myosin moves actin by a swinging of the light chain binding domain, or "neck." To test the hypothesis that the neck functions as a mechanical lever, smooth muscle heavy meromyosin (HMM) mutants were expressed with shorter or longer necks by either deleting or adding light chain binding sites. The mutant HMMs were characterized kinetically and mechanically, with emphasis on measurements of unitary displacements and forces in the laser trap assay. Two shorter necked constructs had smaller unitary step sizes and moved actin more slowly than WT HMM in the motility assay. A longer necked construct that contained an additional essential light chain binding site exhibited a 1.4-fold increase in the unitary step size compared with its control. Kinetic changes were also observed with several of the constructs. The mutant lacking a neck produced force at a somewhat reduced level, while the force exerted by the giraffe construct was higher than control. The single molecule displacement and force data support the hypothesis that the neck functions as a rigid lever, with the fulcrum for movement and force located at a point within the motor domain.

Functional consequences of mutations in the smooth muscle myosin heavy chain at sites implicated in familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is frequently associated with mutations in the beta-cardiac myosin heavy chain. Many of the implicated residues are located in highly conserved regions of the myosin II class, suggesting that these mutations may impair the basic functions of the molecular motor. To test this hypothesis, we have prepared recombinant smooth muscle heavy meromyosin with mutations at sites homologous to those associated with FHC by using a baculovirus/insect cell expression system. Several of the heavy meromyosin mutants, in particular R403Q, showed an increase in actin filament velocity in a motility assay and an enhanced actin-activated ATPase activity. Single molecule mechanics, using a laser trap, gave unitary displacements and forces for the mutants that were similar to wild type, but the attachment times to actin following a unitary displacement were markedly reduced. These results suggest that the increases in activity are due to a change in kinetics and not due to a change in the intrinsic mechanical properties of the motor. In contrast to earlier reports, we find that mutations in residues implicated in FHC affect motor function by enhancing myosin activity rather than by a loss of function.

Biochemical studies of myosin.

This article describes methods for expressing and obtaining purified smooth muscle myosin subfragments using the baculovirus/insect cell expression system, as well as methods for purifying whole myosin from tissue. Protocols for several gel assays that are routinely used with myosin are given, including gels to monitor light chain phosphorylation state and native gels to determine protein homogeneity. Steady-state myosin ATPase and actin-activated ATPase determinations are described, as are some of the more basic transient-state kinetic parameters that can be measured. The in vitro motility assay, in which the rate of actin movement over myosin or its subfragments is quantified, is also presented.

Visualization of head-head interactions in the inhibited state of smooth muscle myosin.

The structural basis for the phosphoryla- tion-dependent regulation of smooth muscle myosin ATPase activity was investigated by forming two- dimensional (2-D) crystalline arrays of expressed unphosphorylated and thiophosphorylated smooth muscle heavy meromyosin (HMM) on positively charged lipid monolayers. A comparison of averaged 2-D projections of both forms at 2.3-nm resolution reveals distinct structural differences. In the active, thiophosphorylated form, the two heads of HMM interact intermolecularly with adjacent molecules. In the unphosphorylated or inhibited state, intramolecular interactions position the actin-binding interface of one head onto the converter domain of the second head, thus providing a mechanism whereby the activity of both heads could be inhibited.

Kinetic characterization of a monomeric unconventional myosin V construct.

An expressed, monomeric murine myosin V construct composed of the motor domain and two calmodulin-binding IQ motifs (MD(2IQ)) was used to assess the regulatory and kinetic properties of this unconventional myosin. In EGTA, the actin-activated ATPase activity of MD(2IQ) was 7.4 +/- 1.6 s(-1) with a K(app) of approximately 1 microM (37 degrees C), and the velocity of actin movement was approximately 0.3 micrometer/s (30 degrees C). Calcium inhibited both of these activities, but the addition of calmodulin restored the values to approximately 70% of control, indicating that calmodulin dissociation caused inhibition. In contrast to myosin II, MD(2IQ) is highly associated with actin at physiological ionic strength in the presence of ATP, but the motor is in a weakly bound conformation based on the pyrene-actin signal. The rate of dissociation of acto-MD(2IQ) by ATP is fast (>850 s(-1)), and ATP hydrolysis occurs at approximately 200 s(-1). The affinity of acto-MD(2IQ) for ADP is somewhat higher than that of smooth S1, and ADP dissociates more slowly. Actin does not cause a large increase in the rate of ADP release, nor does the presence of ADP appreciably alter the affinity of MD(2IQ) for actin. These kinetic data suggest that monomeric myosin V is not processive.

Expression of chicken gizzard RLC complements the cytokinesis and developmental defects of Dictyostelium RLC null cells.

Dictyostelium RLC null cells have defects in cytokinesis and development that can be rescued by expression of either the wild type Dictyostelium RLC or an RLC mutant that cannot be phosphorylated by MLCK (S13A) (Ostrow et al., 1994). The wild type and S13A mutant LCs rescued the cells equally well, despite the fact that RLC phosphorylation increases purified Dictyostelium myosin's activity 5-fold. In this report, we assess the ability of foreign RLCs to rescue the RLC null phenotype. The RLC from smooth muscle myosin, whose activity is tightly controlled by phosphorylation, rescued the null cell phenotype. The purified hybrid myosin had an activity and motility comparable to phosphorylated Dictyostelium myosin. In contrast, cells expressing skeletal muscle RLC were deficient in cytokinesis and development, despite having an activity and motility similar to that of myosin with the unposphorylatable S13A mutant RLC. Neither foreign LC was phosphorylated when expressed in Dictyostelium. These results suggest that the level of actin-activated ATPase activity and motility is not the sole determinant of proper myosin function in vivo. Other heavy chain/light chain interactions, which occur only with the native RLC and smooth muscle RLC, appear to be necessary for optimal function.

Two heads of myosin are better than one for generating force and motion.

Several classes of the myosin superfamily are distinguished by their "double-headed" structure, where each head is a molecular motor capable of hydrolyzing ATP and interacting with actin to generate force and motion. The functional significance of this dimeric structure, however, has eluded investigators since its discovery in the late 1960s. Using an optical-trap transducer, we have measured the unitary displacement and force produced by double-headed and single-headed smooth- and skeletal-muscle myosins. Single-headed myosin produces approximately half the displacement and force (approximately 6 nm; 0.7 pN) of double-headed myosin (approximately 10 nm; 1.4 pN) during a unitary interaction with actin. These data suggest that muscle myosins require both heads to generate maximal force and motion.

Myosin essential light chain isoforms modulate the velocity of shortening propelled by nonphosphorylated cross-bridges.

The differential effects of essential light chain isoforms (LC17a and LC17b) on the mechanical properties of smooth muscle were determined by exchanging recombinant for endogenous LC17 in permeabilized smooth muscle treated with trifluoperazine (TFP). Co-precipitation with endogenous myosin heavy chain verified that 40-60% of endogenous LC17a could be exchanged for recombinant LC17a or LC17b. Upon addition of MgATP in Ca2+-free solution, recombinant LC17 exchange induced slow contractions unaccompanied by regulatory light chain (RLC) phosphorylation only in TFP-treated, but not in untreated, permeabilized smooth muscle; the shortening velocity and rate of force development were approximately 1.5 and 2 times faster, respectively, in response to LC17a than LC17b. Additional incubation with recombinant, thiophosphorylated RLC increased the shortening velocity, independent of the LC17 isoform exchanged. The LC17-induced contractions of TFP-treated muscles were abolished by prior addition of nonphosphorylated RLC. We suggest that LC17 stiffens the lever arm of myosin and, in the absence of regulation by RLC, permits cross-bridge cycling without requiring RLC phosphorylation. Our results are compatible with nonphosphorylated RLC acting as a repressor and with LC17 isoforms modulating the MgADP affinity and, consequently, rate of cooperative cycling of nonphosphorylated cross-bridges.

Crystal structure of a vertebrate smooth muscle myosin motor domain and its complex with the essential light chain: visualization of the pre-power stroke state.

The crystal structures of an expressed vertebrate smooth muscle myosin motor domain (MD) and a motor domain-essential light chain (ELC) complex (MDE), both with a transition state analog (MgADP x AIF4-) in the active site, have been determined to 2.9 A and 3.5 A resolution, respectively. The MDE structure with an ATP analog (MgADP x BeFx) was also determined to 3.6 A resolution. In all three structures, a domain of the C-terminal region, the "converter," is rotated approximately 70 degrees from that in nucleotide-free skeletal subfragment 1 (S1). We have found that the MDE-BeFx and MDE-AIF4- structures are almost identical, consistent with the fact that they both bind weakly to actin. A comparison of the lever arm positions in MDE-AIF4- and in nucleotide-free skeletal S1 shows that a potential displacement of approximately 10 nm can be achieved during the power stroke.

The light chain-binding domain of the smooth muscle myosin heavy chain is not the only determinant of regulation.

Interactions between the dephosphorylated regulatory light chains (RLCs) of smooth muscle myosin are involved in maintaining the enzymatically "off" state. Expressed chimeric smooth muscle heavy meromyosins containing skeletal muscle myosin heavy chain (HC) sequences were used to assess the relative importance of the light chain-binding domain (or "neck") to regulation. Surprisingly, regulation remained intact with a skeletal RLC-binding site. A chimera with the entire alpha-helical neck composed of skeletal HC sequence showed 2-fold regulation of motility and nearly 5-fold regulation of actin-activated ATPase activity. Complete activation of the dephosphorylated state (i.e. complete loss of regulation) occurred when skeletal HC sequence extended from the head/rod junction to the SH1-SH2 helix. Smooth muscle-specific sequences near the motor domain may therefore position the regulatory domain in a way that optimizes RLC-rod-head interactions, thus enabling a completely off state when the RLC is dephosphorylated. Conversely, a chimera that joins the motor domain from unconventional myosin V to the smooth muscle myosin neck and rod showed only 2-fold regulation. The presence of the smooth muscle light chain-binding region and rod is therefore not sufficient to confer complete phosphorylation-dependent regulation upon all motor domains of the myosin family.

Loop I can modulate ADP affinity, ATPase activity, and motility of different scallop myosins. Transient kinetic analysis of S1 isoforms.

The striated muscle myosin of Placopecten moves actin faster in in vitro motility assays and has a higher actin-activated ATPase turnover rate than the myosin of the catch muscle. The heavy chain sequences of the two PlacoS1s are almost identical except at the surface loop 1 near the nucleotide binding pocket, where the two sequences vary significantly. Argopecten striated muscle myosin is 96% identical to Placopecten striated myosin, and both move actin with a similar velocity. To identify the individual kinetic steps which differ between these myosins, we completed a transient kinetic characterization of the three myosin S1s. The two striated S1s have similar rates of nucleotide binding to S1 and to acto.S1. The largest differences between the two are in the rate of ADP dissociation from S1 and affinity of ADP to S1, which differ by a factor of 2. The rates of nucleotide binding, nucleotide dissociation and affinity to nucleotides of the two Placopecten S1s are similar and agree within a factor of 2. In contrast, the affinity of acto.S1 for ADP is nine times weaker for the striated acto.S1 than for the catch acto.S1, compatible with the differences in motility of the Placopectenmyosins. Thus the differences in ADP affinity to acto.S1 and in the in vitro motility can be attributed to the differences in surface loop 1.

A 7-amino-acid insert in the heavy chain nucleotide binding loop alters the kinetics of smooth muscle myosin in the laser trap.

Two smooth muscle myosin heavy chain isoforms differ by a 7-amino-acid insert in a flexible surface loop located near the nucleotide binding site. The non-inserted isoform is predominantly found in tonic muscle, while the inserted isoform is mainly found in phasic muscle. The inserted isoform has twice the actin-activated ATPase activity and actin filament velocity in the in vitro motility assay as the non-inserted isoform. We used the laser trap to characterize the molecular mechanics and kinetics of the inserted isoform ((+)insert) and of a mutant lacking the insert ((-)insert), analogous to the isoform found in tonic muscle. The constructs were expressed as heavy meromyosin using the baculovirus/insect cell system. Unitary displacement (d) was similar for both constructs (approximately 10 nm) but the attachment time (t(on) for the (-)insert was twice as long as for the (+)insert regardless of the [MgATP]. Both the relative average isometric force (Favg(-insert)/Favg(+insert) = 1.1 +/- 0.2 (mean +/- SE) using the in vitro motility mixture assay, and the unitary force (F approximately 1 pN) using the laser trap, showed no difference between the two constructs. However, as under unloaded conditions, t(on) under loaded conditions was longer for the (-)insert compared with the (+)insert construct at limiting [MgATP]. These data suggest that the insert in this surface loop does not affect the mechanics but rather the kinetics of the cross-bridge cycle. Through comparisons of t(on) from d measurements to various [MgATP], we conclude that the insert affects two specific steps in the cross-bridge cycle, that is, MgADP release and MgATP binding.