What type of compound is myosin

Axial spacing of the S-1 heads on the thick filament differs from that of the sites on the thin filament, so that the attachment-detachment cycle takes place asynchronously. Up to the present time, the attachment-detachment cycle shown in Fig. The most crucial step of the attachment-detachment cycle is, of course, conformational changes of the S-1 head attached to the thin filament shown in the middle diagram.

Diagrams of putative attachment-detachment cycle between the S-1 head, extending from the thick filament, and the corresponding site on the thin filament.

For explanations, see text. Molecular mechanism of muscle contraction can be studied biochemically by examining ATPase reaction steps of actin-myosin complex actomyosin in solution. The most probable sequence of actomyosin ATPase reaction taking place in contracting muscle is shown in Fig. M and A represent the myosin S-1 head on the thick filament and the actin monomer on the thin filament, respectively.

The reaction cycle includes attachment of A to, and its detachment from M, thus providing a simple correspondence with the attachment-detachment cycle between actin and myosin in the Huxley contraction model shown in Fig. The most probable sequence of actomyosin ATPase reactions taking place in contracting muscle. Although a number of attempts have been made up to the present time using a variety of experimental methods, the question, as to what makes the filaments slide, is not yet fully answered.

In , A. Huxley and Simmons presented a contraction model, which was central in the field of muscle physiology over many years [ 9 ] Fig. Time-resolved X-ray diffraction studies on contracting muscle also could not detect any appreciable changes in the equatorial reflections in response to a quick decrease in muscle length, which was expected to synchronously rotate the massive S-1 head [ 12 , 13 ].

Thus, the myosin S-1 head tilting model was found to be inconsistent with the experimental observations. Diagram showing the attachment-detachment cycle between the S-1 head and the thin filaments, presented by A. Huxley and Simmons [ 10 ]. Since the S-1 head tilting model is not supported experimentally, the S-1 head structural changes are considered to be limited within a small region in the myosin molecule.

The X-ray S-1 head crystal structure was first obtained by Rayment et al. As shown in Fig. In the intact myosin molecule, the LD is connected to the thick filament via the myosin S Attempts have been made to study possible nucleotide-dependent structural changes of the S-1 head to obtain insight into the mechanism of muscle contraction. The truncated S-1 is nearly globular in shape, and is easy to crystallize.

Myosin S-1 head structure [ 15 ]. For further explanations, see text. Based on this and other results, it has been proposed that the power stroke of the S-1 head, causing myofilament sliding, results from active rotation of the CD around the COD [ 15 ], utilizing chemical energy of ATP hydrolysis taking place in the CD. This is the swinging lever arm hypothesis, which now appears in many textbooks in physiology and biology. It is not certain, however, whether the above nucleotide-dependent structural changes of the S-1 head actually work in muscle contraction or not by the following reasons: 1 It is not clear that the observed rotation of the COD generates torque large enough to cause the filament sliding when the COD is connected to the thick filament via the LD and S-2; 2 It seems possible that the rotation of the COD is an artifact arising from close packing of the S-1 in the crystal, that may make each S-1 in a condition completely different from that in muscle; 3 It seems also possible that the ATP analogs used do not actually mimic intermediate compounds of ATP hydrolysis in muscle; and 4 The hypothesis completely igonores possible roles of the LD as well as the S After the end of power stroke, M remains attached to A B.

In this diagram, both the power and the recovery strokes of M is supposed to result from the swinging lever arm mechanism, so that M does not change its angle of attachment to A throughout the whole cycle, while it swings around a pivot COD, represented by a small circle forward and backward.

The LD is located between M and the pivot, simply serving as a lever arm. Diagram showing the attachment-detachment cycle between myosin S-1 head M , extending from the thick filament, and actin monomer A in the thin filament, based on the actomyosin ATPase reactions [ 16 ].

It is understandable that crystallographists use truncated S-1 head because of easiness in crystallizing it. Another reason for their ignorance of the LD and the S-2 in considering the mechanism of muscle contraction may come from development of so-called in vitro motility assay experiments, in which fluorescently labeled actin filaments are made to slide over myosin molecules or their proteolytic fragments such as HMM and S-1, fixed on a glass surface in the presence of ATP [ 17 ].

Especially, the fact that even the S-1 alone can generate force on actin filaments [ 18 ] seems to have given muscle investigators a belief that only the S-1 head is important in producing muscle contraction. We think that there is no reason to ignore the LD-S-2 boundary in considering the mechanism of the myosin S-1 head power stroke. In the next section, we will present experimental evidence for the essential role of the LD and the S-2, as well as evidence for non-essential role of the COD.

Diagram showing the structural changes of the myosin S-1 and S-2 before solid line and after broken line the power stroke. Our experiments concerning the essential role of the myosin S-2 started in when one of us H. If the velocity values are replotted against forces expressed relative to steady forces, the P-V curves were found to be identical Fig.

Since muscle fiber stiffness changed in parallel with force Fig. A P-V curves obtained before control and 30, 60, and 90min after administration of anti-S-2 antibody. Both velocities and forces are expressed in absolute values.

Myosin is composed of several protein chains: two large "heavy" chains and four small "light" chains. The structures available in the PDB, such as the one shown above, contain only part of the myosin molecule.

In the illustration above, from PDB entry 1b7t , atoms in the heavy chain are colored red on the left-hand side, and atoms in the light chains are colored orange and yellow.

The whole molecule is much larger, as shown on the next page, with a long tail that has been clipped off to allow the molecule to be studied. Fortunately, the crystal structures include most of the "motor" domain, the part of the molecule that performs the power stroke, so we can look at this process in detail.

Illustration of myosin filaments in the muscle sarcomere. Each myosin performs only a tiny molecular motion. It takes about 2 trillion myosin molecules to provide the force to hold up a baseball.

Our biceps have a million times this many, so only a fraction of the myosin molecules need to be exerting themselves at any given time. By working together, the tiny individual power stroke of each myosin is summed to provide macroscopic power in our familiar world. The painting shows how myosin is arranged inside muscle cells. About myosin molecules bind together, with all of the long tails bound tightly together into a large "thick filament.

The many myosin heads extending from the thick filament then reach over to actin filaments , shown in blue and green, and together climb their way up. Power stroke of myosin: pre-power stroke state left and rigor state right. ATP contains a key phosphate-phosphate bond that is difficult to create and is used to power many processes inside cells.

You might be surprised to find, however, that breakage of this phosphate-phosphate bond is not directly responsible for the power stroke in myosin. Instead, it is release of the phosphate left over after ATP is cleaved that powers the stroke. Think of myosin like an arm that can flex towards you or push away. The cleavage of ATP is used in a priming step.

This prepares myosin for the power stroke. The flexed myosin then grabs the actin filament shown in green and blue, from PDB entry 1atn and release of phosphate snaps it into the straight "rigor" form, as shown on the right PDB entry 2mys.

Mammalian cells typically produce in excess of 10, different proteins displaying enzymatic activities. To retain the conformational flexibility required for their catalytic function, most of these enzymes are only marginally thermodynamically stable. External stress brought about by changes in pH, temperature, ionic strength, and the composition and concentration of small-molecule ligands affects the transition to the denatured state. The denatured state is characterized by the loss of enzymatic activity and can be described as an ensemble of states with high conformational entropy Anfinsen and Scheraga, Denatured enzyme molecules are more likely to be affected by adsorption, aggregation, precipitation, and proteolysis.

At the cellular level, their aggregation with one another or with properly functioning proteins leads to deleterious consequences. Their accumulation can interfere with proteasome function, disrupt normal cellular processes by binding critical cell-signaling and cell-trafficking molecules, and trigger signals that result in cell death Tyedmers et al.

Elaborate cellular systems for the maintenance of protein homeostasis proteostasis have evolved to prevent the accumulation of unfolded or misfolded protein aggregates.

These comprise approximately general and specialized chaperone components and ubiquitin-proteasome system UPS and autophagy system components that support protein folding, refolding of stress-denatured enzymes, disaggregation, and proteolytic degradation of irreversibly misfolded proteins Nalepa et al. Myosins are amongst the most abundant proteins in our body. In addition to their role as molecular motors driving the stiffening and contraction of muscles, they contribute to a wide range of functions.

In the case of myosin, the negative consequences of misfolding are amplified by the formation of roadblock-like, strongly-bound complexes with their actin filament tracks. Aberrant myosin activity and proteostasis contribute to hereditary skeletal myopathies, cardiomyopathies, and various disorders of the nervous system Oldfors, ; Walsh et al.

In the course of investigations to identify small-molecule ligands that stabilize myosin against thermal denaturation, we identified the thiadiazinone derivative EMD as a potent mediator of myosin folding. Studies with EMD performed in dogs, with heart failure induced through long-term tachycardia pacing, show potent positive inotropic effects, reduced oxygen cost of contraction, and enhanced diastolic chamber performance Senzaki et al.

Here, we provide evidence indicating that EMD binds to an allosteric pocket in the myosin motor domain near the base of the lever arm. In addition to increasing motility, force production, and thermal stability of native myosin, binding of EMD to heat-inactivated myosin induces refolding and reactivation of ATPase activity and motility.

Our findings demonstrate that small molecules such as EMD can stabilize, enhance the activity of and correct stress-associated misfolding of globular enzymes.

Myosin constructs that bind EMD display increased basal and actin-activated ATPase activity in the presence of the compound. The activation of skeletal muscle myosin-2 and Dd myosin-5 is twofold and fivefold weaker. The initial fast rise of fluorescence reflects the binding of the ATP analogue to the myosin active site, the following plateau phase monitors the duration of the hydrolysis reaction, and the third phase, corresponding to a decrease in fluorescence signal intensity, monitors the rate of product release.

The normalized thermophoresis signals were plotted against the EMD concentration. K D values were obtained by fitting the data to the Hill equation. Errors indicate s. D Fraction of active myosin heads in the absence and presence of saturating concentrations of EMD determined by active site titrations.

The observed turnover of ATP is 1. F EMD mediated activation of motor activity. Filament movement is restarted after the addition of EMD Errors indicate SD.

The number of experimentally accessible active sites is always smaller in preparations of purified enzymes than the number of active sites calculated based on protein concentration. The observed deviation results from incomplete folding, the presence of impurities, and the stress-induced partial loss of function during purification and storage.

In the case of myosin, the amplitude of the fast rise in fluorescence intensity that follows binding of mantATP can be used to estimate the fraction of functional protein by active site titration Tsiavaliaris et al. The increase in the number of binding-competent active sites is followed by a correlated increase in myosin ATPase activity Figure 1D,E.

In addition to the apparent conversion of inactive to catalytically competent myosin, ATPase activities measured in the presence of EMD correlate with the total number of myosin heads rather than the initial number of active myosin heads. To probe the effect of EMD on myosin motor activity, we performed in vitro motility assays. The sliding velocity of actin filaments increased 1. Next, we generated in vitro motility flow cells with actin filaments held at stall force.

The time-dependence of the recovery of motile activity is best fit by a hyperbola. A plateau value of 0. Steady-state kinetic assays performed in the absence and the presence of EMD indicate an allosteric mode of action Figure 2—figure supplement 1.

The mode of binding and the orientation of EMD in this pocket were further analyzed using a flexible, targeted docking procedure. Rearrangements in the side chains of residues Arg29 and Lys34 are predicted to bring these two amino acids into the close vicinity of polar groups in EMD The sets of binding poses differ mainly in the orientation of the dimethylated catechol group, occupying either of the two possible branches of the Y, while the thiadiazinone moiety appears little affected and resides in the root of the Y shaped cavity Figure 2A,B.

Potential residues involved in the binding of the thiadiazinone and tetrahydroquinoline moieties of the compound include Arg23, Asp85, Lys86, Asp, Arg, and Ser Further studies using point mutations that eliminate key residues predicted to interact with EMD are needed to confirm the binding site.

A Predicted binding of EMD near the base of the lever arm as obtained by molecular docking. Two slightly different clusters of binding poses were identified for EMD with comparable binding affinities.

B Close-up of the allosteric binding pocket shown in surface representation. The Y shape of the pocket is outlined and the two residues—Arg29 and Lys34—that were allowed full conformational flexibility during docking are colored red.

For clarity only the best poses for the two identified clusters of binding modes are shown. Hydrogen bonds of EMD to the motor protein are shown in magenta.

To gain a better understanding of its mechanism of action, we examined the effect of EMD binding on the kinetic activity of the myosin motor. The fast process corresponds to the binding of ATP to the active site.

Accordingly, EMD triggers a 3. It can be monitored by the fluorescence increase that is associated with the much faster binding of phosphate to the AC mutant of Escherichia coli phosphate binding protein, labeled at Cys with the thiol reactive coumarin dye MDCC Brune et al.

The observed transients in the absence and presence of EMD were best fitted by single exponential functions. A Kinetic reaction scheme of the actomyosin ATPase cycle. The fast phase of the biphasic transients correspond to ATP binding, the slow phase can be attributed to ATP hydrolysis.

The observed rates for the fast phase show a linear dependence on ATP concentration. Differences in the slopes indicate a threefold increase of the second order rate constants for ATP binding in the presence of EMD The plateau values from a hyperbolic fit of the observed slow components of the fluorescence transients plotted against ATP concentrations define the rate of ATP hydrolysis.

F The rate of phosphate release increased 1. The activation energy for the reaction is 1. Equilibrium binding of ligands leads to a shift of the midpoint of the thermal transition T m , which corresponds to the temperature at which half of all protein molecules are in the native state and the remaining half are in the denatured state.

Typically, the extent of the observed increase in protein thermal stability is proportional to the concentration and affinity of the added ligand. The T m measured for the Dd myosin-2 motor domain corresponds to However, the EMD mediated activation and conversion of inactive to catalytically competent myosin Figure 1C,D and the greatly improved preservation of enzymatic activity during storage Figure 4A indicate that EMD is more potent in preventing the precipitation and irreversible denaturation of the protein than suggested by the shift in T m alone.

The orange columns indicate changes observed in the presence of EMD D Time-dependent recovery of the capacity to bind nucleotide monitored by ATP-induced changes in intrinsic tryptophan fluorescence. Transients obtained after rapid mixing with ATP were measured at the indicated times following the addition of EMD H Determination of the second order rate constant for the EMD mediated refolding reaction.

The observed rate constants for the recovery of ATPase activity and motility were extracted from the data shown in Figure 4F,G. The slope of a linear fit to the data defines k rescue as 0. The y-intercept gives a first order dissociation rate constant for EMD of 0. To elucidate the mode of action of EMD , we submitted myosin constructs in the absence and presence of the compound to heat stress and followed the resulting changes in solubility and functional competence.

This view is supported by experiments that monitor the recovery of basal activity over a 2 hr period Figure 4C.