Myosin's powerstroke transitions define atomic scale movement of cardiac thin filament tropomyosin.

Dynamic interactions between the myosin motor head on thick filaments and the actin molecular track on thin filaments drive the myosin-crossbridge cycle that powers muscle contraction. The process is initiated by Ca2+ and the opening of troponin-tropomyosin-blocked myosin-binding sites on actin. The ensuing recruitment of myosin heads and their transformation from pre-powerstroke to post-powerstroke conformation on actin produce the force required for contraction. Cryo-EM-based atomic models confirm that during this process, tropomyosin occupies three different average positions on actin. Tropomyosin pivoting on actin away from a TnI-imposed myosin-blocking position accounts for part of the Ca2+ activation observed. However, the structure of tropomyosin on thin filaments that follows pre-powerstroke myosin binding and its translocation during myosin's pre-powerstroke to post-powerstroke transition remains unresolved. Here, we approach this transition computationally in silico. We used the myosin helix-loop-helix motif as an anchor to dock models of pre-powerstroke cardiac myosin to the cleft between neighboring actin subunits along cardiac thin filaments. We then performed targeted molecular dynamics simulations of the transition between pre- and post-powerstroke conformations on actin in the presence of cardiac troponin-tropomyosin. These simulations show Arg 369 and Glu 370 on the tip of myosin Loop-4 encountering identically charged residues on tropomyosin. The charge repulsion between residues causes tropomyosin translocation across actin, thus accounting for the final regulatory step in the activation of the thin filament, and, in turn, facilitating myosin movement along the filament. We suggest that during muscle activity, myosin-induced tropomyosin movement is likely to result in unencumbered myosin head interactions on actin at low-energy cost.

MYBPC3-c.772G>A mutation results in haploinsufficiency and altered myosin cycling kinetics in a patient induced stem cell derived cardiomyocyte model of hypertrophic cardiomyopathy.

Approximately 40% of hypertrophic cardiomyopathy (HCM) mutations are linked to the sarcomere protein cardiac myosin binding protein-C (cMyBP-C). These mutations are either classified as missense mutations or truncation mutations. One mutation whose nature has been inconsistently reported in the literature is the MYBPC3-c.772G > A mutation. Using patient-derived human induced pluripotent stem cells differentiated to cardiomyocytes (hiPSC-CMs), we have performed a mechanistic study of the structure-function relationship for this MYBPC3-c.772G > A mutation versus a mutation corrected, isogenic cell line. Our results confirm that this mutation leads to exon skipping and mRNA truncation that ultimately suggests ∼20% less cMyBP-C protein (i.e., haploinsufficiency). This, in turn, results in increased myosin recruitment and accelerated myofibril cycling kinetics. Our mechanistic studies suggest that faster ADP release from myosin is a primary cause of accelerated myofibril cross-bridge cycling due to this mutation. Additionally, the reduction in force generating heads expected from faster ADP release during isometric contractions is outweighed by a cMyBP-C phosphorylation mediated increase in myosin recruitment that leads to a net increase of myofibril force, primarily at submaximal calcium activations. These results match well with our previous report on contractile properties from myectomy samples of the patients from whom the hiPSC-CMs were generated, demonstrating that these cell lines are a good model to study this pathological mutation and extends our understanding of the mechanisms of altered contractile properties of this HCM MYBPC3-c.772G > A mutation.

Protocols for Myosin and Actin-Myosin Assays Using Rapid, Stopped-Flow Kinetics.

Fast transient kinetics using stopped-flow fluorimetry is now a powerful method for defining the ATPase cycle of myosin and its subfragments and has found wide use in defining the difference between myosin isoforms, myosins carrying disease linked mutations, and the effect of small molecules on the ATPase cycle. Here the protocols for completing the classical assays of myosin and actin.myosin using the stopped-flow are described. The assays include ATP and ADP binding to myosin and actin.myosin, displacement of ADP from myosin and actin.myosin, and the cleavage of ATP to ADP and phosphate on myosin. Single and multiple turnover assays are also described.

Danicamtiv Increases Myosin Recruitment and Alters Cross-Bridge Cycling in Cardiac Muscle.

BACKGROUND: Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known that danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. METHODS: Permeabilized porcine cardiac tissue and myofibrils were used for X-ray diffraction and mechanical measurements. A mouse model of genetic dilated cardiomyopathy was used to evaluate the ability of danicamtiv to correct the contractile deficit. RESULTS: Danicamtiv increased force and calcium sensitivity via increasing the number of myosins in the ON state and slowing cross-bridge turnover. Our detailed analysis showed that inhibition of ADP release results in decreased cross-bridge turnover with cross bridges staying attached longer and prolonging myofibril relaxation. Danicamtiv corrected decreased calcium sensitivity in demembranated tissue, abnormal twitch magnitude and kinetics in intact cardiac tissue, and reduced ejection fraction in the whole organ. CONCLUSIONS: As demonstrated by the detailed studies of Danicamtiv, increasing myosin recruitment and altering cross-bridge cycling are 2 mechanisms to increase force and calcium sensitivity in cardiac muscle. Myosin activators such as Danicamtiv can treat the causative hypocontractile phenotype in genetic dilated cardiomyopathy.

Comparing the effects of chemical Ca2+ dyes and R-GECO on contractility and Ca2+ transients in adult and human iPSC cardiomyocytes.

We compared commonly used BAPTA-derived chemical Ca2+ dyes (fura2, Fluo-4, and Rhod-2) with a newer genetically encoded indicator (R-GECO) in single cell models of the heart. We assessed their performance and effects on cardiomyocyte contractility, determining fluorescent signal-to-noise ratios and sarcomere shortening in primary ventricular myocytes from adult mouse and guinea pig, and in human iPSC-derived cardiomyocytes. Chemical Ca2+ dyes displayed dose-dependent contractile impairment in all cell types, and we observed a negative correlation between contraction and fluorescence signal-to-noise ratio, particularly for fura2 and Fluo-4. R-GECO had no effect on sarcomere shortening. BAPTA-based dyes, but not R-GECO, inhibited in vitro acto-myosin ATPase activity. The presence of fura2 accentuated or diminished changes in contractility and Ca2+ handling caused by small molecule modulators of contractility and intracellular ionic homeostasis (mavacamten, levosimendan, and flecainide), but this was not observed when using R-GECO in adult guinea pig left ventricular cardiomyocytes. Ca2+ handling studies are necessary for cardiotoxicity assessments of small molecules intended for clinical use. Caution should be exercised when interpreting small molecule studies assessing contractile effects and Ca2+ transients derived from BAPTA-like chemical Ca2+ dyes in cellular assays, a common platform for cardiac toxicology testing and mechanistic investigation of cardiac disease physiology and treatment.

Danicamtiv increases myosin recruitment and alters the chemomechanical cross bridge cycle in cardiac muscle.

Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Detailed mechanism of action of these agents can help predict potential unwanted affects and identify patient populations that can benefit most from them. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. Using porcine cardiac tissue and myofibrils we demonstrate that Danicamtiv increases force and calcium sensitivity via increasing the number of myosin in the "on" state and slowing cross bridge turnover. Our detailed analysis shows that inhibition of ADP release results in decreased cross bridge turnover with cross bridges staying on longer and prolonging myofibril relaxation. Using a mouse model of genetic dilated cardiomyopathy, we demonstrated that Danicamtiv corrected calcium sensitivity in demembranated and abnormal twitch magnitude and kinetics in intact cardiac tissue. Significance Statement: Directly augmenting sarcomere function has potential to overcome limitations of currently used inotropic agents to improve cardiac contractility. Myosin modulation is a novel mechanism for increased contraction in cardiomyopathies. Danicamtiv is a myosin activator that is currently under investigation for use in cardiomyopathy patients. Our study is the first detailed mechanism of how Danicamtiv increases force and alters kinetics of cardiac activation and relaxation. This new understanding of the mechanism of action of Danicamtiv can be used to help identify patients that could benefit most from this treatment.

Pseudo-phosphorylation of essential light chains affects the functioning of skeletal muscle myosin.

The work aimed to investigate how the phosphorylation of the myosin essential light chain of fast skeletal myosin (LC1) affects the functional properties of the myosin molecule. Using mass-spectrometry, we revealed phosphorylated peptides of LC1 in myosin from different fast skeletal muscles. Mutations S193D and T65D that mimic natural phosphorylation of LC1 were produced, and their effects on functional properties of the entire myosin molecule and isolated myosin head (S1) were studied. We have shown that T65D mutation drastically decreased the sliding velocity of thin filaments in an in vitro motility assay and strongly increased the duration of actin-myosin interaction in optical trap experiments. These effects of T65D mutation in LC1 observed only with the whole myosin but not with S1 were prevented by double T65D/S193D mutation. The T65D and T65D/S193D mutations increased actin-activated ATPase activity of S1 and decreased ADP affinity for the actin-S1 complex. The results indicate that pseudo-phosphorylation of LC1 differently affects the properties of the whole myosin molecule and its isolated head. Also, the results show that phosphorylation of LC1 of skeletal myosin could be one more mechanism of regulation of actin-myosin interaction that needs further investigation.

Functional divergence of the sarcomeric myosin, MYH7b, supports species-specific biological roles.

Myosin heavy chain 7b (MYH7b) is an evolutionarily ancient member of the sarcomeric myosin family, which typically supports striated muscle function. However, in mammals, alternative splicing prevents MYH7b protein production in cardiac and most skeletal muscles and limits expression to a subset of specialized muscles and certain nonmuscle environments. In contrast, MYH7b protein is abundant in python cardiac and skeletal muscles. Although the MYH7b expression pattern diverges in mammals versus reptiles, MYH7b shares high sequence identity across species. So, it remains unclear how mammalian MYH7b function may differ from that of other sarcomeric myosins and whether human and python MYH7b motor functions diverge as their expression patterns suggest. Thus, we generated recombinant human and python MYH7b protein and measured their motor properties to investigate any species-specific differences in activity. Our results reveal that despite having similar working strokes, the MYH7b isoforms have slower actin-activated ATPase cycles and actin sliding velocities than human cardiac ß-MyHC. Furthermore, python MYH7b is tuned to have slower motor activity than human MYH7b because of slower kinetics of the chemomechanical cycle. We found that the MYH7b isoforms adopt a higher proportion of myosin heads in the ultraslow, super-relaxed state compared with human cardiac ß-MyHC. These findings are supported by molecular dynamics simulations that predict MYH7b preferentially occupies myosin active site conformations similar to those observed in the structurally inactive state. Together, these results suggest that MYH7b is specialized for slow and energy-conserving motor activity and that differential tuning of MYH7b orthologs contributes to species-specific biological roles.

Single-molecule imaging reveals how mavacamten and PKA modulate ATP turnover in skeletal muscle myofibrils.

Muscle contraction is controlled at two levels: the thin and the thick filaments. The latter level of control involves three states of myosin heads: active, disordered relaxed (DRX), and super-relaxed (SRX), the distribution of which controls the number of myosins available to interact with actin. How these are controlled is still uncertain. Using fluorescently labeled ATP, we were able to spatially assign the activity of individual myosins within the sarcomere. We observed that SRX comprises 53% of all heads in the C-zone compared with 35% and 44% in the P- and D-zones, respectively. The recently FDA-approved hypertrophic cardiomyopathy drug, mavacamten (mava), significantly decreased DRX, favoring SRX in both the C- and D-zones at 60% and 63%, respectively. Since thick filament regulation is in part regulated by the myosin-binding protein-C (MyBP-C), we also studied PKA phosphorylation. This had the opposite effect as mava, specifically in the C-zone where it decreased SRX to 34%, favoring DRX. These results directly show that excess concentrations of mava do increase SRX, but the effect is limited across the sarcomere, suggesting mava is less effective on skeletal muscle. In addition, we show that PKA directly affects the contractile machinery of skeletal muscle leading to the liberation of repressed heads. Since the effect is focused on the C-zone, this suggests it is likely through MyBP-C phosphorylation, although our data suggest that a further reserve of myosins remain that are not accessible to PKA treatment.

Effect of Myosin Isoforms on Cardiac Muscle Twitch of Mice, Rats and Humans.

To understand how pathology-induced changes in contractile protein isoforms modulate cardiac muscle function, it is necessary to quantify the temporal-mechanical properties of contractions that occur under various conditions. Pathological responses are much easier to study in animal model systems than in humans, but extrapolation between species presents numerous challenges. Employing computational approaches can help elucidate relationships that are difficult to test experimentally by translating the observations from rats and mice, as model organisms, to the human heart. Here, we use the spatially explicit MUSICO platform to model twitch contractions from rodent and human trabeculae collected in a single laboratory. This approach allowed us to identify the variations in kinetic characteristics of α- and ß-myosin isoforms across species and to quantify their effect on cardiac muscle contractile responses. The simulations showed how the twitch transient varied with the ratio of the two myosin isoforms. Particularly, the rate of tension rise was proportional to the fraction of α-myosin present, while the ß-isoform dominated the rate of relaxation unless α-myosin was >50%. Moreover, both the myosin isoform and the Ca2+ transient contributed to the twitch tension transient, allowing two levels of regulation of twitch contraction.

Exploring the super-relaxed state of myosin in myofibrils from fast-twitch, slow-twitch, and cardiac muscle.

Muscle myosin heads, in the absence of actin, have been shown to exist in two states, the relaxed (turnover ∼0.05 s-1) and super-relaxed states (SRX, 0.005 s-1) using a simple fluorescent ATP chase assay (Hooijman, P. et al (2011) Biophys. J.100, 1969-1976). Studies have normally used purified proteins, myosin filaments, or muscle fibers. Here we use muscle myofibrils, which retain most of the ancillary proteins and 3-D architecture of muscle and can be used with rapid mixing methods. Recording timescales from 0.1 to 1000 s provides a precise measure of the two populations of myosin heads present in relaxed myofibrils. We demonstrate that the population of SRX states is formed from rigor cross bridges within 0.2 s of relaxing with fluorescently labeled ATP, and the population of SRX states is relatively constant over the temperature range of 5 °C-30 °C. The SRX population is enhanced in the presence of mavacamten and reduced in the presence of deoxy-ATP. Compared with myofibrils from fast-twitch muscle, slow-twitch muscle, and cardiac muscles, myofibrils require a tenfold lower concentration of mavacamten to be effective, and mavacamten induced a larger increase in the population of the SRX state. Mavacamten is less effective, however, at stabilizing the SRX state at physiological temperatures than at 5 °C. These assays require small quantities of myofibrils, making them suitable for studies of model organism muscles, human biopsies, or human-derived iPSCs.

Acetylation stabilises calmodulin-regulated calcium signalling.

Calmodulin is a conserved calcium signalling protein that regulates a wide range of cellular functions. Amino-terminal acetylation is a ubiquitous post-translational modification that affects the majority of human proteins, to stabilise structure, as well as regulate function and proteolytic degradation. Here, we present data on the impact of amino-terminal acetylation upon structure and calcium signalling function of fission yeast calmodulin. We show that NatA-dependent acetylation stabilises the helical structure of the Schizosaccharomyces pombe calmodulin, impacting its ability to associate with myosin at endocytic foci. We go on to show that this conserved modification impacts both the calcium-binding capacity of yeast and human calmodulins. These findings have significant implications for research undertaken into this highly conserved essential protein.

Alpha and beta myosin isoforms and human atrial and ventricular contraction.

Human atrial and ventricular contractions have distinct mechanical characteristics including speed of contraction, volume of blood delivered and the range of pressure generated. Notably, the ventricle expresses predominantly ß-cardiac myosin while the atrium expresses mostly the α-isoform. In recent years exploration of the properties of pure α- & ß-myosin isoforms have been possible in solution, in isolated myocytes and myofibrils. This allows us to consider the extent to which the atrial vs ventricular mechanical characteristics are defined by the myosin isoform expressed, and how the isoform properties are matched to their physiological roles. To do this we Outline the essential feature of atrial and ventricular contraction; Explore the molecular structural and functional characteristics of the two myosin isoforms; Describe the contractile behaviour of myocytes and myofibrils expressing a single myosin isoform; Finally we outline the outstanding problems in defining the differences between the atria and ventricles. This allowed us consider what features of contraction can and cannot be ascribed to the myosin isoforms present in the atria and ventricles.

Identification of sequence changes in myosin II that adjust muscle contraction velocity.

The speed of muscle contraction is related to body size; muscles in larger species contract at slower rates. Since contraction speed is a property of the myosin isoform expressed in a muscle, we investigated how sequence changes in a range of muscle myosin II isoforms enable this slower rate of muscle contraction. We considered 798 sequences from 13 mammalian myosin II isoforms to identify any adaptation to increasing body mass. We identified a correlation between body mass and sequence divergence for the motor domain of the 4 major adult myosin II isoforms (ß/Type I, IIa, IIb, and IIx), suggesting that these isoforms have adapted to increasing body mass. In contrast, the non-muscle and developmental isoforms show no correlation of sequence divergence with body mass. Analysis of the motor domain sequence of ß-myosin (predominant myosin in Type I/slow and cardiac muscle) from 67 mammals from 2 distinct clades identifies 16 sites, out of 800, associated with body mass (padj < 0.05) but not with the clade (padj > 0.05). Both clades change the same small set of amino acids, in the same order from small to large mammals, suggesting a limited number of ways in which contraction velocity can be successfully manipulated. To test this relationship, the 9 sites that differ between human and rat were mutated in the human ß-myosin to match the rat sequence. Biochemical analysis revealed that the rat-human ß-myosin chimera functioned like the native rat myosin with a 2-fold increase in both motility and in the rate of ADP release from the actin-myosin crossbridge (the step that limits contraction velocity). Thus, these sequence changes indicate adaptation of ß-myosin as species mass increased to enable a reduced contraction velocity and heart rate.

The effect of variable troponin C mutation thin filament incorporation on cardiac muscle twitch contractions.

One of the complexities of understanding the pathology of familial forms of cardiac diseases is the level of mutation incorporation in sarcomeres. Computational models of the sarcomere that are spatially explicit offer an approach to study aspects of mutational incorporation into myofilaments that are more challenging to get at experimentally. We studied two well characterized mutations of cardiac TnC, L48Q and I61Q, that decrease or increase the release rate of Ca2+ from cTnC, k-Ca, resulting in HCM and DCM respectively [1]. Expression of these mutations in transgenic mice was used to provide experimental data for incorporation of 30 and 50% (respectively) into sarcomeres. Here we demonstrate that fixed length twitch contractions of trabeculae from mice containing mutant differ from WT; L48Q trabeculae have slower relaxation while I61Q trabeculae have markedly reduced peak tension. Using our multiscale modelling approach [2] we were able to describe the tension transients of WT mouse myocardium. Tension transients for the mutant cTnCs were simulated with changes in k-Ca, measured experimentally for each cTnC mutant in whole troponin complex, a change in the affinity of cTnC for cTnI, and a reduction in the number of detached crossbridges available for binding. A major advantage of the multiscale explicit 3-D model is that it predicts the effects of variable mutation incorporation, and the effects of variations in mutation distribution within thin filaments in sarcomeres. Such effects are currently impossible to explore experimentally. We explored random and clustered distributions of mutant cTnCs in thin filaments, as well as distributions of individual thin filaments with only WT or mutant cTnCs present. The effects of variable amounts of incorporation and non-random distribution of mutant cTnCs are more marked for I61Q than L48Q cTnC. We conclude that this approach can be effective for study on mutations in multiple proteins of the sarcomere. SUMMARY: A challenge in experimental studies of diseases is accounting for the effect of variable mutation incorporation into myofilaments. Here we use a spatially explicit computational approach, informed by experimental data from transgenic mice expressing one of two mutations in cardiac Troponin C that increase or decrease calcium sensitivity. We demonstrate that the model can accurately describe twitch contractions for the data and go on to explore the effect of variable mutant incorporation and localization on simulated cardiac muscle twitches.

Multiscale modeling of twitch contractions in cardiac trabeculae.

Understanding the dynamics of a cardiac muscle twitch contraction is complex because it requires a detailed understanding of the kinetic processes of the Ca2+ transient, thin-filament activation, and the myosin-actin cross-bridge chemomechanical cycle. Each of these steps has been well defined individually, but understanding how all three of the processes operate in combination is a far more complex problem. Computational modeling has the potential to provide detailed insight into each of these processes, how the dynamics of each process affect the complexity of contractile behavior, and how perturbations such as mutations in sarcomere proteins affect the complex interactions of all of these processes. The mechanisms involved in relaxation of tension during a cardiac twitch have been particularly difficult to discern due to nonhomogeneous sarcomere lengthening during relaxation. Here we use the multiscale MUSICO platform to model rat trabecular twitches. Validation of computational models is dependent on being able to simulate different experimental datasets, but there has been a paucity of data that can provide all of the required parameters in a single experiment, such as simultaneous measurements of force, intracellular Ca2+ transients, and sarcomere length dynamics. In this study, we used data from different studies collected under similar experimental conditions to provide information for all the required parameters. Our simulations established that twitches either in an isometric sarcomere or in fixed-length, multiple-sarcomere trabeculae replicate the experimental observations if models incorporate a length-tension relationship for the nonlinear series elasticity of muscle preparations and a scheme for thick-filament regulation. The thick-filament regulation assumes an off state in which myosin heads are parked onto the thick-filament backbone and are unable to interact with actin, a state analogous to the super-relaxed state. Including these two mechanisms provided simulations that accurately predict twitch contractions over a range of different conditions.

Alternative N-terminal regions of Drosophila myosin heavy chain II regulate communication of the purine binding loop with the essential light chain.

We investigated the biochemical and biophysical properties of one of the four alternative exon-encoded regions within the Drosophila myosin catalytic domain. This region is encoded by alternative exons 3a and 3b and includes part of the N-terminal ß-barrel. Chimeric myosin constructs (IFI-3a and EMB-3b) were generated by exchanging the exon 3-encoded areas between native slow embryonic body wall (EMB) and fast indirect flight muscle myosin isoforms (IFI). We found that this exchange alters the kinetic properties of the myosin S1 head. The ADP release rate (k-D ) in the absence of actin is completely reversed for each chimera compared with the native isoforms. Steady-state data also suggest a reciprocal shift, with basal and actin-activated ATPase activity of IFI-3a showing reduced values compared with wild-type (WT) IFI, whereas for EMB-3b these values are increased compared with wild-type (WT) EMB. In the presence of actin, ADP affinity (KAD ) is unchanged for IFI-3a, compared with IFI, but ADP affinity for EMB-3b is increased, compared with EMB, and shifted toward IFI values. ATP-induced dissociation of acto-S1 (K1k+2 ) is reduced for both exon 3 chimeras. Homology modeling, combined with a recently reported crystal structure for Drosophila EMB, indicates that the exon 3-encoded region in the myosin head is part of the communication pathway between the nucleotide binding pocket (purine binding loop) and the essential light chain, emphasizing an important role for this variable N-terminal domain in regulating actomyosin crossbridge kinetics, in particular with respect to the force-sensing properties of myosin isoforms.

Cryo-EM and Molecular Docking Shows Myosin Loop 4 Contacts Actin and Tropomyosin on Thin Filaments.

The motor protein myosin drives muscle and nonmuscle motility by binding to and moving along actin of thin filaments. Myosin binding to actin also modulates interactions of the regulatory protein, tropomyosin, on thin filaments, and conversely tropomyosin affects myosin binding to actin. Insight into this reciprocity will facilitate a molecular level elucidation of tropomyosin regulation of myosin interaction with actin in muscle contraction, and in turn, promote better understanding of nonmuscle cell motility. Indeed, experimental approaches such as fiber diffraction, cryoelectron microscopy, and three-dimensional reconstruction have long been used to define regulatory interaction of tropomyosin and myosin on actin at a structural level. However, their limited resolution has not proven sufficient to determine tropomyosin and myosin contacts at an atomic-level and thus to fully substantiate possible functional contributions. To overcome this deficiency, we have followed a hybrid approach by performing new cryogenic electron microscopy reconstruction of myosin-S1-decorated F-actin-tropomyosin together with atomic scale protein-protein docking of tropomyosin to the EM models. Here, cryo-EM data were derived from filaments reconstituted with α1-actin, cardiac αα-tropomyosin, and masseter muscle ß-myosin complexes; masseter myosin, which shares sequence identity with ß-cardiac myosin-heavy chain, was used because of its stability in vitro. The data were used to build an atomic model of the tropomyosin cable that fits onto the actin filament between the tip of the myosin head and a cleft on the innermost edge of actin subunits. The docking and atomic scale fitting showed multiple discrete interactions of myosin loop 4 and acidic residues on successive 39-42 residue-long tropomyosin pseudorepeats. The contacts between S1 and tropomyosin on actin appear to compete with and displace ones normally found between actin and tropomyosin on myosin-free thin filaments in relaxed muscle, thus restructuring the filament during myosin-induced activation.

TORC2-Gad8-dependent myosin phosphorylation modulates regulation by calcium.

Cells respond to changes in their environment through signaling networks that modulate cytoskeleton and membrane organization to coordinate cell-cycle progression, polarized cell growth and multicellular development. Here, we define a novel regulatory mechanism by which the motor activity and function of the fission yeast type one myosin, Myo1, is modulated by TORC2-signalling-dependent phosphorylation. Phosphorylation of the conserved serine at position 742 (S742) within the neck region changes both the conformation of the neck region and the interactions between Myo1 and its associating calmodulin light chains. S742 phosphorylation thereby couples the calcium and TOR signaling networks that are involved in the modulation of myosin-1 dynamics to co-ordinate actin polymerization and membrane reorganization at sites of endocytosis and polarised cell growth in response to environmental and cell-cycle cues.

Myosin motor domains carrying mutations implicated in early or late onset hypertrophic cardiomyopathy have similar properties.

Hypertrophic cardiomyopathy (HCM) is a common genetic disorder characterized by left ventricular hypertrophy and cardiac hyper-contractility. Mutations in the ß-cardiac myosin heavy chain gene (ß-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to this disease remain incompletely understood. Predicting the severity of any ß-MyHC mutation is hindered by a lack of detailed examinations at the molecular level. Moreover, because HCM can take ≥20 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations that result in early onset disease would have more severe changes in function than do later onset mutations. Here, we performed steady-state and transient kinetic analyses of myosins carrying one of seven missense mutations in the motor domain. Of these seven, four were previously identified in early onset cardiomyopathy screens. We used the parameters derived from these analyses to model the ATP-driven cross-bridge cycle. Contrary to our hypothesis, the results indicated no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin·myosin·ADP complex at [Actin] = 3 Kapp along with the closely related duty ratio (the fraction of myosin in strongly attached force-holding states), and the measured ATPases all changed in parallel (in both sign and degree of change) compared with wildtype (WT) values. Six of the seven HCM mutations were clearly distinct from a set of previously characterized DCM mutations.