Myosin Motor Domain Research Articles

Exploring novel MYH7 gene variants using in silico analyses in Korean patients with cardiomyopathy

BackgroundPathogenic variants of MYH7, which encodes the beta-myosin heavy chain protein, are major causes of dilated and hypertrophic cardiomyopathy.MethodsIn this study, we used whole-genome sequencing data to identify MYH7 variants in 397 patients with various cardiomyopathy subtypes who were participating in the National Project of Bio Big Data pilot study in Korea. We also performed in silico analyses to predict the pathogenicity of the novel variants, comparing them to known pathogenic missense variants.ResultsWe identified 27 MYH7 variants in 41 unrelated patients with cardiomyopathy, consisting of 20 previously known pathogenic/likely pathogenic variants, 2 variants of uncertain significance, and 5 novel variants. Notably, the pathogenic variants predominantly clustered within the myosin motor domain of MYH7. We confirmed that the novel identified variants could be pathogenic, as indicated by high prediction scores in the in silico analyses, including SIFT, Mutation Assessor, PROVEAN, PolyPhen-2, CADD, REVEL, MetaLR, MetaRNN, and MetaSVM. Furthermore, we assessed their damaging effects on protein dynamics and stability using DynaMut2 and Missense3D tools.ConclusionsOverall, our study identified the distribution of MYH7 variants among patients with cardiomyopathy in Korea, offering new insights for improved diagnosis by enriching the data on the pathogenicity of novel variants using in silico tools and evaluating the function and structural stability of the MYH7 protein.

UNC-45 assisted myosin folding depends on a conserved FX3HY motif implicated in Freeman Sheldon Syndrome

Myosin motors are critical for diverse motility functions, ranging from cytokinesis and endocytosis to muscle contraction. The UNC-45 chaperone controls myosin function mediating the folding, assembly, and degradation of the muscle protein. Here, we analyze the molecular mechanism of UNC-45 as a hub in myosin quality control. We show that UNC-45 forms discrete complexes with folded and unfolded myosin, forwarding them to downstream chaperones and E3 ligases. Structural analysis of a minimal chaperone:substrate complex reveals that UNC-45 binds to a conserved FX3HY motif in the myosin motor domain. Disrupting the observed interface by mutagenesis prevents myosin maturation leading to protein aggregation in vivo. We also show that a mutation in the FX3HY motif linked to the Freeman Sheldon Syndrome impairs UNC-45 assisted folding, reducing the level of functional myosin. These findings demonstrate that a faulty myosin quality control is a critical yet unexplored cause of human myopathies.

Recessive variants in MYO1C as a potential novel cause of proteinuric kidney disease.

Steroid-resistant nephrotic syndrome is the second leading cause of chronic kidney disease among patients < 25years of age. Through exome sequencing, identification of > 65 monogenic causes has revealed insights into disease mechanisms of nephrotic syndrome (NS). To elucidate novel monogenic causes of NS, we combined homozygosity mapping with exome sequencing in a worldwide cohort of 1649 pediatric patients with NS. We identified homozygous missense variants in MYO1C in two unrelated children with NS (c.292C > T, p.R98W; c.2273 A > T, p.K758M). We evaluated publicly available kidney single-cell RNA sequencing datasets and found MYO1C to be predominantly expressed in podocytes. We then performed structural modeling for the identified variants in PyMol using aligned shared regions from two available partial structures of MYO1C (4byf and 4r8g). In both structures, calmodulin, a common regulator of myosin activity, is shown to bind to the IQ motif. At both residue sites (K758; R98), there are ion-ion interactions stabilizing intradomain and ligand interactions: R98 binds to nearby D220 within the myosin motor domain and K758 binds to E14 on a calmodulin molecule. Variants of these charged residues to non-charged amino acids could ablate these ionic interactions, weakening protein structure and function establishing the impact of these variants. We here identified recessive variants in MYO1C as a potential novel cause of NS in children.

A Laing distal myopathy-associated proline substitution in the β-myosin rod perturbs myosin cross-bridging activity.

Proline substitutions within the coiled-coil rod region of the β-myosin gene (MYH7) are the predominant mutations causing Laing distal myopathy (MPD1), an autosomal dominant disorder characterized by progressive weakness of distal/proximal muscles. We report that the MDP1 mutation R1500P, studied in what we believe to be the first mouse model for the disease, adversely affected myosin motor activity despite being in the structural rod domain that directs thick filament assembly. Contractility experiments carried out on isolated mutant muscles, myofibrils, and myofibers identified muscle fatigue and weakness phenotypes, an increased rate of actin-myosin detachment, and a conformational shift of the myosin heads toward the more reactive disordered relaxed (DRX) state, causing hypercontractility and greater ATP consumption. Similarly, molecular analysis of muscle biopsies from patients with MPD1 revealed a significant increase in sarcomeric DRX content, as observed in a subset of myosin motor domain mutations causing hypertrophic cardiomyopathy. Finally, oral administration of MYK-581, a small molecule that decreases the population of heads in the DRX configuration, significantly improved the limited running capacity of the R1500P-transgenic mice and corrected the increased DRX state of the myofibrils from patients. These studies provide evidence of the molecular pathogenesis of proline rod mutations and lay the groundwork for the therapeutic advancement of myosin modulators.

Alternative relay regulates the adenosine triphosphatase activity of Locusta migratoria striated muscle myosin.

Locust (Locusta migratoria) has a single striated muscle myosin heavy chain (Mhc) gene, which contains 5 clusters of alternative exclusive exons and 1 differently included penultimate exon. The alternative exons of Mhc gene encode 4 distinct regions in the myosin motor domain, that is, the N-terminal SH3-like domain, one lip of the nucleotide-binding pocket, the relay, and the converter. Here, we investigated the role of the alternative regions on the motor function of locust muscle myosin. Using Sf9-baculovirus protein expression system, we expressed and purified 5 isoforms of the locust muscle myosin heavy meromyosin (HMM), including the major isoform in the thorax dorsal longitudinal flight muscle (FL1) and 4 isoforms expressed in the abdominal intersegmental muscle (AB1 to AB4). Among these 5 HMMs, FL1-HMM displayed the highest level of actin-activated adenosine triphosphatase (ATPase) activity (hereafter referred as ATPase activity). To identify the alternative region(s) responsible for the elevated ATPase activity of FL1-HMM, we produced a number of chimeras of FL1-HMM and AB4-HMM. Substitution with the relay of AB4-HMM (encoded by exon-14c) substantially decreased the ATPase activity of FL1-HMM, and conversely, the relay of FL1-HMM (encoded by exon-14a) enhanced the ATPase activity of AB4-HMM. Mutagenesis showed that the exon-14a-encoded residues Gly474 and Asn509 are responsible for the elevated ATPase activity of FL1-HMM. Those results indicate that the alternative relay encoded by exon-14a/c play a key role in regulating the ATPase activity of FL1-HMM and AB4-HMM.

The organisation of titin at the centre of the vertebrate striated muscle thick filament.

Striated muscle sarcomeres operate by sliding of the regular arrays of interdigitating thin (actin) and thick (myosin) filaments. Thick filaments are held together at their centre by M-band bridges, maintaining sarcomere structure during contraction and relaxation. The giant sarcomeric protein titin is instrumental in maintaining order, since C-terminal truncations of titin lead to loss of A-band alignment and sarcomere failure. The C-terminal sequence of titin encodes a kinase domain, 10 Ig domains and linker sequences, and is associated with the bare zone at the centre of the sarcomere. Unlike the cross-bridge associated sequence, it does not show any correlation with the organisation of the thick filament bare zone and associated M-band bridges. The precise disposition of titin remains unclear, e.g. whether anti-parallel molecules overlap at centre of the filament. We used superresolution light microscopy with new titin antibodies to establish the position of several titin epitopes in the bare zone, together with key markers for the M-band proteins myomesin and M-protein. We show that the titin kinase domain is at the edge of the bare zone, about 70 nm from the M-band centre at the first myosin motor domains and may therefore account for the first of the accessory protein stripes in the A-band. SAXS data of two titin constructs covering several domains of the bare-zone associated sequence, one from the beginning including the kinase and the other from the end, suggest that they are extended structures but with compact linkers. Conversely, the long insert sequence between M-band Ig-domains 3 and 4 (Ig-162 and Ig-163) appears to be more extensive. Our combined data suggests that in the bare zone, titin molecules from each side of the thick filament overlap each other by the width of the M-band.

Conformational and energetic basis of actomyosin force generation.

Interactions between myosin and actin are essential in producing different types of cellular forces. Two key players in muscle contraction, myosin and actin, slide along each other to produce force using the energy from ATP hydrolysis. Targeting the motor domain of cardiac myosin, several small molecules have been proposed to treat cardiomyopathy. A clear mechanistic picture for the allosteric control in the actomyosin complex can facilitate computer-aided drug discovery. Atomistic simulation approaches such as molecular dynamics (MD) are limited for studying the motor functional cycle owing to the slow timescale as well as a lack of various intermediate structures. Here, integrating comparative modeling and enhanced sampling simulations, we reveal how the human cardiac myosin generates force in the cross-bridge cycle at atomic resolution. Initial conformational ensembles for different actomyosin states are learned from multiple structural templates with Rosetta. This enables us to efficiently sample the energy landscape of the system using Gaussian accelerated MD. Key myosin loop residues, whose substitutions are related to cardiomyopathy, are identified to form stable or transient interactions with actin. We find that the actin-binding cleft closure is allosterically coupled to the myosin core transitions and products release from the active site. Furthermore, a gate between switch I and II is suggested to control phosphate release at the pre-powerstroke state. The predicted metastable states and corresponding energetics are consistent with available experimental data and provide insights into the timing of elementary mechanochemical events.

Preclinical characterization of CK-4021586, a new class of cardiac myosin inhibitors for the treatment of hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common monogenetic heart disease, with an estimated carrier prevalence of ∼1 in 500. Hypercontractility of the cardiac sarcomere is a common driver of the pathological hypertrophic cardiac remodeling that is a hallmark of the disease. Clinical trials of the selective cardiac myosin inhibitors mavacamten and aficamten have demonstrated that inhibition of the biochemical activity of cardiac myosin can induce cardiac remodeling and improve both patient symptoms and exercise capacity in HCM with left ventricular outflow tract obstruction (oHCM). The diverse genetic underpinnings of HCM suggest that it is possible that additional classes of cardiac myosin inhibitors with distinct biochemical mechanisms-of-action may also have a salutary impact on disease expression. While both mavacamten and aficamten bind to the single-headed motor domain of myosin (subfragment-1) and inhibit its ATPase activity, CK-4021586 (CK-586) is a new class of cardiac myosin inhibitor that inhibits the ATPase activity of two-headed heavy meromyosin (HMM) but not single-headed subfragment-1. CK-586 is a partial inhibitor of cardiac myofibrillar ATPase activity (EC50 2.9 µM, maximal inhibition ∼50%) that requires the regulatory light chain. Notably, fractional shortening of electrically-paced adult rat ventricular cardiomyocytes was inhibited almost completely (>80% at 5 µM) without alterations in the calcium (Ca2+) transient. In normal Sprague Dawley rats, CK-586 reduced cardiac fractional shortening in a dose and concentration-dependent manner. In conclusion, CK-586 is a novel, small molecule, cardiac myosin inhibitor that reduces cardiac contractility in vitro and in vivo. CK-586 has a biochemical mechanism of action distinct from both mavacamten and aficamten, providing an additional tool to decrease the number of functionally-available myosin heads and treat the cardiac hypercontractility that forms the pathologic basis of HCM.

Exploring the effect of myosin modulators on the ATP hydrolysis step of human cardiac beta myosin using transition path sampling.

In recent years, cardiac myosin emerged as a potent target for various therapeutic agents termed as myosin modulators in treating genetic cardiomyopathies. Omecamtiv Mecarbil (OM) is one such promising cardiac-specific drug directly binding to an allosteric site of the myosin motor domain. OM is observed to enhance cardiac contractions and affects the various stages of the cross-bridge cycle. Our study investigates the atomistic details of the variations caused by OM on the chemical reaction of ATP to ADP and HPO42- using the method of Transition Path Sampling. We also incorporated the study of mutant forms of myosin causing genetic cardiomyopathies: R712L, P710R and F764L in presence of OM to observe the mutation specific behavior of the same on the hydrolysis step. We also further extended our methodology to study the effect of the recently FDA approved myosin effector Mavacamten on the ATP hydrolysis step as well. Unlike OM, mavacamten is observed to inhibit cardiac contractile performance. Since the crystal structures of mavacamten-bound myosin are not available yet, we employed docking process to develop probable structure of the same. This study will help provide a detailed molecular level understanding of the effect of the two classes of the cardiac-specific myosin effectors on the hydrolysis step and hence, further expand the approach to the other significant steps of the chemomechanical cycle, aiding in targeted applications of the therapeutic agents.

Drug specificity and affinity are encoded in the probability of cryptic pocket opening in myosin motor domains.

The design of compounds that can discriminate between closely related target proteins remains a central challenge in drug discovery. Specific therapeutics targeting the highly conserved myosin motor family are urgently needed as mutations in at least six of its members cause numerous diseases. Allosteric modulators, like the myosin-II inhibitor blebbistatin, are a promising means to achieve specificity. However, it remains unclear why blebbistatin inhibits myosin-II motors with different potencies given that it binds at a highly conserved pocket that is always closed in blebbistatin-free experimental structures. We hypothesized that the probability of pocket opening is an important determinant of the potency of compounds like blebbistatin. To test this hypothesis, we used Markov state models (MSMs) built from over 2 ms of aggregate molecular dynamics simulations with explicit solvent. We find that blebbistatin's binding pocket readily opens in simulations of blebbistatin-sensitive myosin isoforms. Comparing these conformational ensembles reveals that the probability of pocket opening correctly identifies which isoforms are most sensitive to blebbistatin inhibition and that docking against MSMs quantitatively predicts blebbistatin binding affinities (R2=0.82). In a blind prediction for an isoform (Myh7b) whose blebbistatin sensitivity was unknown, we find good agreement between predicted and measured IC50s (0.67 μM vs. 0.36 μM). Therefore, we expect this framework to be useful for the development of novel specific drugs across numerous protein targets.

Protrusion growth driven by myosin-generated force.

Actin-based protrusions extend from the surface of all eukaryotic cells, where they support diverse activities essential for life. Models of protrusion growth hypothesize that actin filament assembly exerts force for pushing the plasma membrane outward. However, membrane-associated myosin motors are also abundant in protrusions, although their potential for contributing, growth-promoting force remains unexplored. Using an inducible system that docks myosin motor domains to membrane-binding modules with temporal control, we found that application of myosin-generated force to the membrane is sufficient for driving robust protrusion elongation in human, mouse, and pig cell culture models. Protrusion growth scaled with motor accumulation, required barbed-end-directed force, and was independent of cargo delivery or recruitment of canonical elongation factors. Application of growth-promoting force was also supported by structurally distinct myosin motors and membrane-binding modules. Thus, myosin-generated force can drive protrusion growth, and this mechanism is likely active in diverse biological contexts.

Myosin-II proteins are involved in the growth, morphogenesis, and virulence of the human pathogenic fungus Mucor circinelloides.

Mucormycosis is an emerging lethal invasive fungal infection. The infection caused by fungi belonging to the order Mucorales has been reported recently as one of the most common fungal infections among COVID-19 patients. The lack of understanding of pathogens, particularly at the molecular level, is one of the reasons for the difficulties in the management of the infection. Myosin is a diverse superfamily of actin-based motor proteins that have various cellular roles. Four families of myosin motors have been found in filamentous fungi, including myosin I, II, V, and fungus-specific chitin synthase with myosin motor domains. Our previous study on Mucor circinelloides, a common pathogen of mucormycosis, showed that the Myo5 protein (ID 51513) belonging to the myosin type V family had a critical impact on the growth and virulence of this fungus. In this study, to investigate the roles of myosin II proteins in M. circinelloides, silencing phenotypes and null mutants corresponding to myosin II encoding genes, designated mcmyo2A (ID 149958) and mcmyo2B (ID 136314), respectively, were generated. Those mutant strains featured a significantly reduced growth rate and impaired sporulation in comparison with the wild-type strain. Notably, the disruption of mcmyo2A led to an almost complete lack of sporulation. Both mutant strains displayed abnormally short, septate, and inflated hyphae with the presence of yeast-like cells and an unusual accumulation of pigment-filled vesicles. In vivo virulence assays of myosin-II mutant strains performed in the invertebrate model Galleria mellonella indicated that the mcmyo2A-knockout strain was avirulent, while the pathogenesis of the mcmyo2B null mutant was unaltered despite the low growth rate and impaired sporulation. The findings provide suggestions for critical contributions of the myosin II proteins to the polarity growth, septation, morphology, pigment transportation, and pathogenesis of M. circinelloides. The findings also implicate the myosin family as a potential target for future therapy to treat mucormycosis.

MYO5B Gene Mutations: A Not Negligible Cause of Intrahepatic Cholestasis of Infancy With Normal Gamma-Glutamyl Transferase Phenotype.

Progressive familial intrahepatic cholestasis is an expanding group of autosomal recessive intrahepatic cholestatic disorders. Recently, next-generation sequencing allowed identifying new genes responsible for new specific disorders. Two biochemical phenotypes have been identified according to gamma-glutamyltransferase (GGT) activity. Mutations of the myosin 5B gene (MYO5B) are known to cause microvillus inclusion disease. Recently, different mutations in MYO5B gene have been reported in patients with low-GGT cholestasis. A multicenter retrospective and prospective study was conducted in 32 children with cryptogenic intrahepatic cholestasis. Clinical, biochemical, histological, and treatment data were analyzed in these patients. DNA from peripheral blood was extracted, and all patients were studied by whole exome sequencing followed by Sanger sequencing. Six patients out of 32 had mutations in the MYO5B gene. Of these six patients, the median age at disease onset was 0.8 years, and the median length of follow-up was 4.2 years. The most common signs were pruritus, poor growth, hepatomegaly, jaundice, and hypocholic stools. Two patients also showed intestinal involvement. Transaminases and conjugated bilirubin were moderately increased, serum bile acids elevated, and GGT persistently normal. At anti-Myo5B immunostaining, performed in liver biopsy of two patients, coarse granules were evident within the cytoplasm of hepatocytes while bile salt export pump was normally expressed at the canalicular membrane. Six variants in homozygosity or compound heterozygosity in the MYO5B gene were identified, and three of them have never been described before. All nucleotide alterations were located on the myosin motor domain except one missense variant found in the isoleucine-glutamine calmodulin-binding motif. We identified causative mutations in MYO5B in 18.7% of a selected cohort of patients with intrahepatic cholestasis confirming a relevant role for the MYO5B gene in low-GGT cholestasis.

Computational interpretation of the effect of omecamtiv mecarbil on the recovery stroke of human cardiac beta myosin, using metadynamics

The small molecule omecamtiv mecarbil (OM) is an allosteric effector binding to the motor domain of the cardiac myosin II, observed to enhance contraction of cardiac muscle. The drug affects the major steps of the myosin mechanochemical cycle central to the contractile function, the recovery stroke being one of them. The recovery stroke primes the lever arm of the myosin leading to transition from the post-rigor state to the pre-powerstroke state, preparing the active residues for catalyzing ATP hydrolysis.

Signatures of allosteric modulator specificity are encoded in myosin motor domain equilibrium fluctuations

Allosteric modulators of myosin motor function have significant potential as treatments for cardiac and muscle diseases. The mechanisms by which known allosteric modulators of myosin function achieve specificity remain unknown because these compounds bind at dynamically changing ‘cryptic’ sites that in some cases are perfectly conserved across isoforms. Here, we modeled the distribution of ligand-free conformations for eight different myosin isoforms to test the hypothesis that differences in cryptic pocket opening might explain the specificity of blebbistatin and CK-571.

PNPT1, MYO15A, PTPRQ, and SLC12A2-associated genetic and phenotypic heterogeneity among hearing impaired assortative mating families in Southern India.

The study was conducted between 2018 and 2020. From a cohort of 113 hearing impaired (HI), five non-DFNB12 probands identified with heterozygous CDH23 variants were subjected to exome analysis. This resolved the etiology of hearing loss (HL) in four South Indian assortative mating families. Six variants, including three novel ones, were identified in four genes: PNPT1 p.(Ala46Gly) and p.(Asn540Ser), MYO15A p.(Leu1485Pro) and p.(Tyr1891Ter), PTPRQ p.(Gln1336Ter), and SLC12A2 p.(Pro988Ser). Compound heterozygous PNPT1 variants were associated with DFNB70 causing prelingual profound sensorineural hearing loss (SNHL), vestibular dysfunction, and unilateral progressive vision loss in one family. In the second family, MYO15A variants in the myosin motor domain, including a novel variant, causing DFNB3, were found to be associated with prelingual profound SNHL. A novel PTPRQ variant was associated with postlingual progressive sensorineural/mixed HL and vestibular dysfunction in the third family with DFNB84A. In the fourth family, the SLC12A2 novel variant was found to segregate with severe-to-profound HL causing DFNA78, across three generations. Our results suggest a high level of allelic, genotypic, and phenotypic heterogeneity of HL in these families. This study is the first to report the association of PNPT1, PTPRQ, and SLC12A2 variants with HL in the Indian population.

When fat meets the engine: implications of dietary rumenic acid on myosin-targeting therapies in heart failure.

When fat meets the engine: implications of dietary rumenic acid on myosin-targeting therapies in heart failure.

Myosin-based regulation of twitch and tetanic contractions in mammalian skeletal muscle.

Time-resolved X-ray diffraction of isolated fast-twitch muscles of mice was used to show how structural changes in the myosin-containing thick filaments contribute to the regulation of muscle contraction, extending the previous focus on regulation by the actin-containing thin filaments. This study shows that muscle activation involves the following sequence of structural changes: thin filament activation, disruption of the helical array of myosin motors characteristic of resting muscle, release of myosin motor domains from the folded conformation on the filament backbone, and actin attachment. Physiological force generation in the 'twitch' response of skeletal muscle to single action potential stimulation is limited by incomplete activation of the thick filament and the rapid inactivation of both filaments. Muscle relaxation after repetitive stimulation is accompanied by a complete recovery of the folded motor conformation on the filament backbone but by incomplete reformation of the helical array, revealing a structural basis for post-tetanic potentiation in isolated muscles.

Allosteric modulation of cardiac myosin mechanics and kinetics by the conjugated omega‐7,9 trans‐fat rumenic acid

Direct binding of rumenic acid to the cardiac myosin-2 motor domain increases the release rate for orthophosphate and increases the Ca2+ responsiveness of cardiac muscle at low load. Physiological cellular concentrations of rumenic acid affect the ATP turnover rates of the super-relaxed and disordered relaxed states of β-cardiac myosin, leading to a net increase in myocardial metabolic load. In Ca2+ -activated trabeculae, rumenic acid exerts a direct inhibitory effect on the force-generating mechanism without affecting the number of force-generating motors. In the presence of saturating actin concentrations rumenic acid binds to the β-cardiac myosin-2 motor domain with an EC50 of 200nM. Molecular docking studies provide information about the binding site, the mode of binding, and associated allosteric communication pathways. Free rumenic acid may exceed thresholds in cardiomyocytes above which contractile efficiency is reduced and interference with small molecule therapeutics, targeting cardiac myosin, occurs. Based on experiments using purified myosin motor domains, reconstituted actomyosin complexes and rat heart ventricular trabeculae, we demonstrate direct binding of rumenic acid, the cis-delta-9-trans-delta-11 isomer of conjugated linoleic acid, to an allosteric site located in motor domain of mammalian cardiac myosin-2 isoforms. In the case of porcine β-cardiac myosin, the EC50 for rumenic acid varies from 10.5μM in the absence of actin to 200nM in the presence of saturating concentrations of actin. Saturating concentrations of rumenic acid increase the maximum turnover of basal and actin-activated ATPase activity of β-cardiac myosin approximately 2-fold but decrease the force output per motor by 23% during isometric contraction. The increase in ATP turnover is linked to an acceleration of the release of the hydrolysis product orthophosphate. In the presence of 5μM rumenic acid, the difference in the rate of ATP turnover by the super-relaxed and disordered relaxed states of cardiac myosin increases from 4-fold to 20-fold. The equilibrium between the two functional myosin states is not affected by rumenic acid. Calcium responsiveness is increased under zero-load conditions but unchanged under load. Molecular docking studies provide information about the rumenic acid binding site, the mode of binding, and associated allosteric communication pathways. They show how the isoform-specific replacement of residues in the binding cleft induces a different mode of rumenic acid binding in the case of non-muscle myosin-2C and blocks binding to skeletal muscle and smooth muscle myosin-2 isoforms.

Conformational Distributions of Isolated Myosin Motor Domains Encode their Mechochemical Properties

Myosin motor domains perform an extraordinary diversity of biological functions despite sharing a common mechanochemical cycle. Motors are adapted to their function, in part, by tuning the thermodynamics and kinetics of steps in this cycle. However, it remains unclear how sequence encodes these differences, since biochemically distinct motors often have nearly indistinguishable crystal structures. We hypothesized that sequences produce distinct biochemical phenotypes by modulating the relative probabilities of an ensemble of conformations primed for different functional roles. To test this hypothesis, we modeled the distribution of conformations for 12 myosin motor domains by building Markov state models (MSMs) from an unprecedented two milliseconds of all-atom, explicit-solvent molecular dynamics simulations. Comparing motors reveals shifts in the balance between nucleotide-favorable and nucleotide-unfavorable P-loop conformations that predict experimentally measured duty ratios and ADP release rates better than sequence or individual structures. This result demonstrates the power of an ensemble perspective for interrogating sequence-function relationships.