The G256E HCM mutation prolongs cardiac muscle relaxation via altered nucleotide handling

The G256E HCM mutation prolongs cardiac muscle relaxation via altered nucleotide handling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The G256E HCM mutation prolongs cardiac muscle relaxation via altered nucleotide handling Michael Regnier, Kerry Kao, Matthew Childers, Divya Pathak, Rama Reddy Goluguri, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7602931/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Mutations in myosin alter its motor functions in diverse ways by affecting different structural and chemo-mechanical events. Multidisciplinary strategies can be used to understand how varying alterations in motor function converge to common phenotypes like hypercontractility and hypertrophic cardiomyopathy (HCM). Here, we combined molecular dynamics (MD) simulations with protein biochemical and myofibril mechanical analyses to study the HCM-causing myosin variant G256E. MD simulations demonstrated that G256E induces structural changes that increase the work required to displace ADP.Mg 2+ from actomyosin complex. Stopped-flow biochemical analysis demonstrated increased ADP affinity for actomyosin and single myofibril mechanics analysis demonstrated increased force generation and reduced ADP sensitivity of the early, slow phase of relaxation. Together, these results demonstrate that slower ADP release from myosin during contraction is a significant contributor to pathological contractile nature of the G256E mutation. This study highlights the importance of detailed chemo-mechanical analysis of mutations associated with hereditary cardiac diseases. Biological sciences/Chemical biology/Mechanism of action Biological sciences/Structural biology/Molecular modelling hiPSC-CMs myosin ADP release molecular dynamics simulations muscle mechanics muscle kinetics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Hypertrophic cardiomyopathy (HCM) affects 1 in 500 people globally and is implicated as a contributing cause of other life-threatening heart diseases such as atrial fibrillation, heart failure, and arrhythmia resulting in sudden death. 1 About 60% of HCM cases are caused by familial inheritance of a single autosomal dominant mutation within a gene encoding a sarcomere protein. 2 In particular, the MYH7 gene encoding the β-myosin heavy chain (MHC) is one of the two most common hotspots for HCM mutations. 3 Myosin is the motor protein responsible for muscle contraction, converting chemical energy produced by ATP hydrolysis and release of products (P i , ADP) into the mechanical work of force production and muscle cell shortening. 4 This involves changes in myosin's structure as it interacts with actin to accelerate the release of ATP hydrolysis products (P i , ADP) in a process known as chemo-mechanical transduction. 5–7 Structural perturbations resulting from disease-associated mutations can disrupt the kinetics and/or mechanics of the chemo-mechanical cycle and can lead to initiation and progression of disease. 8 The G256E MYH7 mutation, associated with HCM, 9 is located in a hairpin turn that connects two strands in the central β-sheet of transducer region of the myosin head. This region conveys information between the nucleotide binding pocket and actin interface of myosin, both critical to chemo-mechanical transduction during contraction and relaxation. 10 In a recent study, we employed CRISPR-Cas9 to edit this mutation into human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to produce a heterozygous (+/-) isogenic model for studying its impact at multiple scales of cardiac muscle organization. 8 A biochemical single ATP turnover assay performed on purified human β-cardiac 25-hep heavy meromyosin (HMM) containing the G256E mutation results in a reduced population of myosin in a low basal ATPase state, often described as the super-relaxed (SRX) state, suggesting a greater portion of the myosin population is available to contribute to muscle contraction. Comparison of the actin-activated ATPase activity of two versions of G256E myosin that differed only in the length of their coiled-coil tail confirmed that the mutation disrupted the ability of myosin to fold back into a sequestered, low ATPase state. This supports the hypothesis that the mutation results in more heads being available to interact with actin at the beginning of contractions. Slower motility in an in vitro F-actin motility assay indicated the mutation results in a higher myosin duty ratio, suggesting that the actomyosin detachment rate may also be affected. This idea was supported by measures of the contraction and relaxation properties of isolated myofibrils from hiPSC-CMs. The kinetics of the slow, early phase of relaxation, which measures the myosin detachment rate from thin filaments, 11 was slower for G256E versus WT myofibrils. Molecular dynamics (MD) simulations of the myosin M.ATP-state suggested that G256E disrupts the shape and stability of the central β-sheet, potentially altering communication between the nucleotide-binding pocket and actin-binding surface. This communication path is critical to open the nucleotide binding cleft that allows ADP release. This, in turn, is required before a new ATP molecule can bind in the nucleotide pocket and lead to the dissociation of myosin from actin. While these findings are suggestive that myosin-actin chemo-mechanics are altered as part of the hypercontractile phenotype of G256E, it is important to understand which particular step(s) of the cross-bridge cycle are affected. The ADP release step is rate limiting for cross-bridge formation in sarcomeres during loaded contractions and is also a determinant of the rate of relaxation. 12 This is supported by biochemical, 13 mechanical, 14 single molecule, 15 and structural 16 evidence, and holds true across multiple classes of myosins from diverse species. Recent studies have shown that various myosin mutations can alter the affinity of myosin for ADP and consequently affect cross-bridge cycling kinetics. 17 Thus, detailed study of how the G256E mutation affects ADP release during chemo-mechanical cycling is merited. Here we report the effects of myosin G256E on ADP release from the nucleotide binding pocket of myosin and its effects on myosin and myofibril chemo-mechanical function. MD simulations of post-powerstroke myosin containing ADP (with Mg 2+ ) complexed with actin (AM.ADP) suggested that G256E significantly alters nucleotide pocket dynamics. Steered MD simulations suggested that the sequence and timing of contact pair disruptions in the pocket are altered and that more energy is required to pull ADP from the pocket. These results provide a structural basis for altered ADP-myosin affinity and dynamics. Stopped-flow kinetics analysis showed that G256E sS1 had a ~30% increase in ADP affinity over wild type (WT) sS1, validating the MD simulation predictions. To determine how this affects the chemo-mechanical function of myofibrils, we measured contraction and relaxation in the absence and presence of elevated ADP. Similar to our initial study, 8 G256E myofibrils generated significantly more force and exhibited a slower, early linear phase kinetics of relaxation ( k REL,slow ) compared to isogenic controls. When ADP was elevated in activating and relaxation solutions there was considerably less effect on k REL,slow for G256E myofibrils, suggesting that ADP release is impaired with the mutation. Combined, these experimental and computational studies support the hypothesis that the G256E mutation alters the structure of the nucleotide binding pocket, resulting in greater ADP affinity for G256E myosin than WT myosin, and this results in slower ADP release to slow crossbridge detachment and prolong contractile relaxation. To our knowledge, this is the first study to examine the reaction pathway and free energy changes associated with release of Mg 2+ -ADP from a post-powerstroke-like state of the actomyosin complex at the atomic scale with molecular simulations. These results highlight the power of combining experimental measures with in-depth computational dynamics to provide detailed structure-based mechanistic insights at the atomistic scale for protein and contractile organelle (myofibril) function. Materials and Methods 3.1 Computational Model Preparation The initial actin+myosin+ADP+Mg 2+ (AM.ADP-Mg 2+ ) structure model was generated based on cryo-EM structures of post-powerstroke actin-bound human cardiac myosin solved by Doran et al. (PDB ID: 8EFH, 3.3 Å resolution; PDB ID: 8EFE, 3.8 Å resolution). 16,18 Our model includes the myosin motor and actin 5-mer from 8EFH; tropomyosin was excluded (chains O, P). Then we grafted the converter domain, tail and essential light chain (ELC) from 8EFE onto the actomyosin complex. Missing disordered heavy atoms were constructed via Modeller 19 . The disordered N-terminal extension of the ELC was not modeled. The final WT model includes actin residues (2-377), myosin residues (1-810), and ELC residues (44-195). A model for the mutant G256E actomyosin complex was generated via in silico mutation of the WT structure with UCSF Chimera. 20,21 The resulting two systems (WT and G256E) were prepared for molecular dynamics (MD) simulation using the AMBER20 simulation package 22 and the ff14SB force field 23 . Water molecules were treated with the TIP3P force field. 24 Metal ions were modeled using the Li and Merz parameter set. 25–27 ADP molecules were treated with the GAFF2 force field. 28 Partial charges for ADP were derived from a restrained electrostatic potential ( resp ) fit to quantum mechanics calculations performed with ORCA. 29 The SHAKE algorithm was used to constrain the motion of hydrogen-containing bonds. 30 Long-range electrostatic interactions were calculated using the particle mesh Ewald (PME) method. Hydrogen atoms were modeled onto the initial structure using the leap module of AMBER, and each protein was solvated with explicit water molecules in a truncated octahedral box and then neutralizing + 120 mM counterions (Na + , Cl - ) were added. Each system was minimized in three stages. First, hydrogen atoms were minimized for 1000 steps in the presence of 100 kcal/mol restraints on all heavy atoms. Second, all solvent atoms were minimized for 1,000 steps in the presence of 25 kcal/mol restraints on all protein atoms. Third, all atoms were minimized for 8,000 steps in the presence of 25 kcal/mol restraints on all backbone heavy atoms (N, O, C, and C α atoms). After minimization, systems were heated to 310 K using the NVT (constant number of particles, volume, and temperature) ensemble and in the presence of 25 kcal/mol restraints on backbone heavy atoms. Next, the systems were equilibrated over five successive stages using the NPT (constant number of particles, pressure, and temperature) ensemble. During the first four stages, the systems were equilibrated for 5 ns in the presence of 25 to 1 kcal/mol restraints on backbone heavy atoms. During the final equilibration stage, the systems were equilibrated for 5 ns in the absence of restraints. A 10 Å nonbonded cutoff was used for all preparation and production simulations. 3.2 Conventional MD Production and Analysis The equilibrated systems were then simulated using conventional molecular dynamics protocols in the NVT ensemble in triplicate for 500 ns each (2 systems, three replicate simulations per system, 500 ns sampling per replicate simulation = 3 µs net sampling) and coordinates were saved every 10 ps. Molecular analyses were performed using cpptraj . 31 C α RMSD values for each simulation were calculated for all residues and relative to the minimized starting structure. C α RMSF values were calculated after aligning the trajectory to the average structure. Residue-residue contacts were counted if at least one pair of heavy atoms between two residues were within 5 Å of one another and contacts are presented as the fraction of simulation time that two residues were interacting. Nucleotide dynamics were evaluated by monitoring 6 dihedral angles that describe ADP structure using internal coordinates, as previously described. 32 The 6 calculated dihedral angles were defined as: θ 1 (atoms: O1B-PB-O3A-PA), θ 2 (PB-O3A-PA-O5′), θ 3 (O3A-PA-O5′-C5′), θ 4 (PA-O5′-C5′-C4′), θ 5 (O5′-C5′-C4′-O4′), and θ 6 (O4′-C1′-N9-C8). Ramachandran-style dihedral angle maps (θ 1 vs θ 2 , θ 3 vs θ 4 , and θ 5 vs θ 6 ) describe the relative orientation of the phosphate groups, the sugar ring, and the purine ring. The orientation of the myosin tail relative to the actin filament was calculated using the ‘vector’ module of cpptraj as previously described. 33 We also calculated interaction energies between myosin residues and either ADP or Mg 2+ using the linear interaction energy module. Electrostatic and van der Waals interaction energies were combined. 3.3 Steered molecular dynamics (sMD) Pre-production protocols were identical to those for the conventional MD simulations except that we employed the hydrogen mass partitioning scheme 34 to enable a 4 fs time step, the total time for the heating and equilibration phases was 1 ns, and 25 kcal/mol restraints were present on all protein heavy backbone atoms, ADP, and Mg 2+ during the entire equilibration. During the production simulations we steered ADP.Mg 2+ 14.4 Å away from the nucleotide binding pockets using a center of mass distance-based reaction coordinate. ADP.Mg 2+ was steered away at 1 Å/nanosecond and so production simulations were 10.8 ns long (WT) or 10 ns long (G256E). 60 WT and 60 G256E simulations were performed. We calculated the average work done to displace ADP, the free energy profiles along the reaction coordinate, the Jarzynski average trajectories, and the average myosin-ADP.Mg 2+ interactions made along the reaction coordinate. 3.4 sS1 protein purification Recombinant WT and G256E human β-cardiac sS1 myosins were purified as described previously. 35 Briefly, human β-cardiac myosin heavy chain ( MYH7) was co-expressed with human ELC ( MYL3) in the differentiated murine myoblast C2C12 cell line (ATCC) using adenovirus generated in HEK293T cells (ATCC) using the AdEasy Vector System (Qbiogene Inc, USA). The sS1 construct is a truncated version of MYH7 (residues 1-808) followed by a flexible GSG (Gly-Ser-Gly) linker and a carboxy-terminal 8-residue (RGSIDTWV) PDZ binding peptide. The MYL3 construct contains an N-terminal FLAG tag followed by a TEV protease site for purification. C2C12 cells were infected with adenovirus constructs 48 hr after differentiation and harvested 4 days after infection in a lysis buffer containing 50 mM NaCl, 20 mM MgCl 2 , 20 mM imidazole pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 3 mM ATP, 1 mM PMSF, 5% sucrose, and Roche protease inhibitors. The harvested cells were immediately flash frozen in liquid nitrogen. The cell pellets were stored up to 2 months at –80°C and thawed at room temperature for purification. Cells were lysed in a dounce homogenizer with 50 strokes and the lysate was clarified by spinning at 30,000rpm in a Ti-60 fixed angle ultracentrifuge rotor for 30 min at 4°C. Supernatant was transferred to a fresh tube and bound to anti-FLAG resin for 1.5hr at 4°C on a nutator. The resin was washed with a buffer (150 mM NaCl, 5 mM MgCl 2 , 20 mM imidazole pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 3 mM ATP, 1 mM PMSF, 10% sucrose, and Roche protease inhibitors) and then nutated overnight with TEV protease at 4°C to cleave the ELC-myosin complex from the resin. The next day, the supernatant was purified using a HiTrap Q HP column on a fast protein liquid chromatography (FPLC) apparatus with a gradient of 0–600 mM NaCl over 20 column volumes in a buffer containing 10 mM imidazole pH 7.5, 4 mM MgCl 2 , 10% sucrose, 1 mM DTT, and 2 mM ATP. Fractions containing human β-cardiac myosin and devoid of mouse skeletal myosin (Coomassie staining on 12.5% SDS PAGE) were pooled and flash frozen in liquid nitrogen. Purified sS1 was buffer exchanged into stopped-flow buffer (120 mM KCl, 5 mM MgCl 2 , 20 mM MOPS, 1 mM DTT, pH 7.0) overnight prior to experiments. Concentration was measured through a Bradford protein assay (5000201, Bio-Rad). 3.5 Pyrene-actin labeling Pyrene-actin was prepared fresh and used within ~1 week. First, G-actin was prepared from lyophilized bovine cardiac muscle as described before. 36 G-actin was diluted to 1 mg/mL before polymerizing with 1 mM ATP, 50 mM KCl, and 2 mM MgCl 2 at 4 °C for 2 hrs. To label, 1.28% w/w N-(1-Pyrene)iodoacetamide (AA00G316, aablocks) was added to F-actin and stirred for 18 hrs at room temperature. Excess N-(1-Pyrene)iodoacetamide was removed by centrifuging at 2,000 x g for 5 mins at 4 °C. Labeled F-actin was pelleted by centrifugation at 92,000 x g for 3 hrs at 4 °C and resuspended in stopped-flow buffer plus minimal ATP (120 mM KCl, 5 mM MgCl 2 , 20 mM MOPS, 1 mM DTT, 200 μM ATP, pH 7.0) in a glass dounce homogenizer. Pyrene-actin was stabilized with 1:1 molar phalloidin (5301, AAT Bioquest) for at least 4 hrs before dialyzing into stopped-flow buffer overnight to remove residual ATP. 3.6 Stopped-flow kinetics Stopped-flow kinetics experiments were performed at 20 °C using a Hi-Tech ‘KinetAsyst’ stopped-flow (SF-61DX2, TgK Scientific, UK). Measurements were made in 120 mM KCl, 5 mM MgCl 2 , 20 mM MOPS, 1 mM DTT, pH 7.0 solution. Pyrene fluorescence was measured by excitation at 365 nm and emission through a GG 400 nm cutoff filter. ADP affinity ( K ADP ) was assessed by competitive binding of ATP and ADP to actomyosin. Briefly, 0.125 μM actomyosin (sS1 + pyrene-actin) was rapidly mixed with 25 µM ATP plus various amounts of ADP (0 – 640 µM). All concentrations reported were post-mixing. An average trace of at least eight individual traces was generated for each condition and fit with a double exponential. The relative k obs of the fast phase ( k rel ) was plotted vs. ADP concentration and fitted with an equation as described previously. 37 3.7 Human induced pluripotent stem cell (hiPSC) culture The parental WTC hiPSC cell line was generated by the Bruce R. Conklin Laboratory at the Gladstone Institutes and University of California, San Francisco (UCSF). 38 Cells were maintained using described methods. 39,40 A detailed cell culture protocol from the Allen Institute for Cell Science can be found on its webpage (http://allencell.org). AICS-0075-085 ACTN2-mEGFP was developed at the Allen Institute for Cell Science (http://allencell.org/cell-catalog) and is available through Coriell. AICS-0097-113 ACTN2-mEGFP MYH7 WT/WT and AICS-0097-141 ACTN2-mEGFP MYH7 WT/G256E were developed at the Allen Institute for Cell Science and are available on request. 3.8 hiPSC-Cardiomyocyte (hiPSC-CM) differentiation and maintenance hiPSCs were differentiated into cardiomyocytes using an established small-molecule protocol 41 with modifications (Fig. 5c). 42 Briefly, hiPSCs were seeded onto Matrigel-coated 12-well tissue culture treated plates at densities ranging from 50 ✕ 10 3 – 125 ✕ 10 3 cells per well and maintained for 2 days with mTeSR1 media (85850, Stemcell Technologies). On day -1, the day prior to differentiation, mTeSR1 media was supplemented with 1 μM CHIR99021 (13122, Cayman Chemical). On day 0, hiPSCs were induced with 5 μM CHIR99021 in a chemically defined media composed of RPMI 1640 (11875-093, Gibco) + 500 μg/mL bovine serum albumin (A9418, Sigma-Aldrich) + 213 μg/mL L-ascorbic acid (A8960, Sigma-Aldrich) hereafter abbreviated RBA. After 48 hours, the media was replaced with 2 μM Wnt-C59 (S7037, Selleck Chem) in RBA media. On day 4, the media was replaced with RBA media. From day 6 onwards, differentiated cells were maintained in RPMI 1640 + 1X B27 plus insulin (A1895601, Gibco) with media changes every 2-3 days. 3.9 Solutions for myofibril mechanics Solutions for isolated myofibril mechanics experiments were prepared as previously described. 43 Experimental solutions were composed of the following: 15 mM total EGTA, 80 mM total MOPS, 5 mM MgATP, 15 mM total creatine phosphate, 83 mM free K, and 52 mM free Na, all free ion concentrations were determined using MAXCHELATOR 44 ). Solutions were adjusted to pH 7.0 with NaOH. Elevated ADP solutions (5.6ADP) were made by replacing 50% of the nucleotide pool with ADP for final concentrations of 2.5 mM ATP and 2.5 mM ADP. 3.10 Isolated myofibril assay hiPSCs were differentiated into cardiomyocytes as described above. On day 14 post-differentiation, hiPSC-CMs were replated at 100k cells/cm 2 . On day 16, hiPSC-CMs were subjected to glucose starvation and lactate enrichment for 4 days in DMEM minus glucose (11966-025, Gibco) media supplemented with 4 mM sodium L-lactate (L7022, Sigma Aldrich). hiPSC-CMs were maintained in RPMI 1640 + 1X B27 plus insulin. On day 30, hiPSC-CMs were replated onto 10 kPa polyacrylamide gels in 6-well glass bottom plates (P06-20-1.5-N, Cellvis) patterned with 15 μm width Matrigel lines prepared by microcontact printing as described previously 45 , and matured for 7 days to promote elongation and formation of cell-to-cell contacts. On day 37, isolated myofibrils for mechanical and kinetic analysis hiPSC-CMs were demembranated in a 1% triton-X pCa 9.0 relaxing solution supplemented with 1:100 protease inhibitor cocktail (P8340, Sigma-Aldrich) for 30 minutes on ice. A cell scraper was then used to gently free isolated myofibrils from permeabilized cells. The supernatant was collected and gently homogenized in a glass dounce homogenizer ~5-7X. Myofibrils were mounted into a custom-built apparatus with rapid Ca 2+ solution switching (15 °C) as previously described. 43,46 Myofibrils were initially suspended in pCa 9.0 relaxing solution before rapidly switching to pCa 4.0 maximal activation solution. Myofibrils were also activated in a pCa 5.6 submaximal activation solution and a pCa 5.6ADP solution containing 50% ADP (2.5 mM ATP, 2.5 mM ADP) solution to challenge myofibrils with ADP. 3.11 Myosin separation gel Cell pellets were flash frozen in liquid nitrogen and stored at -80°C until ready to use. Cell pellets were resuspended in ~3x volume myofibril lysis buffer (5% SDS, 50 mM Tris (pH 8.5), 0.75% sodium deoxycholate, and 1x protease inhibitor cocktail). Sarcomeres and proteins were denatured by boiling samples for 10 minutes. Concentration was measured by blotting for total protein and adjusting loading. Myosin separation gel was made by combining a stacking gel (2.95% Acrylamide with 10% glycerol and pH 8.8) and resolving gel (6% Acrylamide with 10% glycerol and pH 8.8). Separation gels were run for 2 hours and 15 min at 32 mA in a cold room (4°C). Gels were stained for total protein using One-Step Lumitein™ Protein Gel Stain, 1X following manufacturer instructions. Gels were imaged on a BioRad ChemiDoc using SYPRO Ruby protein stain setting. 3.12 Statistics Conventional MD (cMD) simulations were run in triplicate (n=3) for each genotype. Statistics on cMD analyses were performed with a student’s t-test. Steered MD (sMD) simulations were run 60 times (n=60) for each genotype. A student’s t-test was used to compare work required for ADP dissociation for WT vs. G256E. For myofibrils, statistical significance was determined by 2-way ANOVA (WT n=15, G256E n=13). Stopped-flow experiments were replicated 4 times (n=4) and a student’s t-test was used to determine significance. Results 4.1 G256E alters myosin nucleotide binding pocket dynamics with ADP To investigate the possible structural changes within myosin that might result in impaired ADP release we employed conventional molecular dynamics (cMD) to simulate the time-dependent changes of the post-powerstroke state of myosin complexed with actin and ADP (AM.ADP, Fig. 1a), and how G256E alters structure and contacts at the atomic level. In general, G256E simulations had a greater change in global C α RMSD compared to WT simulations (Fig. 1b,c), presumably as a response to the change from Gly to Glu. These changes tended to stabilize by 300 ns (Fig. 1c). We calculated the change in C α RMSF per residue and found that the flexibility of the G256E structure remained very similar to WT except for loop 2 (residues 621-646) and the end of the tail (residues 771-810) (Fig. 1d). In the WT stimulations, ADP primarily formed contacts with the P-loop (residues 179-184) and Purine-ring (residues 126-131) of the nucleotide binding pocket (Fig. 2a,c). In G256E simulations, the introduction of the bulky, negatively charged sidechain (E256) formed a novel salt bridge with R169, as was observed previously in simulations of the G256E M.ATP state. 8 This structural change propagated to the nucleotide pocket. For the majority of G256E simulations, ADP preferred to adopt a conformation that closely associates with Switch-1 (residues 232-244) and the loop that connects the third b-strand of the transducer to the SH1-helix (SH1-loop, residues 673-685). In particular, two non-native contacts formed in the G256E simulations (Fig. 2c S241 & T678) and persisted for greater than 75% of combined simulation time. This new ADP conformation led to the formation of a salt bridge between D239 of Switch-1 and K679 of the SH1-loop, which persisted longer in G256E simulations (average 28.9% of simulation time) compared to WT (average 3.2% of simulation time, p = 0.051). Overall, this results in a loss of contacts between ADP and the P-loop and the Purine-ring (Fig. 2c) and altered dynamics in the nucleotide pocket in a manner that might affect ADP release. The conformation of ADP within the nucleotide binding pocket was assessed by calculation of 6 distinct dihedral angles within ADP: θ 1 (O1B-PB-O3A-PA), θ 2 (PB-O3A-PA-O5′), θ 3 (O3A-PA-O5′-C5′), θ 4 (PA-O5′-C5′-C4′), θ 5 (O5′-C5′-C4′-O4′), and θ 6 (O4′-C1′-N9-C8). The dihedral angles were analyzed via 2D Ramachandran-style plots (Fig. 2e-g). The quantified conformational entropy (Table 1) and percent coverage (Table 2) indicate that ADP is less flexible in the pocket and adopts a more stable conformation for G256E compared to WT simulations. We calculated the interaction energies between myosin and ADP.Mg 2+. Residues with at least 3 kT of interaction energy were considered part of the nucleotide binding pocket (residues 126, 128, 180-190, 224, 228, 232, 233, 238-243, 246, 461, 674, and 679) and most nucleotide binding pocket residues had statistically different interaction energies with ADP.Mg 2+ (Fig. 2d). We also observed a statistically different center of mass distance between ADP.Mg 2+ and the nucleotide binding pocket residues for G256E. The altered conformation of ADP.Mg 2+ in the nucleotide binding pocket led to statistically distinct interaction energies with myosin residues (Fig. 2d). 4.2 ADP release from the post-powerstroke state required more work for G256E simulations compared to WT . We used steered molecular dynamics (sMD) simulations to simulate ADP release from the nucleotide binding pocket of myosin in complex with actin (AM.ADP*). To create a reaction coordinate for steering, we calculated the linear interaction energy (lie) between myosin residues and either ADP or Mg 2+ in the cMD simulations. Myosin residues with an average lie of at least 3 kT with either ADP or Mg 2+ were selected as nucleotide binding pocket residues in the reaction coordinate (Fig. 2d). Next, we calculated the modal distance between ADP.Mg 2+ and the nucleotide binding pocket residues in the cMD simulations (WT: 3.6 Å; G256E: 4.4 Å). We then selected 120 structures from the cMD simulations (approximately 20 structures from each replicate) that had ADP.Mg 2+ - nucleotide binding pocket residue center of mass distances within 0.1 Å of the model results. Then, steered MD simulations were performed (Fig. 3d,e). On average, G256E myosin required 1.5 times as much work (WT: 58 kcal mol -1 G256e: 89 kcal mol -1 , p = 2.4 x 10 -18 ) to displace ADP.Mg 2+ from the binding pocket, indicating a stronger interaction between G256E myosin and ADP.Mg 2+ (Fig. 3a). The Jarzynski averaged trajectories followed this trend (WT: 26 kcal mol -1 G256E: 68 kcal mol -1 , a 1.6-fold difference) (Fig. 3b,c). Note that the free energy values along the reaction coordinate are dependent on the pulling speed in the sMD simulations, and relative differences in free energy along the profile are more meaningful than absolute energies here. 47 The free energy profiles show an initial steep rise along the reaction coordinate that corresponds to movement of the α and β phosphates followed by a shallower rise. More work was required to displace ADP.Mg 2+ from G256E myosin due to the burial of the adenine ring in the SH1-loop in addition to increased interactions of the phosphate groups with Switch-1. We calculated the average interactions that ADP.Mg 2+ made as they were steered out of the nucleotide binding pocket (Fig. 4a,b). We found that ADP.Mg 2+ formed different interactions with WT and G256E myosin as it left the pocket, in large part due to the burial of the adenine ring in the SH1-loop and increased interactions of the phosphates with Switch-1. For both the WT and G256E simulations, ADP was displaced from the nucleotide binding pocket and at the end of the simulations there were no interactions between ADP.Mg 2+ and P-loop or purine binding loop residues. However, at the end of every simulation ADP.Mg 2+ was still interacting with at least E228. Attempts to steer ADP.Mg 2+ further along the reaction coordinate resulted in local unfolding of myosin. We hypothesize this is due to strong interaction with E228 and a secondary reaction coordinate may be necessary to further steer ADP.Mg 2+ from this residue and based on visual inspection we do not predict differences in ADP release from this point onward. Overall, these data suggest increased binding affinity of ADP.Mg 2+ for G256E myosin facilitated through modified ADP coordination that results in an altered release trajectory. 4.3 G256E sS1 has greater affinity for ADP Next, to experimentally determine the ADP affinity of myosin ( K ADP ) we performed an ATP/ADP competitive binding assay using recombinant human β-cardiac short subfragment-1 (β-sS1), a single headed myosin construct containing only the human ventricular essential light chain. Experiments were performed by rapidly mixing 0.125 μM pyrene-actin.β-sS1 with 25 μM ATP + varying amounts of ADP (0, 20, 40, 80, 160, 320, and 640 μM) (all reported concentrations are post-mixing, Fig. 6a). In the absence of ADP, experimental curves were fit with a single-exponential which describes ATP-induced actomyosin dissociation. In the presence of ADP, experimental curves were fit with a double-exponential to account for the slow phase, which describes ADP dissociation from actomyosin. We plotted the relative k obs of the fast phase ( k rel = k obs / k obs,0 ) vs. ADP concentration to assess ADP affinity (Fig. 6b). G256E β-sS1 demonstrated ~30% increased ADP affinity ( K ADP = 33 ± 3 µM, Fig. 6c) compared to WT β-sS1 ( K ADP = 48 ± 11 µM). 4.4 G256E myofibril relaxation is slower and less sensitive to elevated ADP . To determine how the increased ADP binding affinity affects contractile properties, particularly the early, linear phase of relaxation that reflects crossbridge detachment, we used myofibrils from CRISPR/Cas9-edited hiPSC lines containing the heterozygous expression of the G256E mutation ( MYH7 WT/G256E ) and an isogenic control ( MYH7 WT/WT ). The cell lines underwent a series of quality control experiments to confirm genotype and validate that the gene-editing process did not have any off-target effects. 8 We confirmed that our stem cell differentiation and culture conditions (Fig. 5b) provided hiPSC-CMs that express predominantly β-MHC by D30, validating the D37 timepoint was appropriate for experiments (Fig 4c). The mechanical and kinetic properties of hiPSC-CM myofibrils were measured in a custom-built apparatus using a fast solution switching (~5 ms) technique, as previously described. 46 Myofibrils were activated in a submaximal calcium solution (pCa 5.6), relaxed (pCa 9.0), then activated and relaxed with solutions containing 50% ADP (pCa 5.6ADP, 2.5 mM ATP + 2.5 mM ADP) to observe the effects of elevated ADP on contractile properties. Fig. 7a shows representative traces for contraction and relaxation and Fig. 7b shows a higher resolution view of the early, linear phase of relaxation, for measurements of the slope ( k REL,slow ) and duration (t REL,slow ). Force generated at both pCa 5.6 and pCa 5.6ADP was significantly increased for G256E myofibrils by ~2X (Fig. 7c, p < 0.001) compared to WT myofibrils. Activation kinetics ( k ACT ) with elevated ADP were decreased by ~30% (Fig. 7d, p < 0.01) for both G256E and WT myofibrils. For relaxation, the main, fast phase rate ( k REL,fast ) was ~30% slower for G256E myofibrils compared to WT (Fig. 7e, p < 0.05). Addition of ADP slowed k REL,fast for both WT and G256E, but the magnitude of change was greater for WT k REL,fast , such that this rate was no longer significantly different for G256E vs WT myofibrils. For the early, linear phase of relaxation, the duration (t REL,slow ) was prolonged approximately 2-fold by the addition of ADP for both G256E and WT myofibrils, as expected, but was not significantly different between G256E and WT myofibrils at pCa 5.6 or 5.6ADP. However, the slow phase of relaxation ( k REL,slow ) was significantly impaired for G256E vs WT myofibrils with ATP only solutions (Fig. 7f, p < 0.001) at pCa 5.6, similar to what we previously reported. 8 Importantly, elevated ADP had a large effect on k REL,slow in WT myofibrils ( p < 0.0001), greatly slowing this rate, while it resulted in no appreciable change in k REL,slow for G256E myofibrils. This suggests that ADP release is already significantly impaired in G256E myofibrils, such that the response to ADP product inhibition is blunted. Discussion 5.1 Synergy between computation simulations and experiments Here, we simulated ADP release from myosin using enhanced sampling computational techniques. We validated computational results with data collected from biochemical measurements of ADP affinity for sS1 myosin motors and mechanical measurements of hiPSC-CM-derived myofibrils. Our results highlight the synergy between computational simulation and experimental validation in understanding the structure-function basis of the myosin motor and contractile dysfunction resulting from disease associated mutations and this may provide intra-protein targets for more precise therapeutics approaches. In post-powerstroke (AM.ADP) simulations, changing G256 to the E256 mutation forms an enduring salt bridge with R169. This was also observed in the M.ATP state simulations we reported recently. 8 Even though this contact was consistent in simulations of both chemo-mechanical states, there were different consequences. In the M.ATP simulations, the salt bridge led to strain in sheets β5 and β6 of the transducer region and a reduction in the stability of hydrogen bonds between these sheets. However, in post-powerstroke (AM.ADP) simulations, the instability of the transducer region was not observed. This may be due to intrinsic differences in the nucleotide binding pocket in ‘closed’ (i.e. actin-free, ATP/ADP.P i -bound forms) vs ‘open’ (i.e. actin-bound, ADP-bound and nucleotide-free forms) conformations. These findings highlight the importance of simulating myosin in different chemo-mechanical states to obtain a more complete assessment of induced changes in structural dynamics, e.g. a single state structure analysis may result in incomplete understanding or incorrect conclusions. In the case of post-powerstroke simulations, it is possible that the presence of actin stabilized the transducer, as the transducer is known to adopt different conformations in the presence and absence of actin. 48,49 The post-powerstroke simulations ultimately led to the discovery of altered nucleotide pocket dynamics due to the G256E mutation, which was consistent for all replicate simulations of the mutant. In particular, ADP formed novel contacts with the SH1-loop. This loop leads into the SH1 and SH2 helices, which link the converter domain to the rest of the myosin motor domain. In previous experimental studies, SH1-SH2 interactions were found to modulate the affinity of ADP to myosin, and a computational study found electrostatic interactions between the SH1 and SH2 helices alter ADP release. 50,51 SH1 and the relay helix also connect the converter domain to the actin-binding surface, facilitating lever arm movement. 51 Although we did not see any differences in either SH1-SH2 interaction or stability (i.e., secondary structure), it is possible some structural alterations could occur outside of our timescale (i.e., 500 ns). Our sMD simulations represent the first computational study of ADP release from the actin-myosin complex, which was made possible by the existence of a high-resolution atomic structure of post-powerstroke myosin. 16 The sMD simulations revealed that the conformation of ADP within the nucleotide pocket impacts ADP dissociation. In the case of G256E simulations, strong contacts between ADP and Switch-1 and the SH1-loop proved to be an extra energetic barrier inhibiting ADP release. sMD simulations also directed attention to particular residues that are important in ADP dissociation. K184, a positively charged residue in the P-loop lining the bottom of the pocket, is the first-line of defense in preventing ADP release, and the loss of a salt bridge between this residue and the β phosphate is necessary to initiate ADP release. Mutation of this residue to a glutamine (K184Q) has been seen in patients with HCM or left ventricular noncompaction cardiomyopathy and is currently a variant of unknown significance (VUS). 52 Future studies of mutations in this residue and how it relates to ADP release may give insight into its potential pathogenicity. The next residue identified on the reaction coordinate is D239. In WT simulations, this residue forms a salt bridge with K679, acting as an ‘atomic-level staple’ to keep the nucleotide pocket closed. This residue is of particular interest because it has been tied to multiple disease-related mutations. D239N is a likely pathogenic mutation implicated in early-onset HCM. 53,54 Based on our observations we hypothesize that the change from Asp, a negatively charged side chain, to the uncharged polar residue Asn, will disrupt the salt bridge between 239 and 679. This would likely result in faster ADP release. This may explain results from a study demonstrating faster actin gliding velocities and actin-activated ATPase rates in D239N myosin, as ADP release is the rate-limiting step in unloaded actin-myosin assays. 55 Finally, K246 is another residue that is important for P β stabilization. The mutation K246I is currently classified as likely pathogenic by Walsh et al. 56 A change from positive residue to a non-polar one would likely affect coordination of the nucleotide in the pocket through destabilization of P β . 56 In our previous study we found that, for isolated myofibrils from hiPSC-CMs, the kinetics of the slow, early phase of relaxation, k REL,slow following maximal (pCa 4.0) calcium activation was reduced. 8 This could result from either slowed ADP release from myosin or slowed ATP binding to myosin, as both steps that are required for cross-bridge detachment. Here we utilized elevated ADP as a product inhibition method to probe if ADP release from myosin is specifically altered with the G256E mutation. We chose to perform these experiments at a sub-maximal calcium level to make conditions more physiologically relevant, as pCa 5.6 is much closer to the rise in intracellular calcium that occurs during a cardiac twitch. Importantly, k REL,slow was significantly slower for G256E myofibrils at pCa 5.6 (Fig. 7f), a similar result as in our previous study at pCa 4.0. 8 The addition of 2.5 mM ADP to WT myofibrils slowed k REL,slow to a rate that was similar to that for G256E myofibrils in the absence of ADP, and addition of ADP for G256E myofibrils resulted in a significant but smaller further reduction of k REL,slow . The addition of ADP should inhibit ADP release and prolong the kinetics of this phase, which is seen in WT myofibrils but not G256E myofibrils. This suggests that the release of ADP is significantly compromised within G256E myofibrils, explaining in minimal response to ADP product inhibition. These data agree with the conclusions from our computational simulations and myosin biochemical measures regarding ADP.Mg 2+ release and greater ADP.Mg 2+ for myosin, respectively. However, we should not rule out other possible contributions to the relaxation rate, e.g. the rates of ATP binding and actomyosin dissociation. Our preliminary stopped-flow measures of mant-ATP binding to S1 or HMM indicate that the rate of ATP binding is multi-fold faster than k REL,slow for both WT and G256E myofibrils, suggesting ADP release may control myosin detachment and relaxation kinetics. This is supported by measurements of k REL,slow, which was decreased for G256E compared to WT myofibrils at pCa 5.6 (Fig. 7f), and decreased by elevation of ADP for WT, but much less for G256E myofibrils. 5.2 A note on force development . At pCa 5.6, steady state force (Fig. 7c) was approximately doubled for G256E myofibrils compared with WT myofibrils, indicated increased calcium sensitivity of force, while activation kinetics, k ACT (Fig. 7d) were not affected. This contrasts with measurements we reported for maximal activation (pCa 4.0) where both force and k ACT were approximately doubled for G256E myofibrils. 8 This suggests that the rate limiting chemo-mechanical process for the rate of force development at the sub-maximal calcium used (pCa 5.6) is new strong crossbridge formation and the force generating isomerization of myosin associated with inorganic phosphate release, and not ADP release or crossbridge detachment. The elevation of ADP did not affect steady state force but decreased k ACT by a similar amount for both WT and G256E myofibrils. Together this suggests the mutation affects the number of myosins that bind, but not the binding and force development kinetics. 5.3 Limitations of our study Here, we report the first all-atom MD simulations of β-cardiac myosin in the post-powerstroke state. However, our model includes only an actin pentamer and myosin residues 1-810. Thus, there is no effective load in our system and effects on ADP release related to tension (force) or interaction between more complex constituents of the sarcomere are not included. Some studies suggest Mg 2+ may release from myosin prior to ADP release; however, here we have simulated release of ADP.Mg 2+ as a united molecule. We have not yet simulated the rigor actomyosin complex for this mutation, nor have we explored differences in ATP binding or actomyosin attachment for G256E myosin. Additional impacts of thin filament regulatory proteins were also not considered in our current simulation studies. Finally, our models do not account for additional, more complex behavior of myosin, which includes destabilization of the interacting heads motif (IHM) that occurs with myofibril contractile activation and contributes to the hypercontractility of the G256E mutation. 8 Stopped-flow biochemical experiments involve isolated actomyosin constructs in the absence of load which may over-report the effects the G256E mutation on ADP affinity, as load reduces the rate of ADP release from myosin. 57,58 Additionally, shortened myosin constructs are unable to form the IHM found in native thick filaments. As such, we are unable to assess the combined effects or interplay between alterations of myosin recruitment (ON and OFF states) and crossbridge cycling. Myofibrils derived from hiPSC-CMs may contain fetal isoforms of sarcomeric proteins; however, our maturation protocol results in myofibrils that contain predominantly β-myosin, which is the adult ventricular isoform affected by the G256E mutation, and the crossbridge cycling rate is dominated by myosin. To mitigate the limitations of any one technique, our interdisciplinary research strategy used different models to explore the effects of a mutation at different scales to identify the common feature (ADP release) that G256E affects. Conclusions In summary, we have demonstrated that the MYH7 G256E impairs ADP release as well as the previously reported increase in myosin recruitment from the OFF state. 8 Together, these changes contribute to the clinically observed HCM phenotype of hypercontractility and impaired relaxation through delayed cross-bridge detachment and a greater population of myosin in force bearing states. Our conventional MD results suggest that mutations in the transducer region can allosterically affect the nucleotide binding pocket. Using sMD, we developed a predictive method to computationally assess a specific transition step in the cross-bridge cycle–ADP release. Our results were corroborated with experimental data obtained from myofibrils obtained from hiPSC-CMs and recombinant sS1 protein, emphasizing the necessity for both computational approaches and validation against experimental data. Similar studies could help elucidate the mechanisms of other specific mutations in myosin implicated in disease and contribute to developing additional sarcomere targeted therapies. Declarations 7. Acknowledgements This work used the Extreme Science and Engineering Discovery Environment (XSEDE) resource COMET through allocation TG-MCB200100 to M.R. XSEDE was supported by the National Science Foundation grant no. ACI-1548562. Partial funding for MCC was provided by award nos. T32HL007828 and K99HL173646 from the National Heart, Lung, and Blood Institute. Partial support for KYK was provided by T32EB032787. This research was supported by the University of Washington Center for Translational Muscle Research (CTMR) via the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health award no. P30AR074990. This work was initiated and supported by the NIH/NIGMS grant 1RM1 GM131981-03 to JAS, KMR and MR and supported by NHLBI R01HL128368 to MR. This work would not have been possible without the Allen Institute for Cell Science team who contributed with cell line generation. The parental WT unedited hiPSC line, WTC, was provided by the Bruce R. Conklin Laboratory at the Gladstone Institutes and UCSF. The Allen Institute for Cell Science wishes to thank the Allen Institute for Cell Science founder, Paul G. Allen, for his vision, encouragement, and support. The content is solely the responsibility of the authors and does not necessarily represent the official view of the NHLBI or the NIH. The authors have no financial conflicts of interest that may be construed to these data. 8. Author Contributions: Designed research, M.C.C. and M.R. Provided purified protein, D.P. and R.R.G. Performed research, M.C.C., K.K., and T.S.M. Analyzed data, M.C.C. and K.K. Manuscript writing, K.K., M.C.C., and M.R. Resources and Funding, M.R. and J.A.S. References Maron, B. J. & Maron, M. S. Hypertrophic cardiomyopathy. Lancet 381, 242–255 (2013). Marian, A. J. & Braunwald, E. Hypertrophic Cardiomyopathy. Circ. Res. 121, 749–770 (2017). Alfares, A. A. et al. Results of clinical genetic testing of 2,912 probands with hypertrophic cardiomyopathy: expanded panels offer limited additional sensitivity. Genet. Med. 17, 880–888 (2015). Sellers, J. R. Myosins: a diverse superfamily. Biochim. Biophys. Acta (BBA) - Mol. Cell Res. 1496, 3–22 (2000). Pate, E. & Cooke, R. A model of crossbridge action: the effects of ATP, ADP and Pi. J. Muscle Res. Cell Motil. 10, 181–196 (1989). Regnier, M., Lee, D. M. & Homsher, E. ATP Analogs and Muscle Contraction: Mechanics and Kinetics of Nucleoside Triphosphate Binding and Hydrolysis. Biophys. J. 74, 3044–3058 (1998). Regnier, M. & Homsher, E. The Effect of ATP Analogs on Posthydrolytic and Force Development Steps in Skinned Skeletal Muscle Fibers. Biophys. J. 74, 3059–3071 (1998). Lee, S. et al. Incomplete-penetrant hypertrophic cardiomyopathy MYH7 G256E mutation causes hypercontractility and elevated mitochondrial respiration. Proc. Natl. Acad. Sci. 121, e2318413121 (2024). Fananapazir, L. & Epstein, N. D. Genotype-phenotype correlations in hypertrophic cardiomyopathy. Insights provided by comparisons of kindreds with distinct and identical beta-myosin heavy chain gene mutations. Circulation 89, 22–32 (2018). Coureux, P., Sweeney, H. L. & Houdusse, A. Three myosin V structures delineate essential features of chemo-mechanical transduction. EMBO J. 23, 4527–4537 (2004). Tesi, C., Piroddi, N., Colomo, F. & Poggesi, C. Relaxation Kinetics Following Sudden Ca2 + Reduction in Single Myofibrils from Skeletal Muscle. Biophys. J. 83, 2142–2151 (2002). Veigel, C., Molloy, J. E., Schmitz, S. & Kendrick-Jones, J. Load-dependent kinetics of force production by smooth muscle myosin measured with optical tweezers. Nat. Cell Biol. 5, 980–986 (2003). Siemankowski, R. F., Wiseman, M. O. & White, H. D. ADP dissociation from actomyosin subfragment 1 is sufficiently slow to limit the unloaded shortening velocity in vertebrate muscle. Proc. Natl. Acad. Sci. 82, 658–662 (1985). Kovács, M., Thirumurugan, K., Knight, P. J. & Sellers, J. R. Load-dependent mechanism of nonmuscle myosin 2. Proc. Natl. Acad. Sci. 104, 9994–9999 (2007). Capitanio, M. et al. Two independent mechanical events in the interaction cycle of skeletal muscle myosin with actin. Proc. Natl. Acad. Sci. 103, 87–92 (2006). Doran, M. H. et al. Conformational changes linked to ADP release from human cardiac myosin bound to actin-tropomyosin. J. Gen. Physiol. 155, e202213267 (2023). Ujfalusi, Z. et al. Dilated cardiomyopathy myosin mutants have reduced force-generating capacity. J. Biol. Chem. 293, 9017–9029 (2018). Berman, H. M. et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000). Fiser, A., Do, R. K. G. & Šali, A. Modeling of loops in protein structures. Protein Sci. 9, 1753–1773 (2000). Webb, B. & Sali, A. Comparative Protein Structure Modeling Using MODELLER. Curr. Protoc. Bioinform. 54, 5.6.1–5.6.37 (2016). Pettersen, E. F. et al. UCSF Chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004). Case, D. A. et al. Amber 2024. University of California, San Francisco (2024). Maier, J. A. et al. ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J. Chem. Theory Comput. 11, 3696–3713 (2015). Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983). Li, P. & Merz, K. M. Taking into Account the Ion-Induced Dipole Interaction in the Nonbonded Model of Ions. J. Chem. Theory Comput. 10, 289–297 (2014). Li, P., Song, L. F. & Merz, K. M. Systematic Parameterization of Monovalent Ions Employing the Nonbonded Model. J. Chem. Theory Comput. 11, 1645–1657 (2015). Li, P., Song, L. F. & Merz, K. M. Parameterization of Highly Charged Metal Ions Using the 12-6-4 LJ-Type Nonbonded Model in Explicit Water. J. Phys. Chem. B 119, 883–895 (2015). Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A. & Case, D. A. Development and testing of a general amber force field. J. Comput. Chem. 25, 1157–1174 (2004). Neese, F., Wennmohs, F., Becker, U. & Riplinger, C. The ORCA quantum chemistry program package. J. Chem. Phys. 152, 224108 (2020). Miyamoto, S. & Kollman, P. A. Settle: An analytical version of the SHAKE and RATTLE algorithm for rigid water models. J. Comput. Chem. 13, 952–962 (1992). Roe, D. R. & Cheatham, T. E. PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data. J. Chem. Theory Comput. 9, 3084–3095 (2013). Childers, M. C., Geeves, M., Daggett, V. & Regnier, M. Modulation of post-powerstroke dynamics in myosin II by 2′-deoxy-ADP. Arch. Biochem. Biophys. 699, 108733 (2021). Childers, M. C. & Regnier, M. Dynamics of the Pre-Powerstroke Myosin Lever Arm and the Effects of Omecamtiv Mecarbil. Int. J. Mol. Sci. 25, 10425 (2024). Hopkins, C. W., Grand, S. L., Walker, R. C. & Roitberg, A. E. Long-Time-Step Molecular Dynamics through Hydrogen Mass Repartitioning. J. Chem. Theory Comput. 11, 1864–1874 (2015). Nag, S. et al. The myosin mesa and the basis of hypercontractility caused by hypertrophic cardiomyopathy mutations. Nat. Struct. Mol. Biol. 24, 525–533 (2017). Pardee, J. D. & Aspudich, J. Purification of muscle actin. Methods Enzym. 85, 164–181 (1982). Kao, K. & Geeves, M. A. Familial Cardiomyopathies, Methods and Protocols. Methods Mol. Biol. 2735, 191–211 (2023). Kreitzer, F. R. et al. A robust method to derive functional neural crest cells from human pluripotent stem cells. Am. J. stem cells 2, 119–31 (2013). Roberts, B. et al. Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization. Mol. Biol. Cell 28, 2854–2874 (2017). Roberts, B. et al. Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs. Stem Cell Rep. 12, 1145–1158 (2019). Burridge, P. W. et al. Chemically defined generation of human cardiomyocytes. Nat. Methods 11, 855–860 (2014). Bertero, A. et al. Dynamics of genome reorganization during human cardiogenesis reveal an RBM20-dependent splicing factory. Nat. Commun. 10, 1538 (2019). Mohran, S., Steczina, S., Mandrycky, C., Kao, K. & Regnier, M. Familial Cardiomyopathies, Methods and Protocols. Methods Mol. Biol. 2735, 213–233 (2023). Bers, D. M., Patton, C. W. & Nuccitelli, R. Chapter 1 A Practical Guide to the Preparation of Ca2 + Buffers. Methods Cell Biol. 99, 1–26 (2010). Ribeiro, A. J. S. et al. Contractility of single cardiomyocytes differentiated from pluripotent stem cells depends on physiological shape and substrate stiffness. Proc. Natl. Acad. Sci. 112, 12705–12710 (2015). Pioner, J. M. et al. Isolation and Mechanical Measurements of Myofibrils from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Stem Cell Rep. 6, 885–896 (2016). Izrailev, S. et al. Computational Molecular Dynamics: Challenges, Methods, Ideas, Proceedings of the 2nd International Symposium on Algorithms for Macromolecular Modelling, Berlin, May 21–24, 1997. Lect. Notes Comput. Sci. Eng. 39–65 (2011) doi: 10.1007/978-3-642-58360-5_2 . Pospich, S., Sweeney, H. L., Houdusse, A. & Raunser, S. High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism. eLife 10, e73724 (2021). Sweeney, H. L. & Houdusse, A. Structural and Functional Insights into the Myosin Motor Mechanism. Annu. Rev. Biophys. 39, 539–557 (2010). Gargey, A., Iragavarapu, S. B., Grdzelishvili, A. V. & Nesmelov, Y. E. Electrostatic interactions in the SH1-SH2 helix of human cardiac myosin modulate the time of strong actomyosin binding. J. Muscle Res. Cell Motil. 42, 137–147 (2021). Mesentean, S., Koppole, S., Smith, J. C. & Fischer, S. The Principal Motions Involved in the Coupling Mechanism of the Recovery Stroke of the Myosin Motor. J. Mol. Biol. 367, 591–602 (2007). Miller, E. M. et al. Genetic Testing in Pediatric Left Ventricular Noncompaction. Circ.: Cardiovasc. Genet. 10, e001735 (2017). Richards, S. et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 17, 405–423 (2015). Kaski, J. P. et al. Prevalence of Sarcomere Protein Gene Mutations in Preadolescent Children With Hypertrophic Cardiomyopathy. Circ.: Cardiovasc. Genet. 2, 436–441 (2009). Adhikari, A. S. et al. Early-Onset Hypertrophic Cardiomyopathy Mutations Significantly Increase the Velocity, Force, and Actin-Activated ATPase Activity of Human β-Cardiac Myosin. Cell Rep. 17, 2857–2864 (2016). Walsh, R. et al. Reassessment of Mendelian gene pathogenicity using 7,855 cardiomyopathy cases and 60,706 reference samples. Genet. Med. 19, 192–203 (2017). Cremo, C. R. & Geeves, M. A. Interaction of Actin and ADP with the Head Domain of Smooth Muscle Myosin: Implications for Strain-Dependent ADP Release in Smooth Muscle †. Biochemistry 37, 1969–1978 (1998). Greenberg, M. J., Shuman, H. & Ostap, E. M. Inherent Force-Dependent Properties of β-Cardiac Myosin Contribute to the Force-Velocity Relationship of Cardiac Muscle. Biophys. J. 107, L41–L44 (2014). Tables Table 1. Conformational entropy of ADP in WT and G256E simulations. Dihedral pair ADP entropy (cal mol -1 K -1 ) WT G256E θ 1 vs. θ 2 9.63 7.62 θ 3 vs. θ 4 10.00 8.97 θ 5 vs. θ 6 11.25 10.30 Table 2. Percent coverage of dihedral maps sampled by ADP in WT and G256E simulations. Dihedral pair % coverage WT G256E θ 1 vs. θ 2 8.93% 3.88% θ 3 vs. θ 4 21.06% 11.21% θ 5 vs. θ 6 33.49% 17.48% Additional Declarations There is NO Competing Interest. Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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17:36:24","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":147196,"visible":true,"origin":"","legend":"","description":"","filename":"COMMSCHEM250895T0structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7602931/v1/4a56f2ef71db8bc91ea751f6.xml"},{"id":93618988,"identity":"44d43455-d41e-4ccd-a7bf-2a12a9021138","added_by":"auto","created_at":"2025-10-15 17:28:24","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":157448,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7602931/v1/55d268fb86b988dce07d7028.html"},{"id":93618962,"identity":"9af8c7ea-ee11-48c1-9812-12edc3c21474","added_by":"auto","created_at":"2025-10-15 17:28:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":247077,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The post-powerstroke model includes 5 actin monomers (light green, space-filling atoms), myosin motor domain, and essential light chain (ELC, dark green). The functional regions of myosin are colored uniquely: upper 50 kDa domain (cyan), lower 50 kDa domain (blue), transducer (seafoam green), N-terminal domain (yellow), converter domain (dark blue), and tail (purple). E256 (red) is located in a β-hairpin turn between beta strands β\u003csub\u003e6\u003c/sub\u003e and β\u003csub\u003e7\u003c/sub\u003e in the transducer region. (b) Global C\u003csub\u003eα\u003c/sub\u003e RMSD of WT simulations and (c) G256E simulations. On average, G256E simulations had higher global C\u003csub\u003eα\u003c/sub\u003e RMSD than WT simulations. (d) C\u003csub\u003eα\u003c/sub\u003e RMSF difference between WT and G256E simulations by residue. Positive values indicate higher flexibility in WT simulations whereas negative values indicate higher flexibility in G256E simulations. C\u003csub\u003eα\u003c/sub\u003e RMSF mapped onto (e) WT and (f) G256E structures. Darker colors and thicker ribbons indicate higher C\u003csub\u003eα\u003c/sub\u003e RMSF.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7602931/v1/2dcfd534a6a4ada79f9272f4.png"},{"id":93618963,"identity":"1c084c1e-0c59-4a44-8373-20b3d931fdbb","added_by":"auto","created_at":"2025-10-15 17:28:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":309016,"visible":true,"origin":"","legend":"\u003cp\u003eADP occupancy maps for (a) WT and (b) G256E simulations show that ADP adopts a novel conformation in G256E simulations. Occupancy maps cover two standard deviations from the mean ADP occupancy. (c) Changes in ADP contacts with nucleotide pocket residues. In G256E simulations, contacts with the purine-loop and P-loop are lost while new contacts with Switch-1 and the SH-loop are formed. (d) Linear interaction energy analysis for the WT (black) and G256E (red) conventional MD simulations. The bars correspond to the average interaction energy between ADP or MG and myosin residues. Differences with p \u0026lt;= .05 are denoted by *. (e) ADP dihedral angle sampling for (f) WT and (g) G256E simulations. Six dihedral angles were defined: θ\u003csub\u003e1\u003c/sub\u003e (O1B-PB-O3A-PA), θ\u003csub\u003e2\u003c/sub\u003e (PB-O3A-PA-O5′), θ\u003csub\u003e3\u003c/sub\u003e (O3A-PA-O5′-C5′), θ\u003csub\u003e4\u003c/sub\u003e (PA-O5′-C5′-C4′), θ\u003csub\u003e5\u003c/sub\u003e (O5′-C5′-C4′-O4′), and θ\u003csub\u003e6\u003c/sub\u003e (O4′-C1′-N9-C8). These angles were plotted in Ramachandran-style 2-D histogram plots (bin size 5°x5°) to visualize changes in ADP conformation. Each bin is colored according to probability density.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7602931/v1/a0ed7a85e44bf0219ea0f823.png"},{"id":93619303,"identity":"5ad28480-bbf7-4db8-9a0d-819c0778aeb0","added_by":"auto","created_at":"2025-10-15 17:36:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":385338,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Average work required to release ADP from WT (black) or G256E (red) myosin. On average G256E required 1.5x as much work to displace ADP. The free energy profiles of ADP release along our specified reaction coordinate are shown in panels (b) and (c) for the WT and G256E simulations. All 60 profiles are shown and the Jarzynski averaged profile is in bold. These plots show the energy required to move ADP a certain distance away from the nucleotide binding pocket. Snapshots that depict ADP release from myosin from the Jarzynski average trajectories are shown in panels (d) and (e) for the WT and G256E simulations, respectively.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7602931/v1/f5b4cf4b1ac92349ae32bad6.png"},{"id":93620202,"identity":"02f1528d-2af1-482d-b074-5b51768d4e23","added_by":"auto","created_at":"2025-10-15 17:52:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":163173,"visible":true,"origin":"","legend":"\u003cp\u003ePanels (a) and (b) depict the average contacts made between myosin and ADP.Mg\u003csup\u003e2+\u003c/sup\u003e during the steered MD simulations. Contacts are shown only if they appear at least once in at least 90% of the WT or 90% of the G256E simulations. For each contact (y-axis), we averaged the fraction of simulations for which the contact was present at each time in the SMD simulations -- this contact probability corresponds to the color of the dot plots. These plots show that ADP exits WT/G256E myosin via different pathways and highlights residues that form strong interactions with ADP during release (all black lines).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7602931/v1/6aa3dbe27157834fbe103bc8.png"},{"id":93618966,"identity":"d0969df3-1e0a-4092-af70-ae1a7f0b733b","added_by":"auto","created_at":"2025-10-15 17:28:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":110385,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Schematic of experimental and computational workflow synergy. Data from myofibrils derived from CRISPR-edited hiPSCs supported by biochemical experiments with purified β-myosin. Experimental findings are validated by molecular simulations. (b) Cardiac directed differentiation of hiPSCs. hiPSC-CMs are matured through lactate purification and line patterning for seven days before being skinned on D37 for myofibril mechanics. (c) SDS-PAGE myosin separation gel showing the conversion of α-MHC of D14 hiPSC-CMs to β-MHC by D30 and D37. Standards are from adult mouse atria (α-MHC), adult rat ventricle (α/β-MHC), and adult rabbit ventricle (β-MHC).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7602931/v1/7921b15a6a91fae7c4e8c086.png"},{"id":93619307,"identity":"dd857e09-957e-4ad7-8dbb-246c5913b873","added_by":"auto","created_at":"2025-10-15 17:36:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":46946,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Average traces (8-10 individual traces) of ATP-induced pyrene-actin.WT sS1 dissociation. Traces in the absence of ADP (0 µM) were fit with a single exponential. Traces in the presence of ADP (left to right: 20, 40, 80, 160, 320, 640 µM post-mixing) were fit with a double exponential. (b) The relative \u003cem\u003ek\u003c/em\u003e\u003csub\u003eobs\u003c/sub\u003e (\u003cem\u003ek\u003c/em\u003e\u003csub\u003erel\u003c/sub\u003e = \u003cem\u003ek\u003c/em\u003e\u003csub\u003eobs\u003c/sub\u003e/\u003cem\u003e k\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e where \u003cem\u003ek\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e = \u003cem\u003ek\u003c/em\u003e\u003csub\u003eobs\u003c/sub\u003e\u003csup\u003e \u003c/sup\u003ewhen ADP = 0 µM) of the fast phase plotted vs. [ADP]. The data was fit with the equation \u003cem\u003ek\u003c/em\u003e\u003csub\u003erel\u003c/sub\u003e = 1/(1 + [ADP]/\u003cem\u003eK\u003c/em\u003e\u003csub\u003eADP\u003c/sub\u003e) to obtain the apparent affinity. One representative trace each from WT and G256E are shown in (b). (c) G256E caused a ~30% increase in ADP affinity (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eADP \u003c/sub\u003e= 32.82 ± 1.60 μM) compared to WT (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eADP \u003c/sub\u003e= 46.46 ± 3.23 μM). Error bars represent ± SD, n = 4.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7602931/v1/774043fee141ccc78a52522d.png"},{"id":93620060,"identity":"749e4c1d-0063-4e92-be24-977376b1eb93","added_by":"auto","created_at":"2025-10-15 17:44:24","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":139109,"visible":true,"origin":"","legend":"\u003cp\u003e(a \u0026amp; b) Representative activation traces from hiPSC-CM \u003cem\u003eMYH7\u003c/em\u003e\u003csup\u003eWT/G256E\u003c/sup\u003e (red) and isogenic \u003cem\u003eMYH7\u003c/em\u003e\u003csup\u003eWT/WT\u003c/sup\u003e (black) myofibrils with\u003cstrong\u003e \u003c/strong\u003ea close-up of the slow, early phase of relaxation with linear fits (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,slow\u003c/sub\u003e). Traces are an average of 2 sequential activations with activating solutions of 5.6 = pCa 5.6 activation paired with pCa 9.0 relaxation or 5.6ADP = pCa 5.6 activation + 50% ADP (2.5 mM ATP, 2.5 mM ADP) paired with pCa 9.0 + 50% ADP (2.5 mM ATP, 2.5 mM ADP) relaxation. (c) Max force generated\u003cstrong\u003e \u003c/strong\u003eis significantly higher in G256E myofibrils in both 5.6 and 5.6ADP activating solutions. ADP does not inhibit force generation. (d) Activation kinetics (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eACT\u003c/sub\u003e) are unchanged between G256E and WT at both conditions (5.6 and 5.6ADP) and decreased with the addition of ADP. (e) G256E fast phase relaxation kinetics (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,fast\u003c/sub\u003e) are slower at 5.6 compared to isogenic control; ADP inhibition decreases \u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,fast\u003c/sub\u003e significantly. (f) The kinetics of the slow, early phase (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,slow\u003c/sub\u003e) are significantly decreased in G256E myofibrils at 5.6. The addition of ADP decreased \u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,slow \u003c/sub\u003eof WT myofibrils significantly, to the point at which they mimic the kinetics at G256E myofibrils at 5.6/5.6ADP. (g)\u003cstrong\u003e \u003c/strong\u003eThe duration of the slow phase of relaxation (t\u003csub\u003eREL,slow\u003c/sub\u003e) is not significantly different when comparing G256E and WT. The addition of ADP prolongs the duration of this phase. Data are represented as mean ± SD. Statistical significance was determined by 2-way ANOVA. ns \u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, WT n = 15, G256E n = 13.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7602931/v1/a6e51c18512227aa3c8dd568.png"},{"id":93621043,"identity":"a986b7f0-dc6f-45aa-8925-4187b57b45af","added_by":"auto","created_at":"2025-10-15 18:00:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2170384,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7602931/v1/1326687c-bd12-43c1-800d-e5d7cda2e49f.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The G256E HCM mutation prolongs cardiac muscle relaxation via altered nucleotide handling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHypertrophic cardiomyopathy (HCM) affects 1 in 500 people globally and is implicated as a contributing cause of other life-threatening heart diseases such as atrial fibrillation, heart failure, and arrhythmia resulting in sudden death.\u003csup\u003e1\u003c/sup\u003e About 60% of HCM cases are caused by familial inheritance of a single autosomal dominant mutation within a gene encoding a sarcomere protein.\u003csup\u003e2\u003c/sup\u003e In particular, the \u003cem\u003eMYH7\u003c/em\u003e gene encoding the β-myosin heavy chain (MHC) is one of the two most common hotspots for HCM mutations.\u003csup\u003e3\u003c/sup\u003e Myosin is the motor protein responsible for muscle contraction, converting chemical energy produced by ATP hydrolysis and release of products (P\u003csub\u003ei\u003c/sub\u003e, ADP) into the mechanical work of force production and muscle cell shortening.\u003csup\u003e4\u003c/sup\u003e This involves changes in myosin's structure as it interacts with actin to accelerate the release of ATP hydrolysis products (P\u003csub\u003ei\u003c/sub\u003e, ADP) in a process known as chemo-mechanical transduction.\u003csup\u003e5–7\u003c/sup\u003eStructural perturbations resulting from disease-associated mutations can disrupt the kinetics and/or mechanics of the chemo-mechanical cycle and can lead to initiation and progression of disease.\u003csup\u003e8\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe G256E MYH7 mutation, associated with HCM,\u003csup\u003e9\u003c/sup\u003e is located in a hairpin turn that connects two strands in the central β-sheet of transducer region of the myosin head. This region conveys information between the nucleotide binding pocket and actin interface of myosin, both critical to chemo-mechanical transduction during contraction and relaxation.\u003csup\u003e10\u003c/sup\u003e\u0026nbsp; In a recent study, we employed CRISPR-Cas9 to edit this mutation into human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to produce a heterozygous (+/-) isogenic model for studying its impact at multiple scales of cardiac muscle organization.\u003csup\u003e8\u003c/sup\u003e A biochemical single ATP turnover assay performed on purified human β-cardiac 25-hep heavy meromyosin (HMM) containing the G256E mutation results in a reduced population of myosin in a low basal ATPase state, often described as the super-relaxed (SRX) state, suggesting a greater portion of the myosin population is available to contribute to muscle contraction. Comparison of the actin-activated ATPase activity of two versions of G256E myosin that differed only in the length of their coiled-coil tail confirmed that the mutation disrupted the ability of myosin to fold back into a sequestered, low ATPase state. This supports the hypothesis that the mutation results in more heads being available to interact with actin at the beginning of contractions. Slower motility in an \u003cem\u003ein\u0026nbsp;\u003c/em\u003evitro F-actin motility assay indicated the mutation results in a higher myosin duty ratio, suggesting that the actomyosin detachment rate may also be affected. This idea was supported by measures of the contraction and relaxation properties of isolated myofibrils from hiPSC-CMs. The kinetics of the slow, early phase of relaxation, which measures the myosin detachment rate from thin filaments,\u003csup\u003e11\u003c/sup\u003ewas slower for G256E versus WT myofibrils. Molecular dynamics (MD) simulations of the myosin M.ATP-state suggested that G256E disrupts the shape and stability of the central β-sheet, potentially altering communication between the nucleotide-binding pocket and actin-binding surface. This communication path is critical to open the nucleotide binding cleft that allows ADP release. This, in turn, is required before a new ATP molecule can bind in the nucleotide pocket and lead to the dissociation of myosin from actin. \u0026nbsp;While these findings are suggestive that myosin-actin chemo-mechanics are altered as part of the hypercontractile phenotype of G256E, it is important to understand which particular step(s) of the cross-bridge cycle are affected.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; The ADP release step is rate limiting for cross-bridge formation in sarcomeres during loaded contractions and is also a determinant of the rate of relaxation.\u003csup\u003e12\u003c/sup\u003e This is supported by biochemical,\u003csup\u003e13\u003c/sup\u003e mechanical,\u003csup\u003e14\u003c/sup\u003e single molecule,\u003csup\u003e15\u003c/sup\u003e and structural\u003csup\u003e16\u003c/sup\u003e evidence, and holds true across multiple classes of myosins from diverse species. Recent studies have shown that various myosin mutations can alter the affinity of myosin for ADP and consequently affect cross-bridge cycling kinetics.\u003csup\u003e17\u003c/sup\u003e Thus, detailed study of how the G256E mutation affects ADP release during chemo-mechanical cycling is merited. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHere we report the effects of myosin G256E on ADP release from the nucleotide binding pocket of myosin and its effects on myosin and myofibril chemo-mechanical function. \u0026nbsp;MD simulations of post-powerstroke myosin containing ADP (with Mg\u003csup\u003e2+\u003c/sup\u003e) complexed with actin (AM.ADP) suggested that G256E significantly alters nucleotide pocket dynamics. \u0026nbsp;Steered MD simulations suggested that the sequence and timing of contact pair disruptions in the pocket are altered and that more energy is required to pull ADP from the pocket. \u0026nbsp;These results provide a structural basis for altered ADP-myosin affinity and dynamics. Stopped-flow kinetics analysis showed that G256E sS1 had a ~30% increase in ADP affinity over wild type (WT) sS1, validating the MD simulation predictions. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine how this affects the chemo-mechanical function of myofibrils, we measured contraction and relaxation in the absence and presence of elevated ADP. Similar to our initial study,\u003csup\u003e8\u003c/sup\u003eG256E myofibrils generated significantly more force and exhibited a slower, early linear phase kinetics of relaxation (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,slow\u003c/sub\u003e) compared to isogenic controls. \u0026nbsp;When ADP was elevated in activating and relaxation solutions there was considerably less effect on \u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,slow\u003c/sub\u003e for G256E myofibrils, suggesting that ADP release is impaired with the mutation.\u003c/p\u003e\n\u003cp\u003eCombined, these experimental and computational studies support the hypothesis that the G256E mutation alters the structure of the nucleotide binding pocket, resulting in greater ADP affinity for G256E myosin than WT myosin, and this results in slower ADP release to slow crossbridge detachment and prolong contractile relaxation. To our knowledge, this is the first study to examine the reaction pathway and free energy changes associated with release of Mg\u003csup\u003e2+\u003c/sup\u003e-ADP from a post-powerstroke-like state of the actomyosin complex at the atomic scale with molecular simulations. These results highlight the power of combining experimental measures with in-depth computational dynamics to provide detailed structure-based mechanistic insights at the atomistic scale for protein and contractile organelle (myofibril) function.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e3.1\u0026nbsp;\u003c/strong\u003eComputational Model Preparation\u003c/p\u003e\n\u003cp\u003eThe initial actin+myosin+ADP+Mg\u003csup\u003e2+\u003c/sup\u003e (AM.ADP-Mg\u003csup\u003e2+\u003c/sup\u003e) structure model was generated based on cryo-EM structures of post-powerstroke actin-bound human cardiac myosin solved by Doran \u003cem\u003eet al.\u003c/em\u003e (PDB ID: 8EFH, 3.3 \u0026Aring; resolution; PDB ID: 8EFE, 3.8 \u0026Aring; resolution).\u003csup\u003e16,18\u003c/sup\u003e Our model includes the myosin motor and actin 5-mer from 8EFH; tropomyosin was excluded (chains O, P). Then we grafted the converter domain, tail and essential light chain (ELC) from 8EFE onto the actomyosin complex. Missing disordered heavy atoms were constructed via \u003cem\u003eModeller\u003csup\u003e19\u003c/sup\u003e\u003c/em\u003e. The disordered N-terminal extension of the ELC was not modeled. The final WT model includes actin residues (2-377), myosin residues (1-810), and ELC residues (44-195). A model for the mutant G256E actomyosin complex was generated via \u003cem\u003ein silico\u003c/em\u003e mutation of the WT structure with \u003cem\u003eUCSF Chimera.\u003c/em\u003e\u003csup\u003e20,21\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThe resulting two systems (WT and G256E) were prepared for molecular dynamics (MD) simulation using the AMBER20 simulation package\u003csup\u003e22\u003c/sup\u003e and the ff14SB force field\u003csup\u003e23\u003c/sup\u003e. Water molecules were treated with the TIP3P force field.\u003csup\u003e24\u003c/sup\u003e\u0026nbsp; Metal ions were modeled using the Li and Merz parameter set.\u003csup\u003e25\u0026ndash;27\u003c/sup\u003e ADP molecules were treated with the GAFF2 force field.\u003csup\u003e28\u003c/sup\u003e Partial charges for ADP were derived from a restrained electrostatic potential (\u003cem\u003eresp\u003c/em\u003e) fit to quantum mechanics calculations performed with ORCA.\u003csup\u003e29\u003c/sup\u003e\u0026nbsp; The SHAKE algorithm was used to constrain the motion of hydrogen-containing bonds.\u003csup\u003e30\u003c/sup\u003e Long-range electrostatic interactions were calculated using the particle mesh Ewald (PME) method. Hydrogen atoms were modeled onto the initial structure using the \u003cem\u003eleap\u003c/em\u003e module of AMBER, and each protein was solvated with explicit water molecules in a truncated octahedral box and then neutralizing + 120 mM counterions (Na\u003csup\u003e+\u003c/sup\u003e, Cl\u003csup\u003e-\u003c/sup\u003e) were added. Each system was minimized in three stages. First, hydrogen atoms were minimized for 1000 steps in the presence of 100 kcal/mol restraints on all heavy atoms. Second, all solvent atoms were minimized for 1,000 steps in the presence of 25 kcal/mol restraints on all protein atoms. Third, all atoms were minimized for 8,000 steps in the presence of 25 kcal/mol restraints on all backbone heavy atoms (N, O, C, and C\u003csub\u003e\u0026alpha;\u003c/sub\u003e atoms). After minimization, systems were heated to 310 K using the NVT (constant number of particles, volume, and temperature) ensemble and in the presence of 25 kcal/mol restraints on backbone heavy atoms. Next, the systems were equilibrated over five successive stages using the NPT (constant number of particles, pressure, and temperature) ensemble. During the first four stages, the systems were equilibrated for 5 ns in the presence of 25 to 1 kcal/mol restraints on backbone heavy atoms. During the final equilibration stage, the systems were equilibrated for 5 ns in the absence of restraints. A 10 \u0026Aring; nonbonded cutoff was used for all preparation and production simulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2\u0026nbsp;\u003c/strong\u003eConventional MD Production and Analysis\u003c/p\u003e\n\u003cp\u003eThe equilibrated systems were then simulated using conventional molecular dynamics protocols in the NVT ensemble in triplicate for 500 ns each (2 systems, three replicate simulations per system, 500 ns sampling per replicate simulation = 3 \u0026micro;s net sampling) and coordinates were saved every 10 ps.\u003c/p\u003e\n\u003cp\u003eMolecular analyses were performed using \u003cem\u003ecpptraj\u003c/em\u003e.\u003csup\u003e31\u003c/sup\u003e C\u003csub\u003e\u0026alpha;\u003c/sub\u003e RMSD values for each simulation were calculated for all residues and relative to the minimized starting structure. C\u003csub\u003e\u0026alpha;\u003c/sub\u003e RMSF values were calculated after aligning the trajectory to the average structure. Residue-residue contacts were counted if at least one pair of heavy atoms between two residues were within 5 \u0026Aring; of one another and contacts are presented as the fraction of simulation time that two residues were interacting. Nucleotide dynamics were evaluated by monitoring 6 dihedral angles that describe ADP structure using internal coordinates, as previously described.\u003csup\u003e32\u003c/sup\u003e The 6 calculated dihedral angles were defined as: \u0026theta;\u003csub\u003e1\u003c/sub\u003e (atoms: O1B-PB-O3A-PA),\u0026nbsp;\u0026theta;\u003csub\u003e2\u003c/sub\u003e (PB-O3A-PA-O5\u0026prime;),\u0026nbsp;\u0026theta;\u003csub\u003e3\u003c/sub\u003e (O3A-PA-O5\u0026prime;-C5\u0026prime;),\u0026nbsp;\u0026theta;\u003csub\u003e4\u003c/sub\u003e (PA-O5\u0026prime;-C5\u0026prime;-C4\u0026prime;),\u0026nbsp;\u0026theta;\u003csub\u003e5\u003c/sub\u003e (O5\u0026prime;-C5\u0026prime;-C4\u0026prime;-O4\u0026prime;), and\u0026nbsp;\u0026theta;\u003csub\u003e6\u003c/sub\u003e (O4\u0026prime;-C1\u0026prime;-N9-C8).\u0026nbsp;Ramachandran-style dihedral angle maps (\u0026theta;\u003csub\u003e1\u0026nbsp;\u003c/sub\u003evs \u0026theta;\u003csub\u003e2\u003c/sub\u003e, \u0026theta;\u003csub\u003e3\u0026nbsp;\u003c/sub\u003evs \u0026theta;\u003csub\u003e4\u003c/sub\u003e, and \u0026theta;\u003csub\u003e5\u0026nbsp;\u003c/sub\u003evs \u0026theta;\u003csub\u003e6\u003c/sub\u003e) describe the relative orientation of the phosphate groups, the sugar ring, and the purine ring. The orientation of the myosin tail relative to the actin filament was calculated using the \u0026lsquo;vector\u0026rsquo; module of \u003cem\u003ecpptraj\u0026nbsp;\u003c/em\u003eas previously described.\u003csup\u003e33\u003c/sup\u003e\u0026nbsp; We also calculated interaction energies between myosin residues and either ADP or Mg\u003csup\u003e2+\u003c/sup\u003e using the linear interaction energy module. Electrostatic and van der Waals interaction energies were combined.\u003c/p\u003e\n\u003cp id=\"_Toc169003409\"\u003e\u003cstrong\u003e3.3\u0026nbsp;\u003c/strong\u003eSteered molecular dynamics (sMD)\u003c/p\u003e\n\u003cp\u003ePre-production protocols were identical to those for the conventional MD simulations except that we employed the hydrogen mass partitioning scheme\u003csup\u003e34\u003c/sup\u003e\u0026nbsp; to enable a 4 fs time step, the total time for the heating and equilibration phases was 1 ns, and 25 kcal/mol restraints were present on all protein heavy backbone atoms, ADP, and Mg\u003csup\u003e2+\u003c/sup\u003e during the entire equilibration. During the production simulations we steered ADP.Mg\u003csup\u003e2+\u003c/sup\u003e 14.4 \u0026Aring; away from the nucleotide binding pockets using a center of mass distance-based reaction coordinate. ADP.Mg\u003csup\u003e2+\u003c/sup\u003e was steered away at 1 \u0026Aring;/nanosecond and so production simulations were 10.8 ns long (WT) or 10 ns long (G256E). 60 WT and 60 G256E simulations were performed. We calculated the average work done to displace ADP, the free energy profiles along the reaction coordinate, the Jarzynski average trajectories, and the average myosin-ADP.Mg\u003csup\u003e2+\u003c/sup\u003e interactions made along the reaction coordinate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4\u0026nbsp;\u003c/strong\u003esS1 protein purification\u003c/p\u003e\n\u003cp\u003eRecombinant WT and G256E human \u0026beta;-cardiac sS1 myosins were purified as described previously.\u003cstrong\u003e\u003csup\u003e35\u003c/sup\u003e\u003c/strong\u003e\u0026nbsp; Briefly, human \u0026beta;-cardiac myosin heavy chain (\u003cem\u003eMYH7)\u003c/em\u003e was co-expressed with human ELC (\u003cem\u003eMYL3)\u003c/em\u003e in the differentiated murine myoblast C2C12 cell line (ATCC) using adenovirus generated in HEK293T cells (ATCC) using the AdEasy Vector System (Qbiogene Inc, USA). The sS1 construct is a truncated version of MYH7 (residues 1-808) followed by a flexible GSG (Gly-Ser-Gly) linker and a carboxy-terminal 8-residue (RGSIDTWV) PDZ binding peptide. The MYL3 construct contains an N-terminal FLAG tag followed by a TEV protease site for purification. C2C12 cells were infected with adenovirus constructs 48 hr after differentiation and harvested 4 days after infection in a lysis buffer containing 50 mM NaCl, 20 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 20 mM imidazole pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 3 mM ATP, 1 mM PMSF, 5% sucrose, and Roche protease inhibitors. The harvested cells were immediately flash frozen in liquid nitrogen. The cell pellets were stored up to 2 months at \u0026ndash;80\u0026deg;C and thawed at room temperature for purification. Cells were lysed in a dounce homogenizer with 50 strokes and the lysate was clarified by spinning at 30,000rpm in a Ti-60 fixed angle ultracentrifuge rotor for 30 min at 4\u0026deg;C. Supernatant was transferred to a fresh tube and bound to anti-FLAG resin for 1.5hr at 4\u0026deg;C on a nutator. The resin was washed with a buffer (150 mM NaCl, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 20 mM imidazole pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 3 mM ATP, 1 mM PMSF, 10% sucrose, and Roche protease inhibitors) and then nutated overnight with TEV protease at 4\u0026deg;C to cleave the ELC-myosin complex from the resin. The next day, the supernatant was purified using a HiTrap Q HP column on a fast protein liquid chromatography (FPLC) apparatus with a gradient of 0\u0026ndash;600 mM NaCl over 20 column volumes in a buffer containing 10 mM imidazole pH 7.5, 4 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10% sucrose, 1 mM DTT, and 2 mM ATP. Fractions containing human \u0026beta;-cardiac myosin and devoid of mouse skeletal myosin (Coomassie staining on 12.5% SDS PAGE) were pooled and flash frozen in liquid nitrogen. Purified sS1 was buffer exchanged into stopped-flow buffer (120 mM KCl, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 20 mM MOPS, 1 mM DTT, pH 7.0) overnight prior to experiments. Concentration was measured through a Bradford protein assay (5000201, Bio-Rad).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5\u0026nbsp;\u003c/strong\u003ePyrene-actin labeling\u003c/p\u003e\n\u003cp\u003ePyrene-actin was prepared fresh and used within ~1 week. First, G-actin was prepared from lyophilized bovine cardiac muscle as described before.\u003csup\u003e36\u003c/sup\u003e G-actin was diluted to 1 mg/mL before polymerizing with 1 mM ATP, 50 mM KCl, and 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e at 4 \u0026deg;C for 2 hrs. To label, 1.28% w/w N-(1-Pyrene)iodoacetamide (AA00G316, aablocks) was added to F-actin and stirred for 18 hrs at room temperature. Excess N-(1-Pyrene)iodoacetamide was removed by centrifuging at 2,000 x g for 5 mins at 4 \u0026deg;C. Labeled F-actin was pelleted by centrifugation at 92,000 x g for 3 hrs at 4 \u0026deg;C and resuspended in stopped-flow buffer plus minimal ATP (120 mM KCl, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 20 mM MOPS, 1 mM DTT, 200 \u0026mu;M ATP, pH 7.0) in a glass dounce homogenizer. Pyrene-actin was stabilized with 1:1 molar phalloidin (5301, AAT Bioquest) for at least 4 hrs before dialyzing into stopped-flow buffer overnight to remove residual ATP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6\u0026nbsp;\u003c/strong\u003eStopped-flow kinetics\u003c/p\u003e\n\u003cp\u003eStopped-flow kinetics experiments were performed at 20 \u0026deg;C using a Hi-Tech \u0026lsquo;KinetAsyst\u0026rsquo; stopped-flow (SF-61DX2, TgK Scientific, UK). Measurements were made in 120 mM KCl, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 20 mM MOPS, 1 mM DTT, pH 7.0 solution. Pyrene fluorescence was measured by excitation at 365 nm and emission through a GG 400 nm cutoff filter. ADP affinity (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eADP\u003c/sub\u003e) was assessed by competitive binding of ATP and ADP to actomyosin. Briefly, 0.125 \u0026mu;M actomyosin (sS1 + pyrene-actin) was rapidly mixed with 25 \u0026micro;M ATP plus various amounts of ADP (0 \u0026ndash; 640 \u0026micro;M). All concentrations reported were post-mixing. An average trace of at least eight individual traces was generated for each condition and fit with a double exponential. The relative \u003cem\u003ek\u003c/em\u003e\u003csub\u003eobs\u0026nbsp;\u003c/sub\u003eof the fast phase (\u003cem\u003ek\u003c/em\u003e\u003csub\u003erel\u003c/sub\u003e) was plotted vs. ADP concentration and fitted with an equation as described previously.\u003csup\u003e37\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc169003410\"\u003e\u003cstrong\u003e3.7\u0026nbsp;\u003c/strong\u003eHuman induced pluripotent stem cell (hiPSC) culture\u003c/p\u003e\n\u003cp\u003eThe parental WTC hiPSC cell line was generated by the Bruce R. Conklin Laboratory at the Gladstone Institutes and University of California, San Francisco (UCSF).\u003csup\u003e38\u003c/sup\u003e Cells were maintained using described methods.\u003csup\u003e39,40\u003c/sup\u003e A detailed cell culture protocol from the Allen Institute for Cell Science can be found on its webpage (http://allencell.org). AICS-0075-085 ACTN2-mEGFP was developed at the Allen Institute for Cell Science (http://allencell.org/cell-catalog) and is available through Coriell. AICS-0097-113 ACTN2-mEGFP \u003cem\u003eMYH7\u003c/em\u003e\u003csup\u003eWT/WT\u0026nbsp;\u003c/sup\u003eand AICS-0097-141 ACTN2-mEGFP \u003cem\u003eMYH7\u003c/em\u003e\u003csup\u003eWT/G256E\u0026nbsp;\u003c/sup\u003ewere developed at the Allen Institute for Cell Science and are available on request.\u003c/p\u003e\n\u003cp id=\"_Toc169003411\"\u003e\u003cstrong\u003e3.8\u0026nbsp;\u003c/strong\u003ehiPSC-Cardiomyocyte (hiPSC-CM) differentiation and maintenance\u003c/p\u003e\n\u003cp\u003ehiPSCs were differentiated into cardiomyocytes using an established small-molecule protocol\u003csup\u003e41\u003c/sup\u003e with modifications (Fig. 5c).\u003csup\u003e42\u003c/sup\u003e Briefly, hiPSCs were seeded onto Matrigel-coated 12-well tissue culture treated plates at densities ranging from 50 ✕ 10\u003csup\u003e3\u003c/sup\u003e \u0026ndash; 125 ✕ 10\u003csup\u003e3\u003c/sup\u003e cells per well and maintained for 2 days with mTeSR1 media (85850, Stemcell Technologies). On day -1, the day prior to differentiation, mTeSR1 media was supplemented with 1 \u0026mu;M CHIR99021 (13122, Cayman Chemical). On day 0, hiPSCs were induced with 5 \u0026mu;M CHIR99021 in a chemically defined media composed of RPMI 1640 (11875-093, Gibco) + 500 \u0026mu;g/mL bovine serum albumin (A9418, Sigma-Aldrich) + 213 \u0026mu;g/mL L-ascorbic acid (A8960, Sigma-Aldrich) hereafter abbreviated RBA. After 48 hours, the media was replaced with 2 \u0026mu;M Wnt-C59 (S7037, Selleck Chem) in RBA media. On day 4, the media was replaced with RBA media. From day 6 onwards, differentiated cells were maintained in RPMI 1640 + 1X B27 plus insulin (A1895601, Gibco) with media changes every 2-3 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.9\u0026nbsp;\u003c/strong\u003eSolutions for myofibril mechanics\u003c/p\u003e\n\u003cp id=\"_Toc169003412\"\u003eSolutions for isolated myofibril mechanics experiments were prepared as previously described.\u003csup\u003e43\u003c/sup\u003e Experimental solutions were composed of the following: 15 mM total EGTA, 80 mM total MOPS, 5 mM MgATP, 15 mM total creatine phosphate, 83 mM free K, and 52 mM free Na, all free ion concentrations were determined using MAXCHELATOR\u003csup\u003e44\u003c/sup\u003e). Solutions were adjusted to pH 7.0 with NaOH. Elevated ADP solutions (5.6ADP) were made by replacing 50% of the nucleotide pool with ADP for final concentrations of 2.5 mM ATP and 2.5 mM ADP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.10\u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003eIsolated myofibril assay\u003c/p\u003e\n\u003cp\u003ehiPSCs were differentiated into cardiomyocytes as described above. On day 14 post-differentiation, hiPSC-CMs were replated at 100k cells/cm\u003csup\u003e2\u003c/sup\u003e. On day 16, hiPSC-CMs were subjected to glucose starvation and lactate enrichment for 4 days in DMEM minus glucose (11966-025, Gibco) media supplemented with 4 mM sodium L-lactate (L7022, Sigma Aldrich). hiPSC-CMs were maintained in RPMI 1640 + 1X B27 plus insulin. On day 30, hiPSC-CMs were replated onto 10 kPa polyacrylamide gels in 6-well glass bottom plates (P06-20-1.5-N, Cellvis) patterned with 15 \u0026mu;m width Matrigel lines prepared by microcontact printing as described previously\u003csup\u003e45\u003c/sup\u003e, and matured for 7 days to promote elongation and formation of cell-to-cell contacts. On day 37, isolated myofibrils for mechanical and kinetic analysis hiPSC-CMs were demembranated in a 1% triton-X pCa 9.0 relaxing solution supplemented with 1:100 protease inhibitor cocktail (P8340, Sigma-Aldrich) for 30 minutes on ice. A cell scraper was then used to gently free isolated myofibrils from permeabilized cells. The supernatant was collected and gently homogenized in a glass dounce homogenizer ~5-7X. Myofibrils were mounted into a custom-built apparatus with rapid Ca\u003csup\u003e2+\u003c/sup\u003e solution switching (15 \u0026deg;C) as previously described.\u003csup\u003e43,46\u003c/sup\u003e Myofibrils were initially suspended in pCa 9.0 relaxing solution before rapidly switching to pCa 4.0 maximal activation solution. Myofibrils were also activated in a pCa 5.6 submaximal activation solution and a pCa 5.6ADP solution containing 50% ADP (2.5 mM ATP, 2.5 mM ADP) solution to challenge myofibrils with ADP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.11\u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003eMyosin separation gel\u003c/p\u003e\n\u003cp\u003eCell pellets were flash frozen in liquid nitrogen and stored at -80\u0026deg;C until ready to use. Cell pellets were resuspended in ~3x volume myofibril lysis buffer (5% SDS, 50 mM Tris (pH 8.5), 0.75% sodium deoxycholate, and 1x protease inhibitor cocktail). Sarcomeres and proteins were denatured by boiling samples for 10 minutes. Concentration was measured by blotting for total protein and adjusting loading. Myosin separation gel was made by combining a stacking gel (2.95% Acrylamide with 10% glycerol and pH 8.8) and resolving gel (6% Acrylamide with 10% glycerol and pH 8.8). Separation gels were run for 2 hours and 15 min at 32 mA in a cold room (4\u0026deg;C). Gels were stained for total protein using One-Step Lumitein\u0026trade; Protein Gel Stain, 1X following manufacturer instructions. Gels were imaged on a BioRad ChemiDoc using SYPRO Ruby protein stain setting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.12\u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003eStatistics\u003c/p\u003e\n\u003cp\u003eConventional MD (cMD) simulations were run in triplicate (n=3) for each genotype. Statistics on cMD analyses were performed with a student\u0026rsquo;s t-test. Steered MD (sMD) simulations were run 60 times (n=60) for each genotype. A student\u0026rsquo;s t-test was used to compare work required for ADP dissociation for WT vs. G256E. For myofibrils, statistical significance was determined by 2-way ANOVA (WT n=15, G256E n=13). Stopped-flow experiments were replicated 4 times (n=4) and a student\u0026rsquo;s t-test was used to determine significance.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003e\u003cstrong\u003e4.1\u0026nbsp;G256E alters myosin nucleotide binding pocket dynamics with ADP\u0026nbsp;\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eTo investigate the possible structural changes within myosin that might result in impaired ADP release we employed conventional molecular dynamics (cMD) to simulate the time-dependent changes of the post-powerstroke state of myosin complexed with actin and ADP (AM.ADP, Fig. 1a), and how G256E alters structure and contacts at the atomic level. In general, G256E simulations had a greater change in global C\u003csub\u003eα\u003c/sub\u003e RMSD compared to WT simulations (Fig. 1b,c), presumably as a response to the change from Gly to Glu. These changes tended to stabilize by 300 ns (Fig. 1c). We calculated the change in C\u003csub\u003eα\u003c/sub\u003e RMSF per residue and found that the flexibility of the G256E structure remained very similar to WT except for loop 2 (residues 621-646) and the end of the tail (residues 771-810) (Fig. 1d).\u0026nbsp;In the WT stimulations, ADP primarily formed contacts with the P-loop (residues 179-184) and Purine-ring (residues 126-131) of the nucleotide binding pocket (Fig. 2a,c). In G256E simulations, the introduction of the bulky, negatively charged sidechain (E256) formed a novel salt bridge with R169, as was observed previously in simulations of the G256E M.ATP state.\u003csup\u003e8\u003c/sup\u003e\u0026nbsp; This structural change propagated to the nucleotide pocket. For the majority of G256E simulations, ADP preferred to adopt a conformation that closely associates with Switch-1 (residues 232-244) and the loop that connects the third b-strand of the transducer to the SH1-helix (SH1-loop, residues 673-685). In particular, two non-native contacts formed in the G256E simulations (Fig. 2c S241 \u0026amp; T678) and persisted for greater than 75% of combined simulation time. This new ADP conformation led to the formation of a salt bridge between D239 of Switch-1 and K679 of the SH1-loop, which persisted longer in G256E simulations (average 28.9% of simulation time) compared to WT (average 3.2% of simulation time, p = 0.051). Overall, this results in a loss of contacts between ADP and the P-loop and the Purine-ring (Fig. 2c) and altered dynamics in the nucleotide pocket in a manner that might affect ADP release.\u003c/p\u003e\n\u003cp\u003eThe conformation of ADP within the nucleotide binding pocket was assessed by calculation of 6 distinct dihedral angles within ADP: θ\u003csub\u003e1\u003c/sub\u003e (O1B-PB-O3A-PA), θ\u003csub\u003e2\u003c/sub\u003e (PB-O3A-PA-O5′), θ\u003csub\u003e3\u003c/sub\u003e (O3A-PA-O5′-C5′), θ\u003csub\u003e4\u003c/sub\u003e (PA-O5′-C5′-C4′), θ\u003csub\u003e5\u003c/sub\u003e (O5′-C5′-C4′-O4′), and θ\u003csub\u003e6\u003c/sub\u003e (O4′-C1′-N9-C8). The dihedral angles were analyzed via 2D Ramachandran-style plots (Fig. 2e-g). The quantified conformational entropy (Table 1) and percent coverage (Table 2) indicate that ADP is less flexible in the pocket and adopts a more stable conformation for G256E compared to WT simulations. We calculated the interaction energies between myosin and ADP.Mg\u003csup\u003e2+.\u003c/sup\u003e Residues with at least 3 kT of interaction energy were considered part of the nucleotide binding pocket (residues 126, 128, 180-190, 224, 228, 232, 233, 238-243, 246, 461, 674, and 679) and most nucleotide binding pocket residues had statistically different interaction energies with ADP.Mg\u003csup\u003e2+\u003c/sup\u003e (Fig. 2d). We also observed a statistically different center of mass distance between ADP.Mg\u003csup\u003e2+\u003c/sup\u003e and the nucleotide binding pocket residues for G256E.\u0026nbsp;The altered conformation of ADP.Mg\u003csup\u003e2+\u003c/sup\u003e in the nucleotide binding pocket led to statistically distinct interaction energies with myosin residues (Fig. 2d).\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e4.2\u0026nbsp;ADP release from the post-powerstroke state required more work for G256E simulations compared to WT\u003c/strong\u003e.\u003c/h2\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;We used steered molecular dynamics (sMD) simulations to simulate ADP release from the nucleotide binding pocket of myosin in complex with actin (AM.ADP*). To create a reaction coordinate for steering, we calculated the linear interaction energy (lie) between myosin residues and either ADP or Mg\u003csup\u003e2+\u003c/sup\u003e in the cMD simulations. Myosin residues with an average lie of at least 3 kT with either ADP or Mg\u003csup\u003e2+\u003c/sup\u003e were selected as nucleotide binding pocket residues in the reaction coordinate (Fig. 2d). Next, we calculated the modal distance between ADP.Mg\u003csup\u003e2+\u003c/sup\u003e and the nucleotide binding pocket residues in the cMD simulations (WT: 3.6 Å; G256E: 4.4 Å). We then selected 120 structures from the cMD simulations (approximately 20 structures from each replicate) that had ADP.Mg\u003csup\u003e2+\u003c/sup\u003e - nucleotide binding pocket residue center of mass distances within 0.1 Å of the model results. Then, steered MD simulations were performed (Fig. 3d,e). On average, G256E myosin required 1.5 times as much work (WT: 58 kcal mol\u003csup\u003e-1\u003c/sup\u003e G256e: 89 kcal mol\u003csup\u003e-1\u003c/sup\u003e, p = 2.4 x 10\u003csup\u003e-18\u003c/sup\u003e) to displace ADP.Mg\u003csup\u003e2+\u003c/sup\u003e from the binding pocket, indicating a stronger interaction between G256E myosin and ADP.Mg\u003csup\u003e2+\u003c/sup\u003e (Fig. 3a). The Jarzynski averaged trajectories followed this trend (WT: 26 kcal mol\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eG256E: 68 kcal mol\u003csup\u003e-1\u003c/sup\u003e, a 1.6-fold difference) (Fig. 3b,c). Note that the free energy values along the reaction coordinate are dependent on the pulling speed in the sMD simulations, and relative differences in free energy along the profile are more meaningful than absolute energies here.\u003csup\u003e47\u003c/sup\u003e The free energy profiles show an initial steep rise along the reaction coordinate that corresponds to movement of the α and β phosphates followed by a shallower rise. More work was required to displace ADP.Mg\u003csup\u003e2+\u003c/sup\u003e from G256E myosin due to the burial of the adenine ring in the SH1-loop in addition to increased interactions of the phosphate groups with Switch-1. We calculated the average interactions that ADP.Mg\u003csup\u003e2+\u003c/sup\u003e made as they were steered out of the nucleotide binding pocket (Fig. 4a,b). We found that ADP.Mg\u003csup\u003e2+\u003c/sup\u003e formed different interactions with WT and G256E myosin as it left the pocket, in large part due to the burial of the adenine ring in the SH1-loop and increased interactions of the phosphates with Switch-1. For both the WT and G256E simulations, ADP was displaced from the nucleotide binding pocket and at the end of the simulations there were no interactions between ADP.Mg\u003csup\u003e2+\u003c/sup\u003e and P-loop or purine binding loop residues. However, at the end of every simulation ADP.Mg\u003csup\u003e2+\u003c/sup\u003e was still interacting with at least E228. Attempts to steer ADP.Mg\u003csup\u003e2+\u003c/sup\u003e further along the reaction coordinate resulted in local unfolding of myosin. We hypothesize this is due to strong interaction with E228 and a secondary reaction coordinate may be necessary to further steer ADP.Mg\u003csup\u003e2+\u003c/sup\u003e from this residue and based on visual inspection we do not predict differences in ADP release from this point onward. Overall, these data suggest increased binding affinity of ADP.Mg\u003csup\u003e2+\u003c/sup\u003e for G256E myosin facilitated through modified ADP coordination that results in an altered release trajectory.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e4.3\u0026nbsp;G256E sS1 has greater affinity for ADP\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eNext, to experimentally determine the ADP affinity of myosin (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eADP\u003c/sub\u003e) we performed an ATP/ADP competitive binding assay using recombinant human β-cardiac short subfragment-1 (β-sS1), a single headed myosin construct containing only the human ventricular essential light chain. Experiments were performed by rapidly mixing 0.125 μM pyrene-actin.β-sS1 with 25 μM ATP + varying amounts of ADP (0, 20, 40, 80, 160, 320, and 640 μM) (all reported concentrations are post-mixing, Fig. 6a). In the absence of ADP, experimental curves were fit with a single-exponential which describes ATP-induced actomyosin dissociation. In the presence of ADP, experimental curves were fit with a double-exponential to account for the slow phase, which describes ADP dissociation from actomyosin. We plotted the relative \u003cem\u003ek\u003c/em\u003e\u003csub\u003eobs\u003c/sub\u003e of the fast phase (\u003cem\u003ek\u003c/em\u003e\u003csub\u003erel\u003c/sub\u003e = \u003cem\u003ek\u003c/em\u003e\u003csub\u003eobs\u003c/sub\u003e/\u003cem\u003e\u0026nbsp;k\u003c/em\u003e\u003csub\u003eobs,0\u003c/sub\u003e) vs. ADP concentration to assess ADP affinity (Fig. 6b). G256E β-sS1 demonstrated ~30% increased ADP affinity (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eADP\u003c/sub\u003e = 33 ± 3 µM, Fig. 6c) compared to WT β-sS1 (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eADP\u003c/sub\u003e = 48 ± 11 µM).\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e4.4\u0026nbsp;G256E myofibril relaxation is slower and less sensitive to elevated ADP\u003c/strong\u003e.\u003c/h2\u003e\n\u003cp\u003eTo determine how the increased ADP binding affinity affects contractile properties, particularly the early, linear phase of relaxation that reflects crossbridge detachment, we used myofibrils from CRISPR/Cas9-edited hiPSC lines containing the heterozygous expression of the G256E mutation (\u003cem\u003eMYH7\u003c/em\u003e\u003csup\u003eWT/G256E\u003c/sup\u003e) and an isogenic control (\u003cem\u003eMYH7\u003c/em\u003e\u003csup\u003eWT/WT\u003c/sup\u003e). The cell lines underwent a series of quality control experiments to confirm genotype and validate that the gene-editing process did not have any off-target effects.\u003csup\u003e8\u003c/sup\u003e We confirmed that our stem cell differentiation and culture conditions (Fig. 5b) provided hiPSC-CMs that express predominantly β-MHC by D30, validating the D37 timepoint was appropriate for experiments (Fig 4c). The mechanical and kinetic properties of hiPSC-CM myofibrils were measured in a custom-built apparatus using a fast solution switching (~5 ms) technique, as previously described.\u003csup\u003e46\u003c/sup\u003e Myofibrils were activated in a submaximal calcium solution (pCa 5.6), relaxed (pCa 9.0), then activated and relaxed with solutions containing 50% ADP (pCa 5.6ADP, 2.5 mM ATP + 2.5 mM ADP) to observe the effects of elevated ADP on contractile properties. Fig. 7a shows representative traces for contraction and relaxation and Fig. 7b shows a higher resolution view of the early, linear phase of relaxation, for measurements of the slope (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,slow\u003c/sub\u003e) and duration (t\u003csub\u003eREL,slow\u003c/sub\u003e). Force generated at both pCa 5.6 and pCa 5.6ADP was significantly increased for G256E myofibrils by ~2X (Fig. 7c, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) compared to WT myofibrils. Activation kinetics (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eACT\u003c/sub\u003e) with elevated ADP were decreased by ~30% (Fig. 7d, p \u0026lt; 0.01) for both G256E and WT myofibrils. For relaxation, the main, fast phase rate (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,fast\u003c/sub\u003e) was ~30% slower for G256E myofibrils compared to WT (Fig. 7e, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Addition of ADP slowed \u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,fast\u003c/sub\u003e for both WT and G256E, but the magnitude of change was greater for WT \u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,fast\u003c/sub\u003e, such that this rate was no longer significantly different for G256E vs WT myofibrils. For the early, linear phase of relaxation, the duration (t\u003csub\u003eREL,slow\u003c/sub\u003e) was prolonged approximately 2-fold by the addition of ADP for both G256E and WT myofibrils, as expected, but was not significantly different between G256E and WT myofibrils at pCa 5.6 or 5.6ADP. However, the slow phase of relaxation (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,slow\u003c/sub\u003e) was significantly impaired for G256E vs WT myofibrils with ATP only solutions (Fig. 7f, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) at pCa 5.6, similar to what we previously reported.\u003csup\u003e8\u003c/sup\u003e Importantly, elevated ADP had a large effect on \u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,slow\u003c/sub\u003e in WT myofibrils (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001), greatly slowing this rate, while it resulted in no appreciable change in \u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,slow\u003c/sub\u003e for G256E myofibrils. This suggests that ADP release is already significantly impaired in G256E myofibrils, such that the response to ADP product inhibition is blunted.\u003c/p\u003e"},{"header":"Discussion","content":"\u003ch2\u003e\u003cstrong\u003e\u003cem\u003e5.1\u0026nbsp; \u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eSynergy between computation simulations and experiments\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eHere, we simulated ADP release from myosin using enhanced sampling computational techniques. We validated computational results with data collected from biochemical measurements of ADP affinity for sS1 myosin motors and mechanical measurements of hiPSC-CM-derived myofibrils. Our results highlight the synergy between computational simulation and experimental validation in understanding the structure-function basis of the myosin motor and contractile dysfunction resulting from disease associated mutations and this may provide intra-protein targets for more precise therapeutics approaches.\u003c/p\u003e\n\u003cp\u003eIn post-powerstroke (AM.ADP) simulations, changing G256 to the E256 mutation forms an enduring salt bridge with R169. This was also observed in the M.ATP state simulations we reported recently.\u003csup\u003e8\u003c/sup\u003e Even though this contact was consistent in simulations of both chemo-mechanical states, there were different consequences. In the M.ATP simulations, the salt bridge led to strain in sheets β5 and β6 of the transducer region and a reduction in the stability of hydrogen bonds between these sheets. However, in post-powerstroke (AM.ADP) simulations, the instability of the transducer region was not observed. This may be due to intrinsic differences in the nucleotide binding pocket in ‘closed’ (i.e. actin-free, ATP/ADP.P\u003csub\u003ei\u003c/sub\u003e-bound forms) vs ‘open’ (i.e. actin-bound, ADP-bound and nucleotide-free forms) conformations. These findings highlight the importance of simulating myosin in different chemo-mechanical states to obtain a more complete assessment of induced changes in structural dynamics, e.g. a single state structure analysis may result in incomplete understanding or incorrect conclusions. In the case of post-powerstroke simulations, it is possible that the presence of actin stabilized the transducer, as the transducer is known to adopt different conformations in the presence and absence of actin.\u003csup\u003e48,49\u003c/sup\u003e The post-powerstroke simulations ultimately led to the discovery of altered nucleotide pocket dynamics due to the G256E mutation, which was consistent for all replicate simulations of the mutant. In particular, ADP formed novel contacts with the SH1-loop. This loop leads into the SH1 and SH2 helices, which link the converter domain to the rest of the myosin motor domain. In previous experimental studies, SH1-SH2 interactions were found to modulate the affinity of ADP to myosin, and a computational study found electrostatic interactions between the SH1 and SH2 helices alter ADP release.\u003csup\u003e50,51\u003c/sup\u003e SH1 and the relay helix also connect the converter domain to the actin-binding surface, facilitating lever arm movement.\u003csup\u003e51\u003c/sup\u003e Although we did not see any differences in either SH1-SH2 interaction or stability (i.e., secondary structure), it is possible some structural alterations could occur outside of our timescale (i.e., 500 ns).\u003c/p\u003e\n\u003cp\u003eOur sMD simulations represent the first computational study of ADP release from the actin-myosin complex, which was made possible by the existence of a high-resolution atomic structure of post-powerstroke myosin.\u003csup\u003e16\u003c/sup\u003e The sMD simulations revealed that the conformation of ADP within the nucleotide pocket impacts ADP dissociation. In the case of G256E simulations, strong contacts between ADP and Switch-1 and the SH1-loop proved to be an extra energetic barrier inhibiting ADP release. sMD simulations also directed attention to particular residues that are important in ADP dissociation. K184, a positively charged residue in the P-loop lining the bottom of the pocket, is the first-line of defense in preventing ADP release, and the loss of a salt bridge between this residue and the β\u0026nbsp;phosphate is necessary to initiate ADP release. Mutation of this residue to a glutamine (K184Q) has been seen in patients with HCM or left ventricular noncompaction cardiomyopathy and is currently a variant of unknown significance (VUS).\u003csup\u003e52\u003c/sup\u003e Future studies of mutations in this residue and how it relates to ADP release may give insight into its potential pathogenicity. The next residue identified on the reaction coordinate is D239. In WT simulations, this residue forms a salt bridge with K679, acting as an ‘atomic-level staple’ to keep the nucleotide pocket closed. This residue is of particular interest because it has been tied to multiple disease-related mutations. D239N is a likely pathogenic mutation implicated in early-onset HCM.\u003csup\u003e53,54\u003c/sup\u003e Based on our observations we hypothesize that the change from Asp, a negatively charged side chain, to the uncharged polar residue Asn, will disrupt the salt bridge between 239 and 679. This would likely result in faster ADP release. This may explain results from a study demonstrating faster actin gliding velocities and actin-activated ATPase rates in D239N myosin, as ADP release is the rate-limiting step in unloaded actin-myosin assays.\u003csup\u003e55\u003c/sup\u003e Finally, K246 is another residue that is important for P\u003csub\u003eβ\u003c/sub\u003e stabilization. The mutation K246I is currently classified as likely pathogenic by Walsh et al.\u003csup\u003e56\u003c/sup\u003e A change from positive residue to a non-polar one would likely affect coordination of the nucleotide in the pocket through destabilization of P\u003csub\u003eβ\u003c/sub\u003e.\u003csup\u003e56\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eIn our previous study we found that, for isolated myofibrils from hiPSC-CMs, the kinetics of the slow, early phase of relaxation, \u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,slow\u0026nbsp;\u003c/sub\u003efollowing maximal (pCa 4.0) calcium activation was reduced.\u003csup\u003e8\u003c/sup\u003e This could result from either slowed ADP release from myosin or slowed ATP binding to myosin, as both steps that are required for cross-bridge detachment. Here we utilized elevated ADP as a product inhibition method to probe if ADP release from myosin is specifically altered with the G256E mutation. We chose to perform these experiments at a sub-maximal calcium level to make conditions more physiologically relevant, as pCa 5.6 is much closer to the rise in intracellular calcium that occurs during a cardiac twitch. \u0026nbsp;Importantly, \u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,slow\u003c/sub\u003e was significantly slower for G256E myofibrils at pCa 5.6 (Fig. 7f), a similar result as in our previous study at pCa 4.0.\u003csup\u003e8\u003c/sup\u003e The addition of 2.5 mM ADP to WT myofibrils slowed \u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,slow\u003c/sub\u003e to a rate that was similar to that for G256E myofibrils in the absence of ADP, and addition of ADP for G256E myofibrils resulted in a significant but smaller further reduction of \u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,slow\u003c/sub\u003e. The addition of ADP should inhibit ADP release and prolong the kinetics of this phase, which is seen in WT myofibrils but not G256E myofibrils. This suggests that the release of ADP is significantly compromised within G256E myofibrils, explaining in minimal response to ADP product inhibition. These data agree with the conclusions from our computational simulations and myosin biochemical measures regarding ADP.Mg\u003csup\u003e2+\u003c/sup\u003e release and greater ADP.Mg\u003csup\u003e2+\u003c/sup\u003e for myosin, respectively. However, we should not rule out other possible contributions to the relaxation rate, e.g. the rates of ATP binding and actomyosin dissociation. Our preliminary stopped-flow measures of mant-ATP binding to S1 or HMM indicate that the rate of ATP binding is multi-fold faster than \u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,slow\u003c/sub\u003e for both WT and G256E myofibrils, suggesting ADP release may control myosin detachment and relaxation kinetics. This is supported by measurements of \u003cem\u003ek\u003c/em\u003e\u003csub\u003eREL,slow,\u0026nbsp;\u003c/sub\u003ewhich was decreased for G256E compared to WT myofibrils at pCa 5.6 (Fig. 7f), and decreased by elevation of ADP for WT, but much less for G256E myofibrils. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.2 A note on force development\u003c/strong\u003e\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAt pCa 5.6, steady state force (Fig. 7c) was approximately doubled for G256E myofibrils compared with WT myofibrils, indicated increased calcium sensitivity of force, while activation kinetics, \u003cem\u003ek\u003c/em\u003e\u003csub\u003eACT\u003c/sub\u003e (Fig. 7d) were not affected. This contrasts with measurements we reported for maximal activation (pCa 4.0) where both force and \u003cem\u003ek\u003c/em\u003e\u003csub\u003eACT\u003c/sub\u003e were approximately doubled for G256E myofibrils.\u003csup\u003e8\u003c/sup\u003e\u0026nbsp; This suggests that the rate limiting chemo-mechanical process for the rate of force development at the sub-maximal calcium used (pCa 5.6) is new strong crossbridge formation and the force generating isomerization of myosin associated with inorganic phosphate release, and not ADP release or crossbridge detachment. The elevation of ADP did not affect steady state force but decreased \u003cem\u003ek\u003c/em\u003e\u003csub\u003eACT\u003c/sub\u003e by a similar amount for both WT and G256E myofibrils. Together this suggests the mutation affects the number of myosins that bind, but not the binding and force development kinetics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e5.3\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eLimitations of our study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHere, we report the first all-atom MD simulations of β-cardiac myosin in the post-powerstroke state. However, our model includes only an actin pentamer and myosin residues 1-810. Thus, there is no effective load in our system and effects on ADP release related to tension (force) or interaction between more complex constituents of the sarcomere are not included. Some studies suggest Mg\u003csup\u003e2+\u003c/sup\u003e may release from myosin prior to ADP release; however, here we have simulated release of ADP.Mg\u003csup\u003e2+\u003c/sup\u003e as a united molecule. We have not yet simulated the rigor actomyosin complex for this mutation, nor have we explored differences in ATP binding or actomyosin attachment for G256E myosin. Additional impacts of thin filament regulatory proteins were also not considered in our current simulation studies. \u0026nbsp;Finally, our models do not account for additional, more complex behavior of myosin, which includes destabilization of the interacting heads motif (IHM) that occurs with myofibril contractile activation and contributes to the hypercontractility of the G256E mutation.\u003csup\u003e8\u003c/sup\u003eStopped-flow biochemical experiments involve isolated actomyosin constructs in the absence of load which may over-report the effects the G256E mutation on ADP affinity, as load reduces the rate of ADP release from myosin.\u003csup\u003e57,58\u003c/sup\u003e Additionally, shortened myosin constructs are unable to form the IHM found in native thick filaments. As such, we are unable to assess the combined effects or interplay between alterations of myosin recruitment (ON and OFF states) and crossbridge cycling. Myofibrils derived from hiPSC-CMs may contain fetal isoforms of sarcomeric proteins; however, our maturation protocol results in myofibrils that contain predominantly β-myosin, which is the adult ventricular isoform affected by the G256E mutation, and the crossbridge cycling rate is dominated by myosin.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo mitigate the limitations of any one technique, our interdisciplinary research strategy used different models to explore the effects of a mutation at different scales to identify the common feature (ADP release) that G256E affects.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, we have demonstrated that the \u003cem\u003eMYH7\u0026nbsp;\u003c/em\u003eG256E impairs ADP release as well as the previously reported increase in myosin recruitment from the OFF state.\u003cstrong\u003e\u003csup\u003e8\u003c/sup\u003e\u003c/strong\u003e Together, these changes contribute to the clinically observed HCM phenotype of hypercontractility and impaired relaxation through delayed cross-bridge detachment and a greater population of myosin in force bearing states. Our conventional MD results suggest that mutations in the transducer region can allosterically affect the nucleotide binding pocket. Using sMD, we developed a predictive method to computationally assess a specific transition step in the cross-bridge cycle\u0026ndash;ADP release. Our results were corroborated with experimental data obtained from myofibrils obtained from hiPSC-CMs and recombinant sS1 protein, emphasizing the necessity for both computational approaches and validation against experimental data. Similar studies could help elucidate the mechanisms of other specific mutations in myosin implicated in disease and contribute to developing additional sarcomere targeted therapies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e7. \u0026nbsp; Acknowledgements\u003c/p\u003e\n\u003cp\u003eThis work used the Extreme Science and Engineering Discovery Environment (XSEDE) resource COMET through allocation TG-MCB200100 to M.R. XSEDE was supported by the National Science Foundation grant no. ACI-1548562. Partial funding for MCC was provided by award nos. T32HL007828 and K99HL173646 from the National Heart, Lung, and Blood Institute. Partial support for KYK was provided by T32EB032787. This research was supported by the University of Washington Center for Translational Muscle Research (CTMR) via the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health award no. P30AR074990.\u0026nbsp;This work was initiated and supported by the NIH/NIGMS grant 1RM1 GM131981-03 to JAS, KMR and MR and supported by NHLBI R01HL128368 to MR. This work would not have been possible without the Allen Institute for Cell Science team who contributed with cell line generation. The parental WT unedited hiPSC line, WTC, was provided by the Bruce R. Conklin Laboratory at the Gladstone Institutes and UCSF. The Allen Institute for Cell Science wishes to thank the Allen Institute for Cell Science founder, Paul G. Allen, for his vision, encouragement, and support. The content is solely the responsibility of the authors and does not necessarily represent the official view of the NHLBI or the NIH. The authors have no financial conflicts of interest that may be construed to these data.\u003c/p\u003e\n\u003cp\u003e8.\u0026nbsp; \u0026nbsp;Author Contributions:\u003c/p\u003e\n\u003cp\u003eDesigned research, M.C.C. and M.R.\u003c/p\u003e\n\u003cp\u003eProvided purified protein, D.P. and R.R.G.\u003c/p\u003e\n\u003cp\u003ePerformed research, M.C.C., K.K., and T.S.M.\u003c/p\u003e\n\u003cp\u003eAnalyzed data, M.C.C. and K.K.\u003c/p\u003e\n\u003cp\u003eManuscript writing, K.K., M.C.C., and M.R.\u003c/p\u003e\n\u003cp\u003eResources and Funding, M.R. and J.A.S.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMaron, B. J. \u0026amp; Maron, M. S. Hypertrophic cardiomyopathy. \u003cem\u003eLancet\u003c/em\u003e 381, 242\u0026ndash;255 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarian, A. J. \u0026amp; Braunwald, E. Hypertrophic Cardiomyopathy. \u003cem\u003eCirc. Res.\u003c/em\u003e 121, 749\u0026ndash;770 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlfares, A. A. \u003cem\u003eet al.\u003c/em\u003e Results of clinical genetic testing of 2,912 probands with hypertrophic cardiomyopathy: expanded panels offer limited additional sensitivity. \u003cem\u003eGenet. Med.\u003c/em\u003e 17, 880\u0026ndash;888 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSellers, J. R. Myosins: a diverse superfamily. \u003cem\u003eBiochim. Biophys. Acta (BBA) - Mol. Cell Res.\u003c/em\u003e 1496, 3\u0026ndash;22 (2000).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePate, E. \u0026amp; Cooke, R. A model of crossbridge action: the effects of ATP, ADP and Pi. \u003cem\u003eJ. Muscle Res. Cell Motil.\u003c/em\u003e 10, 181\u0026ndash;196 (1989).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRegnier, M., Lee, D. M. \u0026amp; Homsher, E. ATP Analogs and Muscle Contraction: Mechanics and Kinetics of Nucleoside Triphosphate Binding and Hydrolysis. \u003cem\u003eBiophys. J.\u003c/em\u003e 74, 3044\u0026ndash;3058 (1998).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRegnier, M. \u0026amp; Homsher, E. The Effect of ATP Analogs on Posthydrolytic and Force Development Steps in Skinned Skeletal Muscle Fibers. \u003cem\u003eBiophys. J.\u003c/em\u003e 74, 3059\u0026ndash;3071 (1998).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee, S. \u003cem\u003eet al.\u003c/em\u003e Incomplete-penetrant hypertrophic cardiomyopathy MYH7 G256E mutation causes hypercontractility and elevated mitochondrial respiration. \u003cem\u003eProc. Natl. Acad. Sci.\u003c/em\u003e 121, e2318413121 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFananapazir, L. \u0026amp; Epstein, N. D. Genotype-phenotype correlations in hypertrophic cardiomyopathy. Insights provided by comparisons of kindreds with distinct and identical beta-myosin heavy chain gene mutations. \u003cem\u003eCirculation\u003c/em\u003e 89, 22\u0026ndash;32 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCoureux, P., Sweeney, H. L. \u0026amp; Houdusse, A. Three myosin V structures delineate essential features of chemo-mechanical transduction. \u003cem\u003eEMBO J.\u003c/em\u003e 23, 4527\u0026ndash;4537 (2004).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTesi, C., Piroddi, N., Colomo, F. \u0026amp; Poggesi, C. Relaxation Kinetics Following Sudden Ca2\u0026thinsp;+\u0026thinsp;Reduction in Single Myofibrils from Skeletal Muscle. \u003cem\u003eBiophys. J.\u003c/em\u003e 83, 2142\u0026ndash;2151 (2002).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVeigel, C., Molloy, J. E., Schmitz, S. \u0026amp; Kendrick-Jones, J. Load-dependent kinetics of force production by smooth muscle myosin measured with optical tweezers. \u003cem\u003eNat. Cell Biol.\u003c/em\u003e 5, 980\u0026ndash;986 (2003).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSiemankowski, R. F., Wiseman, M. O. \u0026amp; White, H. D. ADP dissociation from actomyosin subfragment 1 is sufficiently slow to limit the unloaded shortening velocity in vertebrate muscle. \u003cem\u003eProc. Natl. Acad. Sci.\u003c/em\u003e 82, 658\u0026ndash;662 (1985).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKov\u0026aacute;cs, M., Thirumurugan, K., Knight, P. J. \u0026amp; Sellers, J. R. Load-dependent mechanism of nonmuscle myosin 2. \u003cem\u003eProc. Natl. Acad. Sci.\u003c/em\u003e 104, 9994\u0026ndash;9999 (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCapitanio, M. \u003cem\u003eet al.\u003c/em\u003e Two independent mechanical events in the interaction cycle of skeletal muscle myosin with actin. \u003cem\u003eProc. Natl. Acad. Sci.\u003c/em\u003e 103, 87\u0026ndash;92 (2006).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDoran, M. H. \u003cem\u003eet al.\u003c/em\u003e Conformational changes linked to ADP release from human cardiac myosin bound to actin-tropomyosin. \u003cem\u003eJ. Gen. Physiol.\u003c/em\u003e 155, e202213267 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUjfalusi, Z. \u003cem\u003eet al.\u003c/em\u003e Dilated cardiomyopathy myosin mutants have reduced force-generating capacity. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e 293, 9017\u0026ndash;9029 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBerman, H. M. \u003cem\u003eet al.\u003c/em\u003e The Protein Data Bank. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e 28, 235\u0026ndash;242 (2000).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFiser, A., Do, R. K. G. \u0026amp; Šali, A. Modeling of loops in protein structures. \u003cem\u003eProtein Sci.\u003c/em\u003e 9, 1753\u0026ndash;1773 (2000).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWebb, B. \u0026amp; Sali, A. Comparative Protein Structure Modeling Using MODELLER. \u003cem\u003eCurr. Protoc. Bioinform.\u003c/em\u003e 54, 5.6.1\u0026ndash;5.6.37 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePettersen, E. F. \u003cem\u003eet al.\u003c/em\u003e UCSF Chimera\u0026mdash;A visualization system for exploratory research and analysis. \u003cem\u003eJ. Comput. Chem.\u003c/em\u003e 25, 1605\u0026ndash;1612 (2004).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCase, D. A. \u003cem\u003eet al.\u003c/em\u003e Amber 2024. \u003cem\u003eUniversity of California, San Francisco\u003c/em\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMaier, J. A. \u003cem\u003eet al.\u003c/em\u003e ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. \u003cem\u003eJ. Chem. Theory Comput.\u003c/em\u003e 11, 3696\u0026ndash;3713 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. \u0026amp; Klein, M. L. Comparison of simple potential functions for simulating liquid water. \u003cem\u003eJ. Chem. Phys.\u003c/em\u003e 79, 926\u0026ndash;935 (1983).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, P. \u0026amp; Merz, K. M. Taking into Account the Ion-Induced Dipole Interaction in the Nonbonded Model of Ions. \u003cem\u003eJ. Chem. Theory Comput.\u003c/em\u003e 10, 289\u0026ndash;297 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, P., Song, L. F. \u0026amp; Merz, K. M. Systematic Parameterization of Monovalent Ions Employing the Nonbonded Model. \u003cem\u003eJ. Chem. Theory Comput.\u003c/em\u003e 11, 1645\u0026ndash;1657 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, P., Song, L. F. \u0026amp; Merz, K. M. Parameterization of Highly Charged Metal Ions Using the 12-6-4 LJ-Type Nonbonded Model in Explicit Water. \u003cem\u003eJ. Phys. Chem. B\u003c/em\u003e 119, 883\u0026ndash;895 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A. \u0026amp; Case, D. A. Development and testing of a general amber force field. \u003cem\u003eJ. Comput. Chem.\u003c/em\u003e 25, 1157\u0026ndash;1174 (2004).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNeese, F., Wennmohs, F., Becker, U. \u0026amp; Riplinger, C. The ORCA quantum chemistry program package. \u003cem\u003eJ. Chem. Phys.\u003c/em\u003e 152, 224108 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMiyamoto, S. \u0026amp; Kollman, P. A. Settle: An analytical version of the SHAKE and RATTLE algorithm for rigid water models. \u003cem\u003eJ. Comput. Chem.\u003c/em\u003e 13, 952\u0026ndash;962 (1992).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoe, D. R. \u0026amp; Cheatham, T. E. PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data. \u003cem\u003eJ. Chem. Theory Comput.\u003c/em\u003e 9, 3084\u0026ndash;3095 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChilders, M. C., Geeves, M., Daggett, V. \u0026amp; Regnier, M. Modulation of post-powerstroke dynamics in myosin II by 2\u0026prime;-deoxy-ADP. \u003cem\u003eArch. Biochem. Biophys.\u003c/em\u003e 699, 108733 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChilders, M. C. \u0026amp; Regnier, M. Dynamics of the Pre-Powerstroke Myosin Lever Arm and the Effects of Omecamtiv Mecarbil. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e 25, 10425 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHopkins, C. W., Grand, S. L., Walker, R. C. \u0026amp; Roitberg, A. E. Long-Time-Step Molecular Dynamics through Hydrogen Mass Repartitioning. \u003cem\u003eJ. Chem. Theory Comput.\u003c/em\u003e 11, 1864\u0026ndash;1874 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNag, S. \u003cem\u003eet al.\u003c/em\u003e The myosin mesa and the basis of hypercontractility caused by hypertrophic cardiomyopathy mutations. \u003cem\u003eNat. Struct. Mol. Biol.\u003c/em\u003e 24, 525\u0026ndash;533 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePardee, J. D. \u0026amp; Aspudich, J. Purification of muscle actin. \u003cem\u003eMethods Enzym.\u003c/em\u003e 85, 164\u0026ndash;181 (1982).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKao, K. \u0026amp; Geeves, M. A. Familial Cardiomyopathies, Methods and Protocols. \u003cem\u003eMethods Mol. Biol.\u003c/em\u003e 2735, 191\u0026ndash;211 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKreitzer, F. R. \u003cem\u003eet al.\u003c/em\u003e A robust method to derive functional neural crest cells from human pluripotent stem cells. \u003cem\u003eAm. J. stem cells\u003c/em\u003e 2, 119\u0026ndash;31 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoberts, B. \u003cem\u003eet al.\u003c/em\u003e Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization. \u003cem\u003eMol. Biol. Cell\u003c/em\u003e 28, 2854\u0026ndash;2874 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoberts, B. \u003cem\u003eet al.\u003c/em\u003e Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs. \u003cem\u003eStem Cell Rep.\u003c/em\u003e 12, 1145\u0026ndash;1158 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBurridge, P. W. \u003cem\u003eet al.\u003c/em\u003e Chemically defined generation of human cardiomyocytes. \u003cem\u003eNat. Methods\u003c/em\u003e 11, 855\u0026ndash;860 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBertero, A. \u003cem\u003eet al.\u003c/em\u003e Dynamics of genome reorganization during human cardiogenesis reveal an RBM20-dependent splicing factory. \u003cem\u003eNat. Commun.\u003c/em\u003e 10, 1538 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMohran, S., Steczina, S., Mandrycky, C., Kao, K. \u0026amp; Regnier, M. Familial Cardiomyopathies, Methods and Protocols. \u003cem\u003eMethods Mol. Biol.\u003c/em\u003e 2735, 213\u0026ndash;233 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBers, D. M., Patton, C. W. \u0026amp; Nuccitelli, R. Chapter 1 A Practical Guide to the Preparation of Ca2\u0026thinsp;+\u0026thinsp;Buffers. \u003cem\u003eMethods Cell Biol.\u003c/em\u003e 99, 1\u0026ndash;26 (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRibeiro, A. J. S. \u003cem\u003eet al.\u003c/em\u003e Contractility of single cardiomyocytes differentiated from pluripotent stem cells depends on physiological shape and substrate stiffness. \u003cem\u003eProc. Natl. Acad. Sci.\u003c/em\u003e 112, 12705\u0026ndash;12710 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePioner, J. M. \u003cem\u003eet al.\u003c/em\u003e Isolation and Mechanical Measurements of Myofibrils from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes. \u003cem\u003eStem Cell Rep.\u003c/em\u003e 6, 885\u0026ndash;896 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIzrailev, S. \u003cem\u003eet al.\u003c/em\u003e Computational Molecular Dynamics: Challenges, Methods, Ideas, Proceedings of the 2nd International Symposium on Algorithms for Macromolecular Modelling, Berlin, May 21\u0026ndash;24, 1997. \u003cem\u003eLect. Notes Comput. Sci. Eng.\u003c/em\u003e 39\u0026ndash;65 (2011) doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-3-642-58360-5_2\u003c/span\u003e\u003cspan address=\"10.1007/978-3-642-58360-5_2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePospich, S., Sweeney, H. L., Houdusse, A. \u0026amp; Raunser, S. High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism. \u003cem\u003eeLife\u003c/em\u003e 10, e73724 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSweeney, H. L. \u0026amp; Houdusse, A. Structural and Functional Insights into the Myosin Motor Mechanism. \u003cem\u003eAnnu. Rev. Biophys.\u003c/em\u003e 39, 539\u0026ndash;557 (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGargey, A., Iragavarapu, S. B., Grdzelishvili, A. V. \u0026amp; Nesmelov, Y. E. Electrostatic interactions in the SH1-SH2 helix of human cardiac myosin modulate the time of strong actomyosin binding. \u003cem\u003eJ. Muscle Res. Cell Motil.\u003c/em\u003e 42, 137\u0026ndash;147 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMesentean, S., Koppole, S., Smith, J. C. \u0026amp; Fischer, S. The Principal Motions Involved in the Coupling Mechanism of the Recovery Stroke of the Myosin Motor. \u003cem\u003eJ. Mol. Biol.\u003c/em\u003e 367, 591\u0026ndash;602 (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMiller, E. M. \u003cem\u003eet al.\u003c/em\u003e Genetic Testing in Pediatric Left Ventricular Noncompaction. \u003cem\u003eCirc.: Cardiovasc. Genet.\u003c/em\u003e 10, e001735 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRichards, S. \u003cem\u003eet al.\u003c/em\u003e Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. \u003cem\u003eGenet. Med.\u003c/em\u003e 17, 405\u0026ndash;423 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaski, J. P. \u003cem\u003eet al.\u003c/em\u003e Prevalence of Sarcomere Protein Gene Mutations in Preadolescent Children With Hypertrophic Cardiomyopathy. \u003cem\u003eCirc.: Cardiovasc. Genet.\u003c/em\u003e 2, 436\u0026ndash;441 (2009).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAdhikari, A. S. \u003cem\u003eet al.\u003c/em\u003e Early-Onset Hypertrophic Cardiomyopathy Mutations Significantly Increase the Velocity, Force, and Actin-Activated ATPase Activity of Human β-Cardiac Myosin. \u003cem\u003eCell Rep.\u003c/em\u003e 17, 2857\u0026ndash;2864 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWalsh, R. \u003cem\u003eet al.\u003c/em\u003e Reassessment of Mendelian gene pathogenicity using 7,855 cardiomyopathy cases and 60,706 reference samples. \u003cem\u003eGenet. Med.\u003c/em\u003e 19, 192\u0026ndash;203 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCremo, C. R. \u0026amp; Geeves, M. A. Interaction of Actin and ADP with the Head Domain of Smooth Muscle Myosin: Implications for Strain-Dependent ADP Release in Smooth Muscle \u0026dagger;. \u003cem\u003eBiochemistry\u003c/em\u003e 37, 1969\u0026ndash;1978 (1998).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGreenberg, M. J., Shuman, H. \u0026amp; Ostap, E. M. Inherent Force-Dependent Properties of β-Cardiac Myosin Contribute to the Force-Velocity Relationship of Cardiac Muscle. \u003cem\u003eBiophys. J.\u003c/em\u003e 107, L41\u0026ndash;L44 (2014).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Conformational entropy of ADP in WT and G256E simulations.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDihedral pair\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 416px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eADP entropy (cal mol\u003csup\u003e-1\u003c/sup\u003e K\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eG256E\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u0026theta;\u003csub\u003e1\u003c/sub\u003e vs. \u0026theta;\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e9.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e7.62\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u0026theta;\u003csub\u003e3\u003c/sub\u003e vs. \u0026theta;\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e10.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e8.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u0026theta;\u003csub\u003e5\u003c/sub\u003e vs. \u0026theta;\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e11.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e10.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Percent coverage of dihedral maps sampled by ADP in WT and G256E simulations.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDihedral pair\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 416px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% coverage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eG256E\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u0026theta;\u003csub\u003e1\u003c/sub\u003e vs. \u0026theta;\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e8.93%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e3.88%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u0026theta;\u003csub\u003e3\u003c/sub\u003e vs. \u0026theta;\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e21.06%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e11.21%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u0026theta;\u003csub\u003e5\u003c/sub\u003e vs. \u0026theta;\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e33.49%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e17.48%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"hiPSC-CMs, myosin, ADP release, molecular dynamics simulations, muscle mechanics, muscle kinetics","lastPublishedDoi":"10.21203/rs.3.rs-7602931/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7602931/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMutations in myosin alter its motor functions in diverse ways by affecting different structural and chemo-mechanical events. Multidisciplinary strategies can be used to understand how varying alterations in motor function converge to common phenotypes like hypercontractility and hypertrophic cardiomyopathy (HCM). Here, we combined molecular dynamics (MD) simulations with protein biochemical and myofibril mechanical analyses to study the HCM-causing myosin variant G256E. MD simulations demonstrated that G256E induces structural changes that increase the work required to displace ADP.Mg\u003csup\u003e2+\u003c/sup\u003e from actomyosin complex. Stopped-flow biochemical analysis demonstrated increased ADP affinity for actomyosin and single myofibril mechanics analysis demonstrated increased force generation and reduced ADP sensitivity of the early, slow phase of relaxation. Together, these results demonstrate that slower ADP release from myosin during contraction is a significant contributor to pathological contractile nature of the G256E mutation. This study highlights the importance of detailed chemo-mechanical analysis of mutations associated with hereditary cardiac diseases.\u003c/p\u003e","manuscriptTitle":"The G256E HCM mutation prolongs cardiac muscle relaxation via altered nucleotide handling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 17:28:19","doi":"10.21203/rs.3.rs-7602931/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-chemistry","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commschem","sideBox":"Learn more about [Communications Chemistry](http://www.nature.com/commschem/)","snPcode":"","submissionUrl":"","title":"Communications Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"46938345-959a-498c-bac5-e46bbcb785a6","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":55669796,"name":"Biological sciences/Chemical biology/Mechanism of action"},{"id":55669797,"name":"Biological sciences/Structural biology/Molecular modelling"}],"tags":[],"updatedAt":"2026-04-17T06:00:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-15 17:28:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7602931","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7602931","identity":"rs-7602931","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}