Figure 1. Motility of the hypertrophic cardiomyopathy mutant, R712L-myosin, is impaired.

(A) Cartoon rendering of the β-cardiac myosin crystal structure (PDB: 5N69). The motor domain (grey, 1–707), converter/lever arm domain (blue, 708–806), and the essential light chain (red) are shown. The box indicates the region expanded to the right showing the E497-R712 salt bridge located at the fulcrum of the lever arm. (B) Distribution of individual filament gliding speeds from motility assays at a concentration of 100 μg⋅ml⁻¹. Wild-type-myosin (black) has a higher average motility rate compared to R712L-myosin (red). (C) Increasing loading concentrations of myosin were added and the average filament speed of fluorescently labelled actin filaments was assessed. Higher concentrations of R712L-myosin were required to achieve motility, and actin filaments were not observed on the surface at concentrations <40 μg⋅ml⁻¹.

Figure 1—figure supplement 1. SDS-PAGE of WT- and R712L-myosin purification.

(A) Representative SDS-PAGE of five independent preparations of recombinantly expressed wild-type- and R712L-myosin (HMM fragments). The yield of purified myosin per unit culture dish was measured for a fixed expression period, using a common infection protocol, comparable multiplicity of infection, and identical purification methods. Expression levels for WT- and R712L-myosin are comparable in the whole cell lysate (WCL) and the triton soluble extract (TSE), the starting point for protein purification of myosin. The level of myosin expression approaches that of the endogenous C2C12 myosin in the cell extract. (B) Purified WT-myosin and the R712L-myosin (HMM fragments) are routinely analyzed by SDS-PAGE. Lane 1, WT-myosin; lane 2, R712L-myosin. Average recovery of WT-myosin was 174 µg per p150 dish (range 100–262) and 157 µg/p150 (range 126–210) for R712L-myosin. SDS-PAGE analysis of each step from each purification from cell lysis through the final purified protein was performed to detect losses from individual steps. Comparable yields of the WT and R712L proteins suggest no difference in the stability in this culture system.

Figure 1—figure supplement 2. E497D-myosin gliding filament assays.

The speeds of individual fluorescently labeled actin filaments were quantified in gliding filament motility assays for E497D-myosin at a loading concentration of 100 μg⋅ml⁻¹ (n = 1408 individual filaments). Wild-type (WT)-myosin is replotted from Figure 1B for comparison.

Figure 1—figure supplement 3. Kinetics of phosphate dissociation from thin filament (TF)-activated β-cardiac myosin.

TF activation of phosphate dissociation was measured via the change in N-[2-(1-maleimidyl)ethyl]−7-(diethylamino)coumarin-3-carboxamide-labeled phosphate binding protein (MDCC-PBP) fluorescence (see Materials and methods). Briefly, 3 µM β-cardiac myosin was first mixed with 2 µM ATP, held in a delay line for 2 s, and then mixed with TFs to accelerate P_i release. Final concentrations in the flow cell were 0.75 µM myosin, 0.5 µM ATP, 0–60 µM actin in native TFs, 5 mM MOPS (pH 7.2), 2 mM MgCl₂, 10 mM KCl, 0.05 mM CaCl₂, 0.25% DMSO, 0 or 50 µM omecamtiv mecarbil (OM), 5 µM MDCC-PBP, 0.1 mM 7-methylguanosine, and 0.01 unit/mL purine nucleoside phosphorylase at 20°C. (A–D) Representative traces showing the change in MDCC-PBP fluorescence with 60 µM actin in TFs in the presence and absence of OM for WT- and R712L-myosin. The data were fit to the sum of two-exponentials for: (A) Wild-type (WT)-myosin (7.8 s⁻¹, 0.52 s⁻¹), (B) WT-myosin with OM (42 s⁻¹, 1.0 s⁻¹), (C) R712L-myosin (17 s⁻¹, 0.78 s⁻¹), and (D) R712L-myosin with OM (17 s⁻¹, 1.0 s⁻¹). (E–F) Kinetics of phosphate release for WT (E) and for R712L-myosin (F) as a function of actin subunit concentration in porcine native TFs. (■) without OM; (□) with OM. Solid lines are hyperbolic fits with constants reported in Table 1.

Figure 1—figure supplement 4. ADP dissociation from actomyosin.

(A) ADP dissociation from actomyosin was measured via light scattering (see Materials and methods). Briefly, stopped flow was used to quickly mix actomyosin and ADP with ATP (30 µM final concentration), and a decrease in light scattering due to dissociation of actomyosin is known to be limited by ADP dissociation in wild-type (WT) myosin. Final concentrations were 1.25 μM myosin, 3 µM actin, 0–800 µM ADP, 30 µM ATP, 2 mM MgCl₂, 25 mM KCl, 0.25% DMSO, and either 0 or 50 µM OM in 5 mM MOPS (pH 7.2). (■) WT-myosin, (□) WT-myosin + 50 µM OM, (●) R712L-myosin, (○) R712L-myosin + 50 µM OM. (B–E) Maximum rates of ADP release were obtained with final concentrations in the cell of 1.25 μM myosin, 3 µM actin, 140 µM ADP, 2 mM ATP, 5 mM MOPS (pH 7.2), 2 mM MgCl₂, 25 mM KCl, 0.25% DMSO, and either 0 or 50 µM OM. Representative traces from (B) WT-myosin (k_obs = 74 s⁻¹), (C) WT-myosin + 50 µM OM (k_obs = 85 s⁻¹), (D) R712L-myosin (k_obs = 141 s⁻¹), and (E) R712L-myosin + 50 µM OM (k_obs = 147 s⁻¹). Average values of k_-AD from multiple experiments are reported in Table 1.

Figure 1—figure supplement 5. ATP binding to β-cardiac myosin measured by intrinsic tryptophan fluorescence.

Myosin was mixed with increasing concentrations of ATP via stopped flow and intrinsic tryptophan fluorescence was monitored. Upon ATP binding, intrinsic tryptophan fluorescence of the myosin increases. (A–D) Representative traces showing the change in fluorescence with 175 µM ATP in the presence and absence of omecamtiv mecarbil (OM) for wild-type (WT)- and R712L-myosin are shown and are best fit by the sum of two exponentials: (A) WT-myosin (139 s⁻¹, 0.48 s⁻¹), (B) WT-myosin with OM (115 s⁻¹, 0.82 s⁻¹), (C) R712L-myosin (80 s⁻¹, 0.77 s⁻¹), (D) R712L-myosin with OM (84 s⁻¹, 0.70 s⁻¹). (E) Rate of ATP binding to myosin as a function of ATP concentration. (■) WT-myosin, (□) WT-myosin + 50 µM OM, (●) R712L-myosin, (○) R712L-myosin + 50 µM OM. Final concentrations in the cell were 1.25 μM myosin, 3.75 µM to 1 mM ATP, 5 mM MOPS (pH 7.2), 2 mM MgCl₂, 25 mM KCl, 0.25% DMSO, and 0 or 50 µM OM. Reactions were carried out at 20°C. Solid lines are hyperbolic fits, with constants reported in Table 1.

Figure 1—figure supplement 6. ATP-induced dissociation of β-cardiac myosin from actin.

A decrease in light scattering due to actomyosin dissociation was observed upon mixing 2.5 µM myosin and 6 µM actin with increasing concentrations of ATP in a stopped flow device. Representative traces showing the change in light scattering in arbitrary units at 50 µM ATP in the presence and absence of omecamtiv mecarbil (OM) for wild-type (WT)- and R712L-myosins. Representative traces for (A) WT-myosin (105 s⁻¹, 19 s⁻¹), (B) WT-myosin + OM (108 s⁻¹, 2.4 s⁻¹), (C) R712L-myosin (155 s⁻¹, 32 s⁻¹), and (D) R712L-myosin + OM (147 s⁻¹, 29 s⁻¹). (E) Rate of actomyosin dissociation as a function of ATP concentration. (■) WT-myosin, (□) WT-myosin + 50 µM OM, (●) R712L-myosin, (○) R712L-myosin + 50 µM OM. Solid lines are fitted hyperbolae with constants given in Table 1. Experimental conditions in the cuvette: 1.25 μM myosin, 3 µM actin, 3.75 µM-1 mM ATP, 5 mM MOPS (pH 7.2), 2 mM MgCl₂, 25 mM KCl, 0.25% DMSO and either 0 or 50 µM OM, 20°C. This figure is related to the data displayed on Table 1.

Figure 1—figure supplement 7. Thin-filament (TF) activation of steady-state ATPase activity.

Steady-state ATPase rates were measured using an NADH-coupled assay at 25°C (5 mM MOPS pH 7.2, 2 mM MgCl₂, 1 mM DTT, 2 mM ATP, 0.01 mM EGTA, 0.1 mM CaCl₂, 0.5% DMSO, and either 0 or 50 µM omecamtiv mecarbil [OM]). 0.02–0.025 µM myosin and 0–25 µM actin concentration in TFs were used. (■) Wild-type (WT)-myosin, (□) WT-myosin + 50 µM OM, (●) R712L, (○) R712L + 50 µM OM. Solid lines are fits of the data by hyperbolic functions, with constants given in Table 1.

Figure 1—figure supplement 8. Minimal kinetic scheme of the ATPase cycle.

M = human β-cardiac myosin HMM fragment, T = ATP, D = ADP, P = phosphate. Positive subscripts denote the binding of the last ligand in the subscript, negative subscripts denote dissociation (e.g., k_-AD is the dissociation of ADP from acto.c-HMM.ADP). This scheme is related to the data displayed on Table 1.