Homologous mutations in β, embryonic, and perinatal muscle myosins have divergent effects on molecular power generation

Figures

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  Figure 1.. Mutations at a highly conserved location in the motor domain of different myosin isoforms cause distinct diseases. a. A homology model structure of β-cardiac myosin motor domain highlights that the arginine residue (R671) is located between the active site and lever arm. b. A multiple sequence alignment of different myosin-Ils expressed in various muscles shows that the mutation site is highly conserved. This paper studies homologous R671 mutations in β (MYH7), embryonic (MYH3), and perinatal (MYH8) myosins implicated in hypertrophic cardiomyopathy (HCM), Freeman-Sheldon syndrome (FSS), and trismus pseudocamptodactyly syndrome (TPS), respectively. Overall, sequence identity and similarity are respectively 79% and 89% between β and embryonic myosin, 81% and 90% between β and perinatal myosin, and 85% and 93% between embryonic and perinatal myosin.

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  Figure 2.. Steady-state actin-activated ATPase rates of myosins measured by solution absorbance. A representative dataset is shown for a single biological replicate of β-WT and R671C (a), EmbWT, R672C, and R672H (b), PeriWT and R674Q (c), with all three WT isoforms shown in (d) where each data point represents the average of three technical replicates at each actin concentration, and error bars represent standard deviations. The data is fit to the Michaelis-Menten equation (solid line) to determine kcat and Km for each myosin. Three independent experiments, each with their own curve fit, were performed at 30°C for each protein. Average values of kcat (e) and Km (f) are plotted as a bar graph with s.e.m., where data points represent the individual measurements of each independent experiment.

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  Figure 3.. Actin sliding velocities of myosins measured by the in vitro motility assay. a-c. Velocities of β S1 WT and R671C (a), embryonic S1 WT, R672C, and R672H (b), and perinatal S1 WT and R674Q (c), each at 23 °C and 30 °C. Reported velocities are “Mean velocities filtered” (see Methods and Supplementary Figures 2–4 for detailed analysis and additional parameters obtained from motility data). Each data point represents one independent experiment. Error bars (most of which are smaller than the lines’ thickness) over the bar graph represent mean ± s.e.m. All relevant pairwise comparisons are statistically significant (Supplementary Table 1). d. Velocities of each myosin are plotted as a function of temperature. The pairs of data points at 23 °C and 30 °C (open circles) are taken from a-c. The data points spanning ~21 °C to 37 °C (black solid circles) are of wild type human β-cardiac short-S1 (residues 1–808) as reported in (Liu C, Ruppel KM, Spudich JA. (2023). Motility assay to probe the calcium sensitivity of myosin and regulated thin filaments. In M Regnier and M Childers (Eds.) Methods in Molecular Biology: Familial Cardiomyopathies. New York, NY: Springer Nature). Error bars…

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  Figure 4.. Single-molecule load-dependent actin-detachment kinetics of myosins measured by optical trapping. a. Setup of the three-bead system in the optical trap (top). In harmonic force spectroscopy (HFS), the stage upon which myosin sits oscillates so that a sinusoidal external force with mean F0 is applied to myosin upon actin binding. Depending on the randomness of when attachment occurs, binding events have different F0’s (bottom). F0>0 represents resistive force, opposite of the direction of stroke, and F0<0 represents assistive force, in the same direction of the stroke. Here, the direction of the lever arm swing and the resulting translation of the actin filament are indicated by the blue curved and straight arrows, respectively. See also Supplementary Figure 5 for illustration of the method of HFS to detect a binding event. b. All events for one example molecule. Time bound tb of each event is plotted against F0. Events binned by force (example bin shown as shaded rectangle) have exponentially distributed bound times, from which the detachment rate at that force is determined by maximum likelihood estimation (MLE) (inset). c. Detachment rates kdet determined across all force bins…

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  Figure 5.. Single-molecule step sizes of myosins measured by optical trapping. a. Determination of step size from HFS data. Left: time traces of three example events before (light gray) and during (dark gray) binding. The amplitude of actin dumbbell oscillations increases upon binding to the myosin which is attached to the oscillating stage (see Supplementary Figure 5 and Methods for details of HFS). Right: hundreds of traces from one molecule are start-aligned, extended in time, and averaged (light and dark gray traces). A fitted sinusoid is then subtracted from the averaged trace to reveal the change in actin position due to myosin binding alone (black). b. Traces of step size from one molecule of each protein studied. c. Step sizes of all molecules measured. Each data point represents one molecule’s step size whose error bars represent uncertainties in the determination of change in actin position. Horizontal lines and shaded rectangles represent mean ± s.e.m. whose values are given in Table 1. All relevant pairwise comparisons are statistically significant except between βS1 WT and EmbS1 WT (Supplementary Table 1).

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  Figure 6.. Single molecule detachment kinetics and step size determine ensemble actin sliding velocity. a. Velocities measured in the unloaded motility assay plotted against the detachment rate at zero force k0 at 23 °C (Table 1). Unlabeled gray data points are from Liu et al. NSMB 201842. Error bars represent s.e.m. As velocity is predominantly detachment limited in our motility assay, linear regression gives a slope of 7.4 nm and R2=0.81. b. Measured velocities V (as in a) plotted against the product of measured step sizes d and measured detachment rates at zero force k0 (Table 1). Data from Liu et al. NSMB 2018 is not shown in b because step size data was not available. Horizontal error bars represent propagated errors in calculating d*k0. The dashed line represents V=d*k0.

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  Figure 7.. Duty ratio, average force, and average power of myosins determined by ensemble ATPase and single molecule measurements. a-c. Duty ratio (a), average force (b), and average power (c) as a function of the load force F, calculated by Equations (2)–(6). All curves are calculated at 30 °C using values of detachment rate kinetics k0,δ, step size d, kcat, and Q10 given in Table 1. The values of duty ratio at zero load are also given in Table 1. Dotted line in (b) represents the average force if duty ratio = 1, in which case the average force equals the load force.

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  Figure 8.. The R672C mutation reduces lever arm priming and ADP pocket opening in molecular dynamics simulations of embryonic myosin. All simulations were started from homology models built from template crystal structures of a pre-powerstroke wild type β-cardiac myosin. a. R671’s hydrogen bonds selectively stabilize the pre-powerstroke state in β myosin. A representative structure of the two hydrogen bonds is shown on the left. The bar chart (right) shows the probability of R671 forming hydrogen bonds with nearby residues T177 (transducer) and Q486 (relay helix) in simulations of the pre-powerstroke and post-rigor states. Data points represent individual trajectories and error bars are standard errors of the mean obtained from bootstrapping. b. Schematic depicting the lever arm angle that was measured in structures from simulations. A reference plane was defined by crystal structures of the pre-powerstroke (PDB: 5N69) and post-rigor (PDB: 6FSA) states. c. Opening and closure of the actin-binding cleft is observed in simulations. The inner cleft distance94 between the α-carbon atoms L277 and S472 (MYH7 numbering) is used as a metric of cleft closure. d. Among cleft closed states, Emb R672C…