The hypertrophic cardiomyopathy mutation G768R makes cardiac myosin a high duty ratio motor

Figures

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  Figure 1.. G768R slows motility velocity to a much greater extent than ATPase of the myosin motor domain. (A) Location of hypertrophic cardiomyopathy-causing mutation G768R at the end of the converter domain shown as pink spheres on cartoon representation of human β-cardiac myosin sS1 (residues 1-808 of PDB structure 8QYU). (B) Multiple sequence alignment of different myosin-IIs expressed in various muscles show that the mutation site is highly conserved. (C) Actin-activated ATPase of WT (black) and G768R (pink) sS1. G768R kcat = 3.92 ± 0.13 s−1,Km= 25.2 ± 1.2μM, WT kcat = 5.37 ± 0.06 s−1, Km= 38.6 ± 2.4μM (see also Fig. S1 and Table S1). (D) Actin sliding velocity of WT and G768R sS1 in the unloaded in vitro actin motility assay. Each data point represents a biological replicate. Each biological replicate represents average of three technical replicates. VWT = 979.6 ± 61 nm/s, VG768R = 194 ± 2 nm/s). Velocities represent MVEL20 from FAST analysis of motility movies (see Methods and Fig. S2). (E) Motility velocities for different mixtures of WT and G768R sS1. Error bars are the standard error of mean from at least six movie replicates. Solid line shows the best quadratic fit to the data.

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  Figure 2.. G768R severely slows actin-detachment kinetics of single myosin molecules to make cardiac myosin a high duty ratio motor. (A) Load-sensitive actin-detachment rates kdet(F0) of WT (black) and G768R (pink) sS1 molecules measured by the harmonic force spectroscopy (HFS) technique in a dual-beam optical trap. Positive forces represent resistive loads, and negative forces represent assistive loads. Each thin line is a fit of Eqn. 1 to data from one molecule, each with a few hundred binding events. Individual data points represent the detachment rates obtained by a MLE (maximum likelihood estimation) of a molecule’s events binned by force. (B) and (C). The fitted parameters k0 (detachment rate at zero load) and δ (load sensitivity) of each molecule corresponding to thin lines in A. Mean and SEM: WT k0= 111 ± 10 s−1, = 0.88 ± 0.08 nm; G768R k0 = 6.7 ± 0.7 s−1, = 0.71 ± 0.06 nm. (D) Averaged, sine-subtracted traces across all binding events of one example molecule each of WT and G768R, from which step size is determined. (E) Step sizes of all WT and G768R molecules. Each point represents one molecule. Error bars represent SEM. **** indicates p≤0.0001. (F) Calculated duty ratio across…

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  Figure 3:. G768R increases the actin-activated ATPase rate of 25-hep HMM β-cardiac myosin by releasing more heads to interact with actin. (A,B) Actin-activated ATPase of 2-hep and 25-hep HMM constructs of WT (A) and G768R (B). (C,D) Data replotted from (A,B) comparing G768R and WT for 2-hep (C) or 25-hep (D). Each data point represents the average of three technical replicates of one biological replicate and error bars represent SDs. See Table S1 and Fig. S1 for fitted kcat and Km values, data from other biological replicates, and statistical analyses. Curves are fitted to Michaelis-Menten kinetics, and shaded areas depict the 95% CI of the fits. Arrows with percentages represent changes relative to 2-hep (A,B) or WT (C,D).

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  Figure 4.. The G768R mutation alters lever arm priming and reduces ADP pocket opening in MD simulations of β-cardiac myosin. (A) Initial structural models of the human WT and mutant β-cardiac myosin were built from a pre-powerstroke (PPS) (PDB: 8QYU) and a post-rigor (PR) (6FSA) WT structure. The models are comprised of the heavy chain head (residues 2-810) and the essential light chain (ELC). The motor domain is colored white; the converter domain and lever arm are colored pink in PPS and purple in PR models. Residue 768 is shown as a cyan sphere. (B) Probability distributions of the lever arm angle (as defined in (D)) in all PPS simulations (top) and conditioned on inner cleft closed (≤ 1.1 nm) (bottom). Among all frames, G768R has an overall population shift towards larger angles compared to WT. Among cleft-closed states, the mutant also exhibits a smaller angle population suggesting loss in lever arm priming. (C) Opening and closing of the actin binding cleft is observed in the simulations of models in the PPS conformation. The inner cleft distance, between the α-carbons of L277 and S472, is used as a metric of cleft closure ( Liu et al., 2024). (D) The lever arm angle was defined as…