Research ArticleCLINICAL MEDICINE

Biophysical properties of human β-cardiac myosin with converter mutations that cause hypertrophic cardiomyopathy

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Science Advances  10 Feb 2017:
Vol. 3, no. 2, e1601959
DOI: 10.1126/sciadv.1601959
  • Fig. 1 Structure of a homology-modeled human β-cardiac sS1 domain and intrinsic force measurements for the three converter domain HCM mutant forms of the protein.

    (A) Structure of the homology-modeled (see Materials and Methods) human β-cardiac sS1 containing residues 1 to 808 of the MyHC (dark gray) and the ELC (beige) based on the structure of Winkelmann et al. (56). Positions of the HCM mutations R719W (blue), R723G (red), and G741R (green) in the converter domain (white). (B) Blowup of the converter domain shown in (A), with all residues shown in stick form except for the three mutated residues. (C) Intrinsic force measurements using a dual-beam laser trap. More than three independent protein preparations were made for each mutant and for WT human β-cardiac sS1; 800 to 900 binding events of six to seven individual molecules were analyzed. P = 0.0067, one-way analysis of variance (ANOVA). The error bars show the SEM. (D) Cumulative probability distributions from the same intrinsic force measurements shown in (C).

  • Fig. 2 Actin-activated ATPase activity of recombinant human β-cardiac sS1 with mutations in the converter domain compared with WT human β-cardiac sS1.

    (A) Representative actin-activated sS1 ATPase curves. (B) Mutant human β-cardiac sS1 kcat values relative to WT (error bars: 95% confidence interval).

  • Fig. 3 In vitro motility data for the three mutant human β-cardiac sS1 proteins compared to WT.

    (A) An example of automatic analysis of an in vitro motility movie with FAST (11) (available for download at http://spudlab.stanford.edu). Scatterplot of actin filament velocities as a function of filament length (gray, all velocity points for each filament; blue, maximum velocity of each filament). The dashed line indicates the TOP5% of filament velocities. At the top right, two histograms of velocities are shown: a histogram of all filament velocities with its MVEL marked as a black line and a histogram of velocities with tolerance filtering to eliminate intermittently moving filaments with a velocity dispersion higher than 20% of their mean within a five-frame window (MVEL20). At the bottom right, the effects of various levels of tolerance filtering on the TOP5% and the MVELs are shown. (B) The TOP5% velocities relative to WT for gliding actin filaments (left) or RTFs (in 10−5 M Ca2+) (right) driven by the three mutant human β-cardiac sS1 proteins. Each mutant protein was normalized against its matching WT protein prepared and assayed on the same days. Each bar graph is a mean of relative velocity (error bars represent ±95% confidence interval). (C) The percent mobile filaments for the velocity measurements using WT and mutant human β-cardiac sS1 proteins, for actin (left) and RTFs (right). Data are presented as means ± SEM. (D) In vitro motility assays with mixtures of WT and mutant protein at varying ratios.

  • Fig. 4 Loaded in vitro motility assays for the three mutant proteins compared to WT controls.

    The percent time mobile (11) is a measure of the relative effectiveness of the load molecule utrophin to overcome the Fensemble of the sS1 on the surface (see Materials and Methods) (11). The effect of utrophin concentration on the velocity of actin (left) and RTFs (in 10−5 M Ca2+) (right) is shown. For WT and each mutant protein, ≥5 independent protein preparations and ≥5 separate sets of curves were combined.

  • Fig. 5 Homology-modeled structures of human β-cardiac short heavy meromyosin (MyHC ending at residue 961) in the open and closed states.

    (A) Illustration of the equilibrium between open and closed states. (B) The closed state, shown enlarged and slightly rotated to the left about its vertical axis compared to that in (A). The interface of the blocked head (dark gray) and the converter (white) of the free head (light gray) is seen from the side. (C) Face-on view of the converter domain surface involved in the S1-S1 interaction. This view was obtained by rotating the molecule in (B) 90° to the left about its vertical axis and removing the blocked head from the image. (D) Blowup of the binding interface of the converter in (C), showing the positions of the three converter domain HCM mutations studied here.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/2/e1601959/DC1

    fig. S1. sS1 purification and ATPase measurements.

    fig. S2. Comparison of kcat (relative to WT) for R723G human β-cardiac sS1 with and without a FLAG tag on the ELC.

    fig. S3. Unloaded MVEL measurements for actin and RTF.

    fig. S4. Structure of the part of the converter domain showing G741R (green), R719W (blue), and R723G (red), neighboring residues whose conflict with the G741R conversion (residues I736 and P731; magenta) and whose interactions with R719 may be lost by the R719W conversion.

    table S1. Summary of kcat and Km values from actin-activated ATPase assays.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. sS1 purification and ATPase measurements.
    • fig. S2. Comparison of kcat (relative to WT) for R723G human β-cardiac sS1 with and without a FLAG tag on the ELC.
    • fig. S3. Unloaded MVEL measurements for actin and RTF.
    • fig. S4. Structure of the part of the converter domain showing G741R (green), R719W (blue), and R723G (red), neighboring residues whose conflict with the G741R conversion (residues I736 and P731; magenta) and whose interactions with R719 may be lost by the R719W conversion.
    • table S1. Summary of kcat and Km values from actin-activated ATPase assays.

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