Divergent molecular phenotypes in point mutations at the same residue in beta-myosin heavy chain lead to distinct cardiomyopathies

Figures

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  Figure 1.. Actin-activated ATPase Activity of myosin constructs. (A) ATPase activity of myosin S1 constructs was measured over time with increasing actin concentrations. Data points were fitted using Michaelis Menten kinetics to obtain maximum ATPase activity (kcat) and actin affinity (KM). (B) Measured values of kcat and KM for HCM-linked β-MHC I467V and LVNC-linked β-MHC I467T S1 mutations. Neither mutation significantly altered maximal ATPase activity nor actin affinity. Data are represented as mean ± SEM. A one-way ANOVA was used to assess statistical significance (p<0.05) compared to β-MHC WT S1. N=6–11 curves per myosin S1 construct.

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  Figure 2.. Single turnover ATPase assay of myosin S1. (A) Representative double-exponential fluorescence decay curves from the single-turnover mant-ATP dependent ATPase assay for myosin S1 constructs (WT: blue, β-I467V: red, β-I467T: green). (B) The disordered (DRX, solid bars) and super-relaxed (SRX, dashed lines) states were calculated from the double-exponential fit of decay curves. (C) Area under the curve (AUC) was calculated for each decay curve. (D) Summary table of the single turnover assay measurements. ATPase rates associated with the DRX and SRX states (kDRX and kSRX, respectively) were calculated from the double exponential decay fits. Values are calculated from 5–7 decay curves per mutation. Data are represented as mean ± SEM. A one-way ANOVA was used to assess statistical significance (* = p<0.05 vs. β-WT S1) and adjusted with Tukey’s multiple comparisons to assess differences between mutations (# = p<0.05 vs. β-I467V S1). N= 5–7 curves per myosin S1 construct.

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  Figure 3.. Unloaded Actin Sliding Velocity. (A) In vitro motility rates for myosin S1 constructs were measured using TRITC-labeled actin filaments. Average actin sliding velocity was calculated from actin filaments that were ≥1 μm in length and mobile in >15 frames per video. (B) Summary values for In vitro motility actin sliding velocity assay. Values are an average of three separate purifications per S1 construct, and 7–9 videos per construct. % motile was calculated from the ratio of actin filaments moving in >15 frames relative to total number of actin filaments tracked. Values are reported as average ± standard error. A one-way ANOVA was used to assess statistical significance (p<0.05) compared to β-MHC WT S1. N = 7–8 videos per myosin S1 construct.

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  Figure 4.. I467T disrupts interactions between ADP and the nucleotide-binding pocket in molecular dynamics simulations. (A) Homology model structure of human beta-cardiac myosin, (B) focused in on the ADP-Pi binding site, with ADP depicted in yellow and orange, Pi depicted in orange, and Mg2+ in green. The red dotted line indicates a hydrogen bond between adenine and Y134 that is seen in nucleotide-bound crystal structures. (C) A native hydrogen bond the ADP base (ADP-N6) makes with a binding site residue (TYR134-OH) is disrupted in I467T. Additionally, the inset in (C) shows that the probability of a contact between ADP and TYR134 is significantly lower in I467T (p < 0.0001). (D) The contact probability between W130 and V186, two residues that form a lid over the adenine nucleobase, is significantly lower in I467T (p = 0.0011).