β-Cardiac myosin hypertrophic cardiomyopathy mutations release sequestered heads and increase enzymatic activity

Abstract

Hypertrophic cardiomyopathy (HCM) affects 1 in 500 people and leads to hyper-contractility of the heart. Nearly 40 percent of HCM-causing mutations are found in human β-cardiac myosin. Previous studies looking at the effect of HCM mutations on the force, velocity and ATPase activity of the catalytic domain of human β-cardiac myosin have not shown clear trends leading to hypercontractility at the molecular scale. Here we present functional data showing that four separate HCM mutations located at the myosin head-tail (R249Q, H251N) and head-head (D382Y, R719W) interfaces of a folded-back sequestered state referred to as the interacting heads motif (IHM) lead to a significant increase in the number of heads functionally accessible for interaction with actin. These results provide evidence that HCM mutations can modulate myosin activity by disrupting intramolecular interactions within the proposed sequestered state, which could lead to hypercontractility at the molecular level.

Fig. 1

Location of mutated residues on folded back model of human β-cardiac myosin. a Model of the back-side view of the human β-cardiac myosin IHM (MS03 homology model), with the alpha-carbon backbones of the two S1 heads and the light chains shown as lines, and the S2 tail region represented by sticks. The four residues mutated in this study are represented by spheres. b Close up of region outlined by the dashed box in figure a showing the mutated residues R249Q (blue) and H251N (light blue) at the head-tail interface and D382Y (red) and R719W (blue) at the head–head interface

Fig. 2

Mutations R719W and D382Y do not affect proximal S2 binding. Microscale thermophoresis of WT (black), R719W (blue), and D382Y (red) 2-hep HMM titrated with proximal S2. Representative data from one preparation of myosin, and three experimental repeats. Error bars represent the SEM. Source data are provided as a Source Data file

Fig. 3

WT 25-hep HMM has lower ATPase activity than WT 2-hep HMM. a Mant-ATP single turnover kinetics of WT 2-hep HMM (black solid line and filled circles) and 25-hep HMM (red solid line and empty squares). Representative data from one preparation of each protein. Data is fit to a double exponential [Eq. (1)), with the slow phase in the 25-hep HMM clearly visible. Inset shows models of the 2-hep and 25-hep HMM, along with an SDS-PAGE gel showing the human heavy chain, the human ELC and the human RLC for both the 2-hep and 25-hep HMM (for uncropped image, see Supplementary Fig. 4). b Actin-activated ATPase activity of WT 2-hep HMM (black solid line and filled circles) and WT 25-hep HMM (red solid line and empty squares). Data is combined from two independent protein preparations with two experimental replicates for each preparation. Each point is an average with the error bar representing the SEM. Source data are provided as a Source Data file

Fig. 4

Effect of mesa mutations R249Q and H251N on HMM ATPase kinetics. In all cases, black solid line and filled circles are 2-hep HMM data; red solid line and empty squares are 25-hep HMM data. a Mant-nucleotide single turnover kinetics for R249Q 2-hep HMM and 25-hep HMM. b Actin-activated ATPase data for R249Q 2-hep and 25-hep HMM. c Mant-nucleotide single turnover kinetics for H251N 2-hep and 25-hep HMM. d Actin-activated ATPase data for H251N 2-hep and 25-hep HMM. a, c show representative data from one preparation each. Each of panels b and d are combined data from three independent preparations of HMMs with three experimental replicates for each preparation. Each point is an average with the error bar representing the SEM. Source data are provided as a Source Data file

Fig. 5

Effect of PHHIS-converter domain interaction mutations R719W and D382Y on HMM ATPase kinetics. In all cases, black solid line and filled circles are 2-hep HMM data; red solid line and empty squares are 25-hep HMM data. a Mant-nucleotide single turnover kinetics for R719W 2-hep and 25-hep HMM. b Actin-activated ATPase data for R719W 2-hep and 25-hep HMM. c Mant-nucleotide single turnover kinetics for D382Y 2-hep and 25-hep HMM. d Actin-activated ATPase data for D382Y 2-hep and 25-hep HMM. a, c show representative data from one preparation each. Each of panels b and d are combined data from two independent preparations of HMMs with three experimental replicates for each preparation. Each point is an average with the error bar representing the SEM. Source data are provided as a Source Data file

Fig. 6

Effect of double switch mutation D382R/R719D on HMM ATPase kinetics. In all cases, black solid line and filled circles are 2-hep HMM data; Red solid line and empty squares are 25-hep HMM data. a Mant-nucleotide single turnover kinetics for D382R/R719D 2-hep and 25-hep HMM. b Actin-activated ATPase data for D382R/R719D 2-hep and 25-hep HMM. a Shows representative data from one preparation each. Panel b is combined from two independent preparations of HMMs with three experimental replicates for each preparation. Each point is an average with the error bar representing the SEM. c Model of the front-side view of the human β-cardiac myosin IHM (MS03 homology model, downloadable at https://spudlab.stanford.edu/homology-models/), with the two S1 heads and the light chains shown as lines, and the S2 tail region represented by sticks. The Arg719 residue (blue) is part of the free head converter (purple) and the Asp382 (red) residue is part of the blocked head PHHIS. The heavy blue curved line indicates the generally positive surface of the free head converter that interacts with the generally negative blocked head PHHIS surface, marked by the heavy red curved line. d The image in c rotated 90° counterclockwise about the vertical axis defining the binding interface. The free head converter-binding interface, shown in vacuum-electrostatics mode in PyMOL, is generally positively charged. e The image in c rotated 90° clockwise about the vertical axis defining the binding interface. The blocked head PHHIS-binding interface, shown in vacuum-electrostatics mode in PyMOL, is generally negatively charged. Source data are provided as a Source Data file

Fig. 7

Effect of I457T mutation on HMM ATPase kinetics and in vitro motility velocity. The I457T mutation (green spheres) shown on the MS03 homology model of the IHM. The mutation is located on the transducer region (pink ribbons) and is not close to the proposed interfaces within the IHM. b Close up of transducer region from panel a. c Mant-nucleotide release kinetics for I457T 2-hep and 25-hep HMM. d Actin-activated ATPase activity of I457T 2-hep and 25-hep HMM. e Bar plot showing the actin-activated ATPase activity of WT and I457T 2-hep HMM. f Bar plot showing the actin gliding velocity as measured by the in vitro motility assay of WT and I457T 2-hep HMM. In panels e andand f individual data points are denoted by closed circles. c Shows representative data from one preparation of each protein. Panel d is combined data from two independent protein preparations with three experimental replicates for each preparation. For panels e and f, each point is an average of two independent experiments (each from a different myosin preparation). Error bars represent the SEM. Source data are provided as a Source Data file