A FRET assay to quantitate levels of the human β-cardiac myosin interacting heads motif based on its near-atomic resolution structure

Abstract

In cardiac muscle, many myosin molecules are in a resting or "OFF" state with their catalytic heads in a folded structure known as the interacting heads motif (IHM). Many mutations in the human β-cardiac myosin gene that cause hypertrophic cardiomyopathy (HCM) are thought to destabilize (decrease the population of) the IHM state. The effects of pathogenic mutations on the IHM structural state are often studied using indirect assays, including a single-ATP turnover assay that detects the super-relaxed (SRX) biochemical state of myosin functionally. Here we develop and use a fluorescence resonance energy transfer (FRET) based sensor for direct quantification of the IHM state in solution. The FRET sensor was able to quantify destabilization of the IHM state in solution, induced by (a) increasing salt concentration, (b) altering proximal S2 tail length, or (c) introducing the HCM mutation P710R, as well as stabilization of the IHM state by introducing a dilated cardiomyopathy-causing mutation (E525K). Our FRET sensor conclusively showed that these perturbations indeed alter the structural IHM state. These results establish that the structural IHM state is one of the structural correlates of the biochemical SRX state in solution.

Figure 1.. Distances monitored by the AF488 RLC-Cy3ATP or FRET sensor.

(A) Schematic diagram showing the open state and the IHM state of β-cardiac HMM. The AF488 donor fluorophore is at the C-terminus of the human ventricular RLC, labeled “D”, and the acceptor Cy3-ATP location is labeled “A”. The distances monitored by the FRET sensor are shown with dashed lines. (B) Near-atomic resolution structure of the IHM conformation of human β-cardiac 15-hep HMM (PDB ID: 8ACT) showing the two distances probed by the FRET sensor. (C) Structure of IHM state showing the distances sampled by linkers of Donor and Acceptor dyes. The range of distances sampled by the donor AF488, which has a ~15 Å-long C5 linker, are shown in green circles. The range of distances sampled by the acceptor Cy3-ATP, which has a ~30 Å-long linker, are shown in red circles.

Figure 2.. Steady state measurements with the RLC AF488-Cy3 ATP FRET sensor reports on structural changes in human β-cardiac 15-hep HMM due to changes in salt (KOAc) concentration.

(A) Fluorescence emission spectra of 15-hep myosin with FRET sensor in the presence of 0 mM salt (blue curve) and 150 mM salt (red curve). (B) FRET efficiency calculated from the fluorescence emission spectra obtained at different salt concentrations is plotted along with the % SRX state obtained from the single-turnover assay.

Figure 3.. An 8-hep human β-cardiac HMM construct that cannot form the IHM state shows lower time-resolved FRET efficiency that is independent of salt concentration.

(A) Schematic diagram showing difference between the 15-hep and 8-hep HMM constructs. The 15-hep HMM construct has been shown to form the folded back state while the 8-hep HMM construct cannot form the folded back state. (B) FRET efficiencies measured by time-resolved fluorescence for 8- and 15-hep β-cardiac HMM at 0 mM and 150 mM salt (KOAc) concentrations. The data for 15-hep HMM was collected from 3 different protein preparations measured across 4 different experiments and the data for 8-hep HMM was obtained from 2 different protein preparations across two different experiments.

Figure 4.. FRET sensor demonstrates altered SRX and FRET efficiencies due to cardiomyopathy-causing mutations in β-cardiac myosin.

(A) Plot showing % SRX quantified by the SRX assay at different salt concentrations. Data shown is for 15-hep HMM constructs of WT human β-cardiac HMM (blue), HCM mutant P710R (red), and DCM mutant E525K (green). (B, C) Comparison of FRET efficiency measured by time-resolved fluorescence for WT, P710R and E525K 15hep β-cardiac HMM at 0 mM (B) and 150 mM (C) salt concentrations.