In cardiac muscle, myosin molecules exist in multiple structural states as they transit through their ATPase cycle, including an off-cycle resting or OFF-state with their catalytic heads in a folded structure known as the interacting-heads motif (IHM). The blocked head configuration (BHC) of the IHM is unusual because its light chain binding region is held in an exaggerated prestroke angle stabilized by interactions with its own S2 tail. An additional partial OFF-state, where the second head of the IHM is not folded back onto the blocked head, has been proposed, which still has the blocked head interacting with S2. Many mutations in the human β-cardiac myosin gene that cause hypertrophic cardiomyopathy are thought to destabilize (decrease the population of) the OFF-states. The effects of pathogenic mutations on the folded back structural states are often studied using indirect assays, including a single-ATP turnover assay that detects the biochemical state of myosin functionally. Here, we use a fluorescence resonance energy transfer (FRET) based sensor for direct quantification in solution of the myosin BHC state. Using the FRET sensor, we provide evidence that the myosin tail acts as an activator of the recovery stroke transition after ATP binding to poststroke state apomyosin and that BHC formation is rapid after ATP binding and depends on the formation of the prestroke state. We propose that the positively charged loop 2 of the prestroke state head interacts with the Ring 2 cluster of negatively charged residues on the S2 tail to form a preBHC state that facilitates BHC state formation.
Goluguri et al. (Sun,) studied this question.