Dissecting the structural origin of relaxation in skeletal muscle

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How muscle contracts has been a long-standing question. Despite major advances in this area, how muscle relaxes is still not fully understood. Contraction occurs by the sliding of myosin-containing thick past actin-containing thin filaments, powered by myosin heads, motors that produce sliding force, fueled by ATP. Relaxation occurs when thin filaments are switched off so heads cannot bind to produce force, leaving the idling heads to organize themselves helically in the thick filament. What is currently known about the role of thick filaments in relaxation? On the structural side, low-resolution models of cardiac (mouse, human) and skeletal (tarantula) thick filaments have been achieved, but their atomic structure remains unsolved. On the energetics side, the energy consumption of relaxed skeletal muscle revealed a surprising phenomenon, so-called super- relaxation (SRX) that greatly reduces ATP consumption. A widely accepted view associates this ubiquitous and fundamental energy-saving state with the unique way myosin’s two heads fold together in the relaxed tarantula filament—the so-called interacting-heads motif (IHM), found across the animal kingdom, which structurally inhibits both heads, switching off their activity. Regardless of its appeal, this SRX=IHM hypothesis has not been proved, and recent ATP turnover results suggest, instead, association of SRX with a specific myosin head conformation. Elucidating this puzzle is crucial to understanding how muscle relaxes, how it malfunctions in disease and how therapeutic drug treatments work. The solution requires determination by cryo-EM of the atomic structures of the thick filament and myosin molecules from muscle. Here, we propose to determine the structures of skeletal myosin molecules and filaments, far less studied than cardiac. This will allow us to dissect how key IHM interactions constrain activity of the two heads, shutting them off, thus conserving ATP in relaxation. We will use single particle EM and cryo-EM to define the structural basis of the SRX state at near-atomic level in thick filaments and myosin molecules from rabbit skeletal muscle. By comparing with tarantula, which shows tenfold- greater energy-saving (hyper-relaxation, HRX), we will gain deeper insight into the mechanism of ATPase inhibition. And we will use EM and X-ray diffraction to investigate how therapeutic drugs alter the IHM. Aim 1 will define the structural basis of SRX in skeletal thick filaments by revealing their near-atomic cryo-EM structures. Aim 2 will define the structural basis of SRX in skeletal myosin heads and heavy meromyosin molecules by assessing: (A) if SRX results from a specific head conformation, and (B) if the IHM correlates with the SRX state. Aim 3 will reveal the structural impact of drugs on skeletal thick filaments and myosin molecules. Despite the vital role of SRX in skeletal muscle relaxation, its structural basis and relation to the IHM and to other thick filament proteins (MyBP-C, titin) remains unknown. Our studies will reveal the IHM structure in skeletal thick filaments and myosin molecules, clarify its association with the SRX state and with MyBP-C and titin, and provide critical insights into the molecular basis of relaxation and the influence of therapeutic drugs.