Heart failure and other muscle-related chronic diseases, including muscular dystrophy, are often associated with high levels of circulating catecholamines and with chronic activation of adrenergic signaling pathways in many tissues. The resulting chronic adrenergic stimulation-induced PKA phosphorylation and oxidation of ryanodine receptors (RyRs)/calcium release channels at Ser2843 in the skeletal muscle RyR1 (Ser2809 in the cardiac RyR2). These protein modifications typically cause intracellular calcium leak that leads to muscle pathology including cardiac arrhythmias and skeletal muscle weakness.
To better understand the molecular mechanism of calcium release, RyR-related calcium leak, and the pathology caused by RyRs mutations in skeletal and cardiac muscles, complete models of the molecules at different states, including pathological states, are required.
Single particle cryo-EM reconstruction is a powerful tool to solve large macromolecular complexes. About 80% of the skeletal muscle RyR1 ordered structure was recently solved to ~4 Å resolution but did not allow unambiguous sequence assignment or side-chains orientation in most of the molecule map. Furthermore, 818 residues, at critical domains associated with disease-causing mutations were completely unresolved leaving us with a partial model that is missing valuable data. Completing the RyR1 model was and still is an important scientific goal not only to better understand cardiac and skeletal muscle diseases but also to help understand and better design drugs that can prevent fatal calcium leak, including rycals and dantrolene that specifically target RyRs.
The overall objectives were first to complete the model of the RyR1 and to understand the conformational changes that follow RyR1 PKA phosphorylation and pathological oxidation and that may lead to calcium leak and skeletal muscle damage and weakness.
Our laboratory aim to utilize, implement and develop advanced techniques to promote the understanding of mechanisms EC-coupling at the molecular and atomic level resolution and explore novel methods for understanding calcium release regulation and pathology. The study has allowed deeper understandings of how the RyR/calcium release channels are regulated, and how drugs that fix the calcium leak bind to the channels. This has important implications for developing novel therapies for heart and skeletal muscle diseases. To my knowledge, this is the first time cryo-EM was used to unambiguously draw small molecules (including drugs) binding sites. In this study we are very close to demonstrate that cryo-EM can serve as an alternative tool to crystallography for structure-based drug design, particularly targeting large protein complexes where crystallography is notoriously complicated. The strong biomedical and the translational nature of the study will make it feasible for me to rationally develop treatments targeting RyRs. As we achieved complete models of RyRs, this new information enables the determination of domain-domain interfaces and the location and structural and the functional effects of disease-causing RyRs mutations.
