Structural identification of the RY12 domain of RyR1 as an ADP sensor and the target of the malignant hyperthermia therapeutic dantrolene

Malignant hyperthermia (MH) is a pharmacogenetic condition triggered by volatile anesthetics, which activate MH susceptible mutants of ryanodine receptor 1 (RyR1). The precise binding sites and mechanisms of the therapeutic dantrolene and MH triggers have remained unclear. Here, we present structures of RyR1 bound to dantrolene and the MH trigger 4-chloro-m-cresol (4CmC), revealing the dantrolene and 4CmC binding sites in atomic detail. Dantrolene binds stacked with ATP or ADP in the RY12 domain at the corner of the receptor, inducing a conformational change. Functional analyses confirm ATP or ADP is required for dantrolene inhibition, and a single point mutation disrupting this peripheral ATP site abolishes this ATP/ADP-dependent inhibition. Strikingly, in the absence of dantrolene, this site selectively binds two ADP molecules even in the presence of excess ATP, indicating a role in ATP/ADP ratio sensing. Additionally, we identified four 4CmC binding sites, revealing one primary binding site in an amphipathic cavity in the core solenoid, and three auxiliary binding sites in the EF-hand, transmembrane, and RY12 domains. These findings offer insights into the binding modes and mechanisms of MH-linked molecules, and reveal the RY12 domain's regulatory role as a promiscuous sensor for adenine nucleotides and small ligands.