Cooperative roles of W882 and W996 in state-dependent dantrolene stabilization within the RyR1 P1 domain

Dantrolene is a clinically approved therapeutic used to suppress abnormal calcium release associated with malignant hyperthermia linked to RyR1 mutations. Cryo-electron microscopy studies have revealed a putative dantrolene-binding cavity within the RyR1 P1 domain and highlighted several residues associated with ligand recognition. However, the dynamic molecular interactions governing ligand stabilization within this region remain incompletely understood. MM/GBSA calculations, conformational entropy analyses, and potential of mean force (PMF) profiling were employed to investigate the local binding dynamics of dantrolene within isolated RyR1 P1-domain complexes. Comparative analyses of the P1 domain from open- and closed-state conformations of RyR1 revealed that the closed-state complex provides a more stable binding environment characterized by lower backbone fluctuations, enhanced ligand-protein contacts, stabilization of a distal loop region (residues 1006-1028), and higher ligand dissociation barriers. Per-residue energetic decomposition and interaction analyses identified W882 and W996 as major contributors to ligand stabilization through cooperative hydrogen bonding and π-mediated interactions. Alanine substitution of these residues increased ligand conformational flexibility and substantially reduced the energetic barriers associated with ligand dissociation, whereas R1000 exhibited comparatively smaller contributions to overall binding stability. Overall, these computational findings are consistent with previous experimental observations and provide atomistic insight into the interaction networks and structural determinants underlying state-dependent stabilization of dantrolene in a cryo-EM-derived RyR1 P1-domain binding model.