Aromatic amino acids in the orthosteric region regulate cannabinoid receptor 1 conformation transitions

Cannabinoid receptor 1 (CB1) is a class A G protein-coupled receptor that exhibits constitutive activity by spontaneously sampling both active and inactive states in the absence of ligand binding. Structural studies have identified a domain of aromatic residues, F2003.36, W3566.48, and W2795.43, that modulates receptor function and ligand engagement; however, their mechanistic roles remain unclear. Here, we use equilibrium and targeted molecular dynamics simulations to investigate how alanine substitutions at these positions alter CB1 dynamics. Our results show that mutations, especially the F200A+W356A combination, enhanced conformational flexibility and shifted the receptor toward inactive-like states. W3566.48 emerged as a structural pivot essential for active-state stability, F2003.36 acted as a late-stage kinetic barrier to inactivation, and W2795.43 modulated orthosteric pocket packing and flexibility, amplifying destabilization when combined with F2003.36 or W3566.48 mutations. Targeted simulations showed that mutation of this aromatic domain lowers energetic barriers and rewires the inactivation pathway. These findings define the structural logic of CB1 toggle-switch control and provide a mechanistic framework for designing modulators that tune basal activity in CB1.