Conformational signatures of native ligand and pharmacochaperone binding in rhodopsin

Misfolding often underlies rhodopsin-linked retinitis pigmentosa, and small-molecule pharmacochaperones represent a promising therapeutic strategy. However, the mechanisms by which these compounds stabilize rhodopsin remain incompletely understood. We combine amide- and histidine-specific hydrogen-deuterium exchange (HDX) mass spectrometry, protein structure network analysis, molecular docking, and functional spectroscopy to define ligand-induced conformational signatures in rhodopsin elicited by quercetin, myricetin, and chromenone, and compare them with those of native chromophore. Binding of 11-cis-retinal to opsin produces a benchmark orthosteric signature characterized by backbone protection across TM4-TM7, suppression of EX1-like exchange at TM1 and TM4 N-termini, and reframing of residue interaction networks. All three non-retinoid ligands induce partially overlapping HDX footprints consistent with interaction within the orthosteric site, but with ligand-specific differences. Quercetin most closely resembles the 11-cis-retinal pattern, whereas myricetin and chromenone show reduced and redistributed protection. These findings define structural determinants of ligand-induced opsin stabilization and provide a framework for optimizing small-molecule opsin stabilizers.