Molecular basis underlying specific interaction of mammalian melanopsins with an antagonist AA92593

Melanopsin functions in intrinsically photosensitive retinal ganglion cells of mammals to regulate circadian clock and pupil constriction. The opsinamide AA92593 has been reported to specifically inhibit mouse and human melanopsin functions as a competitive antagonist against retinal; however, the molecular mechanisms underlying its specificity have not been resolved. In this study, we attempted to identify amino acid residues responsible for the specific interaction of AA92593 with mammalian melanopsins. Our cell-based assays confirmed that AA92593 effectively inhibited the light-induced cellular responses of mammalian melanopsins, but not those of non-mammalian vertebrate and invertebrate melanopsins. These results suggest that amino acid residues specifically conserved among mammalian melanopsins are important for the antagonistic effect of AA92593, and we noticed Phe-94, Ser-188, and Ser-269 as candidate residues. Substitutions of these residues reduced the antagonistic effect of AA92593. We conducted docking and molecular dynamics simulations based on the AlphaFold-predicted melanopsin structure. The simulations indicated that Phe-94, Ser-188, and Ser-269 are located at the AA92593-binding site, and additionally identified Trp-189 and Leu-207 interacting with the antagonist. Substitutions of Trp-189 and Leu-207 affected the antagonistic effect of AA92593. Furthermore, substitutions of these amino acid residues converted AA92593-insensitive melanopsins susceptible to the antagonist. Based on experiments and molecular simulations, five amino acid residues, at positions 94, 188, 189, 207, and 269, were found to be responsible for the specific interaction with AA92593 in mammalian melanopsins.