Mapping Putative Allosteric Sites in Rhodopsin and Elucidating the Structural Basis of Retigabine Modulation

Rhodopsin (Rho), a prototypical G protein–coupled receptor (GPCR) is the sensory receptor essential for dim-light vision. Its misfolding is a major cause of retinitis pigmentosa (RP), a group of inherited retinal degenerative diseases. Pharmacological chaperones that stabilize misfolded Rho represent a promising therapeutic strategy, yet systematic approaches to identify such modulators remain limited. In this study, we present the results of a computational study aimed to identify and characterize potential allosteric binding sites in Rho. For this purpose, extensive molecular dynamics (MD) simulations were performed to capture the conformational plasticity of Rho, followed by clustering analysis to obtain representative receptor conformations. Fragment-based mapping (FTMap) of these structures revealed nine transient pockets (P1–P9) across the receptor surface. The characterized sites were used to elucidate the binding mode of the small-molecule modulator retigabine. Ensemble docking and MD simulations permitted to identify a combined P1+P2 site as a prospective binding location. To validate the prediction, experimental studies were conducted using recombinant wild-type (WT) Rho and the Q36 1.31 A Rho mutant. Spectroscopic analyses showed that retigabine does not significantly alter the photobleaching and acidification behaviour of either protein. However, fluorescence-based assays revealed that retigabine accelerates Meta II decay and retinal release in WT Rho but not in the Q36 1.31 A Rho mutant, supporting the involvement of Gln 36 in ligand binding. Moreover, retigabine treatment attenuated the increase of intrinsic fluorescence observed in illuminated WT Rho, whereas no significant effect was detected in the Q36 1.31 A mutant, consistent with the proposed role of Gln 36 as a key residue within the prospective P1+P2 retigabine-binding site. The results of the present work establish a framework for the rational discovery of novel allosteric modulators targeting rhodopsin, with potential therapeutic implications for retinitis pigmentosa.