Flavonoids enhance rod opsin stability, folding, and self-association by directly binding to ligand-free opsin and modulating its conformation

Abstract

Rhodopsin (Rho) is a visual G protein–coupled receptor expressed in the rod photoreceptors of the eye, where it mediates transmission of a light signal into a cell and converts this signal into a nerve impulse. More than 100 mutations in Rho are linked to various ocular impairments, including retinitis pigmentosa (RP). Accordingly, much effort has been directed toward developing ligands that target Rho and improve its folding and stability. Natural compounds may provide another viable approach to such drug discovery efforts. The dietary polyphenol compounds, ubiquitously present in fruits and vegetables, have beneficial effects in several eye diseases. However, the underlying mechanism of their activity is not fully understood. In this study, we used a combination of computational methods, biochemical and biophysical approaches, including bioluminescence resonance energy transfer, and mammalian cell expression systems to clarify the effects of four common bioactive flavonoids (quercetin, myricetin, and their mono-glycosylated forms quercetin-3-rhamnoside and myricetrin) on rod opsin stability, function, and membrane organization. We observed that by directly interacting with ligand-free opsin, flavonoids modulate its conformation, thereby causing faster entry of the retinal chromophore into its binding pocket. Moreover, flavonoids significantly increased opsin stability, most likely by introducing structural rigidity and promoting receptor self-association within the biological membranes. Of note, the binding of flavonoids to an RP-linked P23H opsin variant partially restored its normal cellular trafficking. Together, our results suggest that flavonoids could be utilized as lead compounds in the development of effective nonretinoid therapeutics for managing RP-related retinopathies.

The Retinoid and Non-Retinoid Ligands of the Rod Visual G Protein-Coupled Receptor

“…As suggested, anthocyanins accommodate the cytoplasmic allosteric binding site in rod opsin [28,29], while flavonoids bind rather to the extracellular region of this receptor [17,24]. As noted, flavonoids also improve stability, folding and membrane targeting of RP-linked specific rod opsin mutants [20]. The beneficial effects of polyphenolic compounds enhancing night vision have also been reported [30].…”

“…Indeed, retinal-free opsin is highly unstable and prone to unspecific aggregation and degradation. The temperature of melting of opsin (Tm = 54.4 • C) is significantly lower than that of retinal-bound rhodopsin (Tm = 71.9 • C), indicating that the interaction network between retinal and the specific residues in the binding pocket is critical for the stability of this visual receptor [20,41]. adRP mutations can perturb the chromophore-binding site leading to various degrees of impairments in the binding of retinal, which then can result in improper folding and inefficient membrane integration of rhodopsin.…”

“…Despite the natural ligand 11-cis-retinal, this hydrophobic cavity also fits other retinoid analogs [16][17][18]. Moreover, the non-retinoid ligands consisting of aromatic rings can accommodate the retinal-binding pocket and interact with the surrounding residues via van-der-Waals and π-π stacking interactions [19,20]. The interaction profiles of rod opsin with YC-001 (Table 1), a recently identified small molecule chaperone that improves membrane targeting of retinitis pigmentosa (RP)-linked P23H opsin mutant, and flavonoids (Table 1) emerged from our in silico analyses are shown in Figure 3A and B, respectively.…”

“…However, the interaction pattern and the types of interactions that occur in the orthosteric site are different for these compounds. The molecular docking of YC-001 or quercetin to bovine rod opsin (PDB ID: 3CAP) was performed using VINA/VegaZZ 3.1.0.21 software as described in Ortega et al [20] with 30 iterations for each compound. The resulting complexes were visualized with the Biovia Discovery Studio Visualizer 17.2.0 software and 2D diagrams were obtained by selecting the main atoms that interact with the ligand.…”

“…Recent studies probing the interaction between flavonoids and rod opsin revealed that specific flavonoids such as quercetin and myricetin (Table 1) can bind to ligand-free opsin and act as its structural modulators [20]. Computational analysis of the available crystal structures for the dark state, 11-cis-retinal-bound rhodopsin (PDB ID: 1GZM) [49], photoactivated rhodopsin (PDB ID: 4A4M) [50] and ligand-free opsin (PDB ID: 3CAP) [60] detected potential binding modes for flavonoids only in the ligand-free opsin structure.…”