Flavonoid allosteric modulation of mutated visual rhodopsin associated with retinitis pigmentosa

Dietary flavonoids exhibit many biologically-relevant functions and can potentially have beneficial effects in the treatment of pathological conditions. In spite of its well known antioxidant properties, scarce structural information is available on the interaction of flavonoids with membrane receptors. Advances in the structural biology of a specific class of membrane receptors, the G protein-coupled receptors, have significantly increased our understanding of drug action and paved the way for developing improved therapeutic approaches. We have analyzed the effect of the flavonoid quercetin on the conformation, stability and function of the G protein-coupled receptor rhodopsin, and the G90V mutant associated with the retinal degenerative disease retinitis pigmentosa. By using a combination of experimental and computational methods, we suggest that quercetin can act as an allosteric modulator of opsin regenerated with 9-cis-retinal and more importantly, that this binding has a positive effect on the stability and conformational properties of the G90V mutant associated with retinitis pigmentosa. These results open new possibilities to use quercetin and other flavonoids, in combination with specific retinoids like 9-cis-retinal, for the treatment of retinal degeneration associated with retinitis pigmentosa. Moreover, the use of flavonoids as allosteric modulators may also be applicable to other members of the G protein-coupled receptors superfamily.

receptors were normalized to equal amounts and mixed with protein loading buffer 4x (Tris 0.0625 M, 2% SDS, 10% glycerol, 0.4M DTT and 0.1% bromophenol blue). The prepared samples and 6 µl of the protein molecular weight marker were loaded into the corresponding wells and the gel was run at 100V for 2 h. The gel was stained overnight using Generon quick Coomassie stain. The stained gels were distained with water until the protein bands could be visualized.
After separating the proteins using SDS-PAGE, the proteins from the gel were transferred onto a nitrocellulose membrane using the Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad). For opsins immunodetection, Rho-1D4 (1:10000 in TBS (8g NaCl, 1.121g Tris, 0.4 ml HCl in 1 L ddH 2 O, pH 8.0) buffer) primary antibody and goat antimouse secondary IgG antibody (1:5000 in TBS buffer) were used.

Quercetin (Q) identification by HPLC-ESI-MS/MS
For Q extraction, 200 µl of 80% ethanol acidified with 0.1% formic acid was added to the sample. The mixture was vortexed for 1min and then sonicated for 5 min on ice. Full-scan data were acquired by scanning from m/z 100 to 800 in profile mode using a cycle time of 1s.

Molecular modeling studies
Docking studies of Q using Glide (Schrödinger) were carried out on the crystallographic structure of rhodopsin (Rho) bound to 11-cis-retinal and to Rho bound to 9-cis-retinal retrieved from the Protein Data Bank. The structures were prepared for docking studies (optimization of hydrogen bonds, protonation states, etc.) using the protein preparation wizard tool of the Schrodinger software. The structure of Q used in the present study 3 was downloaded from the PubChem website and prepared for docking studies using the LigPrep tool, also from Schrodinger.

Western blot
The WT (A) and G90V (B) mutant were characterized by Western blot (Fig. S1)  In the case of the samples obtained from the third elution of G90V 9CR and G90V 9CR-Q at pH 6, they also were analyzed by Western blot (Fig. S2). The electrophoretic pattern of the G90V 9CR-Q mutant showed low intensity in the band corresponding to truncated protein, indicating that Q presence could help in the folding process of this mutant.

Figure S2. Western blot of immunopurified G90V 9CR and G90V 9CR-Q. The
presence of Q decreased the amount corresponding to truncated protein (~27 kDa).

Q identification by HPLC-ESI-MS/MS
The G90V 9CR-Q mutant was analyzed by HPLC-ESI-MS/MS. From the G90V CR-Q mutant sample, Q was isolated as described under Methods. For the mass spectrometry study, a Q standard was run at a concentration of 1 ppm. A product ion scan of both the sample and the standard was done (Fig. S3). In this experiment the ions characteristics 5 of Q could be found in the sample, and the concentration detected was 0.0035 ppm (0.0115 µM).

Molecular modeling studies
The superimposition of the structures of Rho bound to 9-cis-retinal (Fig. S4 A, in green) and 11-cis-retinal (Fig. S4 A, in blue) show that the ligands do not change significantly the transmembrane region, but the two structures show slight differences in the extracellular loop 2 (ECL2) region (Fig. S4 A). The results of the docking study  revealed that Q binds differentially to both structures. Specifically, it binds to a site involving the ECL2 in isoRho (Fig. S4 B) that is not found on Rho. Figure S4. Docking of Q on Rho. A, Superimposition of the structures of Rho bound to 9-cis-retinal (in green) and 11-cis-retinal (in blue). B, structure of Q (orange) bound to the 9-cis-Rho (in green).