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. 2017 Sep 11;7(1):11167.
doi: 10.1038/s41598-017-11391-x.

Flavonoid allosteric modulation of mutated visual rhodopsin associated with retinitis pigmentosa

María Guadalupe Herrera-Hernández  1   2 , Eva Ramon  1 , Cecylia S Lupala  3 , Mercè Tena-Campos  1 , Juan J Pérez  3 , Pere Garriga  4
Affiliations

Flavonoid allosteric modulation of mutated visual rhodopsin associated with retinitis pigmentosa

María Guadalupe Herrera-Hernández et al. Sci Rep. .

Abstract

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.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Absorption spectra of the first elution of immunopurified WT and G90V mutant regenerated with 11-cis-retinal (11CR) and 9-cis-retinal (9CR). After the immunopurification the receptors were characterized by UV-vis spectroscopy. Solid line represents the receptor without treatment, dotted line represents the receptor with 1 µM Q treatment. Samples were eluted with 100 µM of 9-mer peptide in PBS pH 7.4 and 0.05% DM. Spectra were recorded at 20 °C. (A) WT 11CR. (B) WT 9CR. (C) G90V 11CR. (D) G90V 9CR.
Figure 2
Figure 2
Q is identified in the third elution of immunopurified G90V 9CR mutant sample. During the experiments, a second protein elution was performed to recover as much protein as possible. This second elution was done in PBS pH 6 and used in the regeneration experiments. When it was possible, up to a third elution was carried out in the samples that showed a higher yield, as was the case in the treatments with Q in the receptors regenerated with 9CR. Solid line represents the third elution of G90V 9CR without Q treatment. Dashed line represents the third elution of G90V 9CR-Q with treatment of 1 µM Q. Dotted line represents the difference spectra of G90V 9CR-Q minus G90V 9CR. Samples were eluted with 100 µM of 9-mer peptide in PBS pH 7.4 and 0.05% DM.
Figure 3
Figure 3
UV-vis characterization of WT 11CR, G90V 11CR with and without Q treatment and G90V 9CR-Q. Dark state (solid line), photobleaching (dotted line) and acidification (dashed line). Samples in PBS pH 7.4 and 0.05% DM. Spectra were recorded at 20 °C. (A) WT 11CR. (B) G90VT 11CR. (C) G90V 11CR-Q. (D) G90V 9CR-Q.
Figure 4
Figure 4
Chemical stability of the immunopurified WT and G90V mutant. Immunopurified protein samples in PBS pH 7.4 and 0.05% DM, after Q treatment, were incubated with 50 mM hydroxylamine, pH 7 and the decrease of Amax was recorded over time at 20 °C. (A) WT 11CR with (⚪) and W/O 1 µM Q (●). (B) WT 9CR with (⚪) and W/O 1 µM Q (●). (C) G90V 11CR with (⚪) and W/O 1 µM Q (●). (D) G90V 9CR with (⚪) and W/O 1 µM Q (●). Mean value and standard error (SE) obtained in independent purifications (n = 3).
Figure 5
Figure 5
Initial rates of chromophore regeneration of the immunopurified WT and G90V mutant with 1 µM Q treatment. (A) 2.5 molar excess fold of 11CR or 9CR (0.925 µM) (dotted line) with regard to Rho concentration (0.37 µM) (solid line) was added to the immunopurified WT and the G90V mutant, in the different buffers containing 0.05% DM + 1 µM Q, samples were illuminated with light of >495 nm to avoid photobleaching of the free retinal (dashed line), and successive spectra were recorded at 20 °C in the dark until no further increase in Amax was detected (inset). (B) The normalized absorbance values at Amax were plotted as a function of time, the data were fit to a single exponential function and the initial rates derived. (C) Initial rates of chromophore regeneration of WT 11CR and WT 9CR. (D) Initial rates of chromophore regeneration of G90V 11CR and G90V 9CR. Mean value and standard error (SE) obtained in independent purifications (n = 3).
Figure 6
Figure 6
Meta II decay of the immunopurified WT and G90V mutant regenerated with 11CR or 9CR with or W/O 1 µM Q treatment. Samples were incubated at 20 °C, and after a steady base line was obtained, they were photobleached and the Trp fluorescence was monitored over time. The fluorescence increase was fit to a single exponential function and the t1/2 calculated. Mean value and standard error (SE) obtained from independent purifications (n = 3).
Figure 7
Figure 7
Gt activation by WT and G90V mutant regenerated with 11CR or 9CR with or W/O 1 µM Q treatment. Gt activity was measured by means of a radionucleotide filter-binding assay in Gt buffer. The reaction was initiated by the addition of the WT or mutant, and samples were filtrated at different times in the dark and after illumination. (A) WT 11CR with (⚪) and W/O 1 µM Q (●). (B) WT 9CR with (⚪) and W/O 1 µM Q (●). (C) G90V 11CR with (⚪) and W/O 1 µM Q (●). (D) G90V 9CR with (⚪) and W/O 1 µM Q (●). Mean value and standard error (SE) obtained in independent purifications (n = 3).
Figure 8
Figure 8
Pictorial view of the putative binding sites identified using SiteMap in: (A) opsin (B), rhodopsin (11CR), (C) isorhodopsin (9CR), and (D) Meta II.
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