Specificity of the chromophore-binding site in human cone opsins

Abstract

The variable composition of the chromophore-binding pocket in visual receptors is essential for vision. The visual phototransduction starts with the cis-trans isomerization of the retinal chromophore upon absorption of photons. Despite sharing the common 11-cis-retinal chromophore, rod and cone photoreceptors possess distinct photochemical properties. Thus, a detailed molecular characterization of the chromophore-binding pocket of these receptors is critical to understanding the differences in the photochemistry of vision between rods and cones. Unlike for rhodopsin (Rh), the crystal structures of cone opsins remain to be determined. To obtain insights into the specific chromophore-protein interactions that govern spectral tuning in human visual pigments, here we harnessed the unique binding properties of 11-cis-6-membered-ring-retinal (11-cis-6mr-retinal) with human blue, green, and red cone opsins. To unravel the specificity of the chromophore-binding pocket of cone opsins, we applied 11-cis-6mr-retinal analog-binding analyses to human blue, green, and red cone opsins. Our results revealed that among the three cone opsins, only blue cone opsin can accommodate the 11-cis-6mr-retinal in its chromophore-binding pocket, resulting in the formation of a synthetic blue pigment (B6mr) that absorbs visible light. A combination of primary sequence alignment, molecular modeling, and mutagenesis experiments revealed the specific amino acid residue 6.48 (Tyr-262 in blue cone opsins and Trp-281 in green and red cone opsins) as a selectivity filter in human cone opsins. Altogether, the results of our study uncover the molecular basis underlying the binding selectivity of 11-cis-6mr-retinal to the cone opsins.

Keywords: 11-cis-6mr-retinal; G protein-coupled receptor (GPCR); membrane protein; photoreceptor; protein structure; retinal pigment; retinoid; rhodopsin; vision.

Figure 1.

Binding of 11-cis-retinal chromophore and its locked 11-cis-6mr-retinal analog to human blue, green, and red cone opsins and bovine rod opsin. a, UV-visible absorption spectra of reconstituted blue cone opsin, Rh, and green and red cone opsins with the native chromophore 11-cis-retinal (blue, black, green, and red dashed lines, respectively) and UV-visible absorption spectra of blue cone opsin, Rh, and green and red cone opsins bound with 11-cis-6mr-retinal analog (blue, black, green, and red solid lines, respectively). b, partial amino acid sequence alignment of human blue, green, and red cone opsins and bovine Rh (black). Conserved residues in the chromophore-binding pocket are shown. The amino acid numbers are based on bovine Rh sequence.

Figure 2.

Comparison of the retinal-binding pockets in WT and mutated cone opsins. a, WT blue cone opsin (dark blue) and the Y262W mutant (light blue) (top panel), WT green cone opsin (dark green) and the W281Y mutant (light green) (bottom panel), and Tyr residues (yellow sticks) and Trp residues (orange sticks) are shown. 11-cis-6mr-Retinal was modeled into the binding pocket of WT and mutated cone opsins and is shown with light gray sticks in WT blue and green cone opsins and with dark gray sticks in mutated blue and green cone opsins. b, magnified view of 11-cis-6mr-retinal conformations in the binding pocket of WT and mutated blue and green cone opsins (top and bottom panels, respectively) relative to the residues of interest, Tyr and Trp, are shown. The same 11-cis-6mr-retinal orientations as in the merged panels of a and after 60° rotation along the y axis are shown.

Figure 3.

Binding of 11-cis-retinal chromophore and its locked 11-cis-6mr-retinal analog to green cone opsin and the W281Y mutant. a, SDS-polyacrylamide gel of regenerated and purified pigments. Pigment regeneration was performed in HEK-293T cell pellets, which then were purified by 1D4 immunoaffinity chromatography. Proteins were separated on an SDS-polyacrylamide gel (2 μg was loaded in each lane), and the gel was stained with Coomassie Blue. Proteins were deglycosylated with peptide:N-glycosidase F before loading onto the gel. b, UV-visible absorption spectra of reconstituted W281Y green cone opsin regenerated with 11-cis-retinal (11-cis) (dark green), spectra of W281Y green cone opsin regenerated with 11-cis-6mr-retinal (6mr) (light green), and UV-visible absorption spectra of reconstituted WT green cone opsin regenerated with 11-cis-retinal (WT (11-cis)) (dark green dashed line) and with 11-cis-6mr-retinal (WT (6mr)) (light green dashed line) are shown.

Figure 4.

The crystal structure of Rh6mr (PDB code 5TE5 (21)) is shown on the left. The magnified region of Rh6mr displaying 11-cis-6mr-retinal in the chromophore-binding pocket is shown on the right. 11-cis-6mr-Retinal is shown with black sticks. Trp-265^6.48 and Ala-117^3.32 are shown with orange and cyan sticks and balls, respectively. H, helix.

Figure 5.

Spectral properties of blue cone opsin reconstituted with 11-cis-6mr-retinal compared with rod opsin reconstituted with 11-cis-6mr-retinal. a, UV-visible absorption spectra of blue cone opsin regenerated with 11-cis-6mr-retinal (B6mr) in dark conditions (black spectrum) and after illumination for 5 (orange spectrum) and 30 min (red spectrum). Sample illuminated for 30 min was then kept for 1200 min in the dark (red dotted spectrum). b, UV-visible absorption spectra of rod opsin regenerated with 11-cis-6mr-retinal (Rh6mr) in dark conditions (black spectrum) and after illumination for 1 min (red spectrum). Sample illuminated for 30 min was then kept for 1200 min in the dark (black dotted spectrum). c, HPLC elution profile of retinoid oximes extracted from dark-state B6mr (black line) or from B6mr illuminated for 5 min (red line). d, HPLC elution profile of retinoid oximes extracted from dark-state Rh6mr (black line) or from Rh6mr illuminated for 1 min (red line). e, photosensitivity of B6mr. Samples were illuminated with light from a 150-W Fiber-Lite source delivered through a 400–440-nm band pass interference filter at 20 °C. The percentage of residual pigment was plotted against the incident photon count and fitted with an exponential function. The slope of the fitting line corresponds to the relative photosensitivity of the pigment at the irradiating wavelength. Error bars represent standard deviation (S.D.). f, accessibility of the bulk solvent to the protonated Schiff base in blue cone opsin regenerated with 11-cis-retinal (Blue opsin; light blue) or 11-cis-6mr-retinal (B6mr; dark blue). The UV-visible absorption spectra were recorded every 2 min at 20 °C. The percentage of residual pigment was plotted as a function of time and fitted with an exponential function. Error bars represent S.D. φ, quantum yield.

Figure 6.

Thermal stability and acidification of blue cone opsin reconstituted with 11-cis-6mr-retinal. a, UV-visible absorption spectra of dark-state B6mr (blue line), light-activated Meta-II–like B6mr (red line), and Meta-II–like B6mr after acid denaturation (yellow line). The pH of the sample was adjusted to 2 with H₂SO₄ to protonate the Schiff base. Inset in a, the difference spectrum of acidified sample indicates that the Schiff base in Meta-II-like B6mr is in a deprotonated state. b, the thermal stability of purified Rh6mr or B6mr was determined. Samples were incubated at 37 °C in the dark, and their absorbance spectra were recorded every 2 min for 40 min. The change in the absorbance maximum was calculated as a percentage of residual pigment, assuming the absorbance at the initial point as 100%, and plotted as a function of time. These plots were used to calculate the half-lives (t_½) of chromophore release. Each experiment was performed in triplicate.

Figure 7.

Photochemical modeling of B6mr compared with Rh6mr. B6mr and R6mr feature the red-shifted λ_max relative to WT blue cone opsin and Rh (at 440 and 505 nm, respectively). Upon prolonged light illumination, B6mr is converted to the active Meta-II–like state, and 11-cis-6mr-retinal (6mr) is released from the binding pocket, whereas Rh6mr displays reversible photochemical behavior, resulting in back-conversion of its Meta-II–like state to the ground R6mr state. Calculated quantum yields (φ) of B6mr and R6mr were 0.0047 and 0.027, respectively. * indicates the photoactivated (Meta II-like) state of B6mr and Rh6mr.