Amino acid residue at position 188 determines the UV-sensitive bistable property of vertebrate non-visual opsin Opn5

Abstract

Opsins are G protein-coupled receptors specialized for photoreception in animals. Opn5 is categorized in an independent opsin group and functions for various non-visual photoreceptions. Among vertebrate Opn5 subgroups (Opn5m, Opn5L1 and Opn5L2), Opn5m and Opn5L2 bind 11-cis retinal to form a UV-sensitive resting state, which is inter-convertible with the all-trans retinal bound active state by photoreception. Thus, these opsins are characterized as bistable opsins. To assess the molecular basis of the UV-sensitive bistable property, we introduced comprehensive mutations at Thr188, which is well conserved among these opsins. The mutations in Opn5m drastically hampered 11-cis retinal incorporation and the bistable photoreaction. Moreover, T188C mutant Opn5m exclusively bound all-trans retinal and thermally self-regenerated to the original form after photoreception, which is similar to the photocyclic property of Opn5L1 bearing Cys188. Therefore, the residue at position 188 underlies the UV-sensitive bistable property of Opn5m and contributes to the diversification of vertebrate Opn5 subgroups.

Fig. 1. Binding selectivity of retinal isomers in wild-type and W167F, L212F, and T188C mutant Opn5m proteins.

a Different amino acid residues around the retinal between Opn5m and Opn5L1 subgroups. A structural model was constructed based on the crystal structure of squid rhodopsin (PDB: 2z73) visualized using UCSF Chimera. Squid rhodopsin has serine and phenylalanine at positions 188 and 212 (based on the bovine rhodopsin numbering system) (Supplementary Fig. 1), which were replaced by threonine and leucine in this model. b-e (left) Absorption spectra of Opn5m wild-type (b) and W167F (c), L212F (d), and T188C (e) mutant proteins purified after the addition of 11-cis or all-trans retinal to the collected cell membranes. The spectra were recorded at 0 °C in the dark (curve 1) and after yellow light (>500 nm) irradiation (curve 2). Spectral change by light irradiation is shown in left panels of Supplementary Fig. 2a–h. (right) Retinal configuration changes of Opn5m wild-type (b) and W167F (c), L212F (d), and T188C (e) mutant proteins purified after the addition of 11-cis or all-trans retinal to the collected cell membranes. The retinal isomers before and after yellow light (>500 nm) irradiation were analyzed with HPLC after extraction of the chromophore as retinal oximes (right panels of Supplementary Fig. 2a–h). The negative peak in the visible region of the difference spectrum calculated before and after yellow light irradiation of the wild-type (Supplementary Fig. 2e) corresponds to λmax of the all-trans retinal bound form (474 nm). λmax of the all-trans retinal bound form of the mutant proteins calculated from their difference spectra (Supplementary Fig. 2f–h) are 470 nm (W167F), 475 nm (L212F), and 470 nm (T188C).

Fig. 2. Binding selectivity of retinal isomers in Opn5m Thr188 mutant proteins.

a–f (left) Absorption spectra of Opn5m T188S (a), T188G (b), T188A (c), T188N (d), T188M (e), and T188Q (f) mutant proteins purified after the addition of 11-cis retinal to the collected cell membranes. The spectra were recorded at 0 °C in the dark (curve 1) and after yellow light irradiation (curve 2). Spectral change by light irradiation is shown in Supplementary Fig. 3. (right) Retinal configuration changes of Opn5m T188S (a), T188G (b), T188A (c), T188N (d), T188M (e), and T188Q (f) mutant proteins purified after the addition of 11-cis retinal to the collected cell membranes. The retinal isomers before and after yellow light (>500 nm) irradiation were analyzed with HPLC after extraction of the chromophore as retinal oximes. (Supplementary Fig. 4a–f). g–l (left) Absorption spectra of Opn5m T188S (g), T188G (h), T188A (i), T188N (j), T188M (k), and T188Q (l) mutant proteins purified after the addition of all-trans retinal to the collected cell membranes. The spectra were recorded at 0 °C in the dark (curve 1) and after yellow light irradiation (curve 2). Spectral change by light irradiation is shown in Supplementary Fig. 3. (right) Retinal configuration changes of Opn5m T188S (g), T188G (h) T188A (i), T188N (j), T188M (k), and T188Q (l) mutant proteins purified after the addition of all-trans retinal to the collected cell membranes. The retinal isomers before and after yellow light (>500 nm) irradiation were analyzed with HPLC after extraction of the chromophore as retinal oximes. (Supplementary Fig. 4g–l).

Fig. 3. Spectral changes of wild-type and Thr188 mutant Opn5m proteins.

Absorption spectra of Opn5m wild-type (a) and T188S (b), T188G (c), T188A (d), T188N (e), T188M (f), T188Q (g), and T188C (h) mutant proteins purified after the addition of all-trans retinal to the medium of the transfected cultured cells. The spectra were recorded at 0 °C in the dark (curve 1), after yellow light (>500 nm) irradiation (curve 2), after subsequent UV light (360 nm) irradiation (curve 3), after yellow light re-irradiation (curve 4), after UV light re-irradiation (curve 5) and after yellow light re-irradiation (curve 6). Spectral changes by each light irradiation are shown in Supplementary Fig. 5.

Fig. 4. Molecular characteristics of T188C mutant Opn5m protein.

a, b Absorption spectra of Opn5m T188C mutant protein purified after the addition of all-trans retinal to the medium of the transfected cultured cells. The spectra were recorded at 10 °C (a) in the dark (curve 1) and 0, 4.33, 16.5, 37.5, and 116 min after yellow light (>500 nm) irradiation (curve 2–6). Spectral changes observed during the incubation in the dark after light irradiation are shown in Supplementary Fig. 6a. The spectra of T188C mutant protein were also recorded at 37 °C (b) in the dark (curve 1) and 0.0016, 0.032, 0.10, 3.26, 1995 s after yellow flash light (>460 nm) irradiation (curve 2–6). Spectral changes observed during the incubation in the dark after light irradiation are shown in Supplementary Fig. 6b. c Absorption change of Opn5m T188C mutant protein at 470 nm after light irradiation at 37 °C. Time course of the absorbance at 470 nm was plotted according to the data in Supplementary Fig. 6b and was fitted with a double-exponential function (τ = 0.11 and 39.2 s). These time constants are more than 200 times faster than that of chicken Opn5L1 (τ = 175 min) as described previously. d Retinal configuration analysis of Opn5m T188C mutant protein. (Left) Retinal isomers of Opn5m T188C mutant protein purified after reconstitution with all-trans retinal were analyzed with HPLC after extraction of the chromophore from the samples before irradiation (black), after yellow light irradiation (red) and after subsequent incubation for 2 h in the dark (blue) as retinal oximes. AT, all-trans retinal; 11, 11-cis retinal; 13, 13-cis retinal. (Right) Isomeric compositions of retinal before light irradiation, after light irradiation, and after subsequent incubation.