Phylogenetic tree, selected amino acid residues, absorption spectra, and light-induced Ca2+ responses of Acropora tenuis opsins belonging to the ASO-II group.

(A) Maximum-likelihood (ML) tree of animal opsins including A. tenuis opsins in the ASO-II group. Seven opsins in the ASO-II group that were identified and cloned from A. tenuis in this study are shown in bold and the three members for which we obtained absorption spectra are highlighted in red. Numbers at the nodes represent support values of each ML branch estimated by 1000 bootstrap samplings (≥ 70% are indicated). Scale bar = 0.6 substitutions per site. All branches and support values are provided in Fig. S1. (B) Selected residues near the Schiff base in opsins of the ASO-II group and other animal opsins. Animal opsins typically have an acidic residue acting as counterion at one of three established sites (yellow): E94 (e.g., jellyfish opsin), E113 (e.g., bovine rhodopsin), or E181 (e.g., jumping spider Rh1). Remarkably, opsins in the ASO-II group lack an acidic residue at any of these positions, but instead feature an acidic residue at position 292 (red). The retinal-binding lysine, Lys296, is shown in black. A more detailed sequence alignment is provided in Fig. S2. Residues are numbered according to bovine rhodopsin. (C) Absorption spectra in the dark of three A. tenuis opsins in the ASO-II group (Antho2a, Antho2c and Antho2e). The absorption spectra were measured at 0°C in 140 mM NaCl at pH 6.5. The number in each graph shows the λmax value. (D) Results of the aequorin-based bioluminescent reporter assay for monitoring light-induced changes in Ca2+ in HEK293S cells expressing the same three opsins in the ASO-II group as in panel C. In each graph, luminescence values were normalized to the baseline. Black circles with error bars indicate the means ± SEMs (n = 3) of the measured relative luminescence. Black arrowheads at time 0 indicate the timing of one-minute irradiation with green (495 nm; for Antho2a and Antho2c) or UV (395 nm; for Antho2e) light.

Absorption spectra of wild type and the E292A mutant of A. tenuis Antho2a.

(A, B) Absorption spectra of the dark state (curve 1, black) and the photoproduct (curve 2, red) of the wild type (Antho2a WT, A) and the E292A mutant (Antho2a E292A, B) at 140 mM NaCl and pH 6.5. The samples were kept at 0°C during the spectroscopic measurements. (C, D) Absorption spectra of the dark state of Antho2a WT (C) and Antho2a E292A (D) prepared in Cl−-depleted conditions, before (curve 1, black) and after (curve 2, blue) adding Cl− (see Materials and Methods for details). In the Cl−-depleted condition, the pigments were solubilized in 70 mM Na2SO4, which reportedly does not access to the Cl− binding site in the chicken red-sensitive cone visual pigment iodosin (Shichida et al., 1990), to moderate protein denaturation. (E, F) Effect of halide anions on the absorption spectra of wild type Antho2a (E) and the Antho2a E292A mutant (F) at pH 6.5 and 0°C. The graphic shows the normalized absorption spectra of the pigments prepared in 140 mM NaCl (black curves), 140 mM NaBr (blue curves), and 140 mM NaI (red curves).

Effects of pH and Cl− concentration on the absorption spectra of the dark states of wild type Antho2a and the Antho2a E292A mutant.

(A, B) Changes in the absorbance at λmax as a function of pH for (A) wild type Antho2a and (B) the E292A mutant at different Cl− concentrations. The absorbance values at “visible λmax” (mean absorbance at 503 ± 5 nm for the wild type and 505 ± 5 nm for the E292A mutant, respectively) were normalized for each Cl− concentration to those at the lowest pH, in which the Schiff base is assumed to be fully protonated (“Rel. abs. at visible λmax” in the y-axes). Solid and dashed lines represent sigmoid fits to the experimental data for each Cl− concentration (indicated by different colors). The pH-dependent change of wild type Antho2a at 140mM NaCl is also shown in panel B (dotted grey line). The full absorption spectra used to generate these plots are provided in Fig. S6 (for wild type Antho2a) and Fig. S8 (for the E292A mutant). (C) Changes in the absorbance at λmax for wild type Antho2a (black open circles) and the E292A mutant (red solid circles) as a function of Cl− concentration. The absorbance values at visible λmax were normalized to those at 500 mM NaCl for both the wild type and the E292A mutant. The lines in the graph were generated by fitting the Hill equation to the experimental data. The full absorption spectra used to generate these plots are provided in Fig. S9.

QM/MM structural model of wild type Antho2a in the dark state (A) and detailed views of the retinal binding pocket with a protonated (neutral) Glu292 (B), a deprotonated (negatively charged) Glu292 (C), and the E292A mutant (D).

The retinal protonated Schiff base (PSB) and the binding pocket residues are shown as sticks (including polar hydrogens) and the Cl− ion as a sphere with its coordination shown as dashes. “Wat” indicates a water molecule. Residues in the QM region are marked in bold.

Comparison of the light-evoked intracellular Ca2+ levels between wild type Antho2a and the E292A mutant.

The graph shows the mean ± S.E.M (n = 4) of the measured relative luminescence values (luminescence values normalized to the baseline) for wild type Antho2a (black) and the E292A mutant (red) at pH7.0. The green vertical line indicates the time of cell illumination with green light (510 nm, for 1 s, 1.65 × 1015 photons/cm2/s).

pH-dependent changes in the absorption spectra of Antho2c and Antho2e at different Cl− concentrations at 0°C.

(A–C) Absorption spectra of purified wild type Antho2c pigment at (A) 0 mM, (B) 0.093 mM, and (C) 9.3 mM NaCl concentrations. The corresponding pH values are indicated on each curve in the graphs. (D) Summary of the spectral changes for wild type Antho2c across different Cl− concentrations at neutral pH (pH 6.5). (E, F) Absorption spectra of (E) wild type Antho2e (Antho2e WT) and (F) its R113A mutant (Antho2e R113A) at different Cl− concentrations at pH 6.5 at 0°C. Each color indicates a different Cl− concentration.

Maximum-likelihood (ML) tree of animal opsins, with non-opsin GPCRs included as an outgroup.

(a simplified version of the ML tree is shown in Fig. 1A). The tree includes opsins belonging to the eight main groups (see main text) as well as of opsins from the recently identified subgroups xenopsins and chaopsins (Ramirez MD et al. Genome Biol Evol 8:3640–3652, 2016). The sixteen A. tenuis opsins (eight opsins in the cnidopsin group, one opsin in the ASO-I group, and seven opsins in the ASO-II group) that were identified in this study are shown in bold. The three opsins in the ASO-II group for which absorption spectra were successfully obtained are highlighted in red. Numbers at nodes represent support values for the ML branch estimated by 1000 bootstrap samplings (≥ 70% are indicated). Scale bar = 0.6 substitutions per site.

Key residues in opsins of the ASO-II group and other animal opsins.

Residues near the Schiff base were selected with reference to the crystal structure of bovine rhodopsin (PDB ID: 1U19) and the homology model of Antho2a (shown in Fig. 4). Position 292 contains an acidic residue (D/E) only in opsins of the ASO-II group (red). In addition, functionally important residues (such as the retinal binding Lys296 (black), the three established counterion sites (yellow), and two highly conserved motifs in Class A GPCRs, E(D)RY on TM3 (green) and NPxxY on TM7 (blue)) are also shown. Residues are numbered according to bovine rhodopsin.

Photo- and thermal reactions of wild type Antho2a.

(A) Absorption spectra of purified wild type Antho2a were measured at 0°C in the dark (curve 1, black), after orange light irradiation (> 560 nm, curve 2 and curve 3, deep and pale orange), and after subsequent violet light irradiation (420 nm, curve 4, purple). Irradiation of wild type Antho2a with orange light shifted the absorption maximum from 503 nm in the dark (curve 1, black) to 476 nm in the photoproduct (curve 2 and curve 3, orange). Upon subsequent irradiation with 420 nm light (while the photoproduct was stable), the λmax of the photoproduct stayed at 476 nm, with only a slight decrease of the peak absorbance (curve 4, purple) possibly resulting from degradation of the photoproducts upon light irradiation. (B) The configuration of retinal in wild type Antho2a before (black) and after (orange) irradiation with orange light (> 560 nm) was analyzed with HPLC. Retinal was extracted in its oxime form. AT, all-trans retinal; 11, 11-cis retinal. (C) Difference spectra calculated from the absorption spectra recorded before and after sequential irradiations with 500 nm and 420 nm light for crude extracts of detergent-solubilized cell membranes expressing wild type Antho2a. Curve 1: Difference spectrum of after minus before 500 nm irradiation, showing a blue-shifted spectral change indicative of photoproduct formation. Curves 2 and 3: Difference spectra of after minus before subsequent 420-nm light irradiation (curve 2), and after minus before a second 500 nm light irradiation, following the 420 nm irradiation (curve 3). Neither condition resulted in spectral changes consistent with regeneration of the dark state, indicating that Antho2a is not bistable under these conditions. (D) Changes in the absorption spectra of the purified pigment of wild type Antho2a after irradiation with orange (>560 nm) light (with the sample kept in the dark at 0°C). Each colored curve corresponds to a different incubation time after the light irradiation. (E) Difference spectra obtained by subtracting the spectrum of wild type Antho2a in the dark from the spectra measured at different time points after irradiation (shown in D). (F, G) Acid denaturation of pigments before (dark state, F) and after light irradiation (G). (F) Absorption spectra of wild type Antho2a in the dark were measured immediately after sample preparation at pH 6.5 (curve 1), and after incubation over night at 0°C (curve 2, pH 6.5) with subsequent addition of HCl to pH 1.9 (curve 3). (G) Absorption spectra of wild type Antho2a incubated for 20 hours at 0°C following irradiation with orange light (> 560 nm), measured at pH 6.5 (curve 1) and after acidification to pH 1.9 (curve 2). When retinal binds to opsin via a Schiff base (protonated or deprotonated), acid denaturation traps the chromophore as a protonated Schiff base, exhibiting absorption at λmax ∼440 nm. Acid denaturation of irradiated Antho2a (incubated for 20 hours post-irradiation) did not yield a product with λmax at ∼440 nm (G, curve 2), whereas the dark state pigment after acidification displayed absorption at ∼440 nm (F, curve 3). These results indicate that Antho2a gradually releases retinal after light irradiation. (H) Absorption spectra of purified wild type Antho2a at pH 8.0, measured at 0°C before (dark state; curve 1, black) and 0, 10, and 30 minutes after irradiation with orange light (> 560 nm; red, green, and yellow curves, respectively).

(A, B) Effect of pH on the absorption spectra of the dark state and photoproduct of (A) wild type Antho2a and (B) the E292A mutant at 140 mM NaCl.

Each graph shows spectra before (curves 1, black) and after (curves 2, red) irradiation with orange (> 560 nm) light.

Effect of pH and NaCl concentration on absorption spectra of the dark states and photoproducts of Antho2a.

(A) Absorption spectra of wild type Antho2a at 0.28 mM NaCl under different pH conditions (pH 6.5 and pH 7.3). (B) Absorption spectra of wild type Antho2a at pH 6.5 with varying NaCl concentrations (2.8 mM, 8 mM, and 800 mM). (C) Absorption spectra of the E292A mutant of Antho2a at 800 mM NaCl and different pH conditions (pH 4.8, pH 6.6, and pH 7.6). Each graph shows spectra before (curves 1, black) and after (curves 2, deep purple/red, and 3, pale purple/pink) irradiation with UV light (< 410 nm, A) or orange light (> 550 nm or > 560 nm, B and C). Curves 2 and 3 in each graph represent, respectively, the first measurement (immediately after irradiation) and a subsequent measurement (< 5 min. after irradiation). The minimal change in the absorption spectra over this time scale indicates that the spectra remain stable after irradiation.

pH-dependent changes in the absorption spectra of Antho2a WT at (A) 0.28 mM, (B) 2.8 mM, (C) 28 mM, (D) 140 mM, and (E) 500 mM Cl− at 0°C.

The pH values of the solution, measured right after each spectroscopic measurement, are indicated on the corresponding curves in each graph.

pH-dependent changes in the absorption spectra of Antho2a WT and Antho2a E292A at 0 mM NaCl (containing 70 mM Na2SO4) at 0°C.

The pH values at which the absorption spectra were measured are indicated on the corresponding curves.

pH-dependent changes in the absorption spectra of Antho2a E292A at (A) 2.8 mM, (B) 28 mM, (C) 140 mM, and (D) 500 mM Cl− at 0°C.

The pH values are indicated on the corresponding curves in each graph.

Absorption spectra of (A) wild type Antho2a and (B) the Antho2a E292A mutant under different Cl− concentrations at pH 6.5 and 0°C.

Each color indicates a different concentration of Cl−.

Schematic drawing of the environment of the protonated Schiff base depicting the counterion switch from a chloride ion (top) to Glu292 (bottom) upon light activation.

Expression levels and normalized Ca2+ responses of wild type and E292A Antho2a.

(A) The relative absorbances at λmax (± 5 nm) of wild type (grey) and E292A (red) Antho2a are used as indicators of expression levels. The proteins were expressed and purified under identical conditions, and absorbance values of the E292A mutant were normalized to those of the wild type. Spectra were measured at 0°C, pH 6.5, and 140 mM NaCl. Each bar represents mean ± S.E.M. of n = 4 replicates from separate transfections in cultured cells. (B) Normalized maximum Ca2+ responses of HEK293S cells expressing wild-type Antho2a and the E292A mutant after irradiation with green light (510 nm). Responses were normalized to the expression levels shown in panel A. Each bar represents mean ± S.E.M. of n = 4 replicates from separate transfections in cultured cells. Statistical evaluation of the normalized Ca2+ responses for wild type Antho2a versus the E292A mutant was conducted using a Welch’s t-test (two-sided). ***P < 0.001.

Vertical excitation energies (ΔEcalc) and oscillator strengths (f) computed by quantum mechanics/molecular mechanics (QM/MM) calculations using different QM methods with the cc-pVTZ basis set.