Crystal structure of heliorhodopsin

Abstract

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics1. They are widely present in bacteria, archaea, algae and algal viruses2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the β-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.

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Fig. 1: Overall structure of T. archaeon HeR.
Fig. 2: Dimer interface and HS-AFM analysis.
Fig. 3: Intramolecular interactions.
Fig. 4: Lateral fenestration.

Data availability

Coordinates and structure factors have been deposited in the Protein Data Bank, under the accession number 6IS6. The X-ray diffraction images are also available at the Zenodo data repository (https://doi.org/10.5281/zenodo.3333323).

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Acknowledgements

The diffraction experiments were performed at SPring-8 BL32XU (proposal 2017B2578). We thank the members of the Nureki laboratory and the beamline staff at BL32XU of SPring-8 (Sayo, Japan) for technical assistance during data collection, R. Nakamura and for technical assistance, S. Sugita for help with confocal microscopy. This research was partially supported by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under grant number JP19am0101070 (support number 1627). This work was supported by JSPS KAKENHI grants 16H06294 (O.N.), 17J30010, 30809421 (W.S.), 25104009, 18H03986, 19H04959 (H.K.), 17H03007 (K. Inoue), 19H05389, 18H04512, 18H01837 (T.U.), 18K06109 (S.P.T.) and by JST PRESTO (JPMJPR15P2 to K. Inoue and JPMJPR1688 to S.P.T.) and CREST (JPMJCR1753 to H.K. and JPMJCR17N5 to Y.F.). O.B. is supported by the Louis and Lyra Richmond Memorial Chair in Life Sciences.

Author information

Affiliations

Authors

Contributions

W.S. screened the homologues of the HeRs, and purified and crystallized T. archaeon HeR, with assistance from T.I. and S.O. T.I. performed the SEC–MALLS analysis. A.H. purified the McHeR for spectroscopic analysis. K.Y. solved and refined the structure. K. Inoue, M. Singh, M.K., M.H., R.M., S.T., R.A.-Y., K.K., Y.F. and H.K. performed spectroscopic analyses. M.K., S.H., Y.Y. and S.P.T. determined the topology. K.K. and M. Singh performed the scrambling assay. K. Ikeda, M. Shibata and T.U. performed the AFM analysis. A.P. and O.B. performed bioinformatics analyses. The manuscript was mainly prepared by W.S., K. Inoue, H.K. and O.N. H.K. and O.N. supervised the research.

Corresponding authors

Correspondence to Hideki Kandori or Osamu Nureki.

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The authors declare no competing interests.

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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Peer review information Nature thanks Klaus Gerwert and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Phylogenetic tree and biochemical characterization.

a, Phylogenetic tree of HeRs and representative type-1 rhodopsins. HeR 48C12, the first-discovered HeR shows 12.6% identity and 35.4% similarity with bacteriorhodopsin (BR), and 15.0% identity and 36.0% similarity with green-absorbing proteorhodopsin (GPR). b, Ion-transport assays of TaHeR, HeR 48C12, and GPR. This experiment was performed twice, and the representative data are shown. c, ATR–FTIR spectra on the exchange between Cl and Br in the solvent for TaHeR and the Cl pump (NpHR). d, ATR–FTIR spectra on the exchange between Na+ and K+ in the solvent for TaHeR and the Na+ pump (KR2). This experiment was performed twice, and the representative data are shown. e, HPLC pattern of chromophores extracted from TaHeR. HPLC pattern of retinal extracted from TaHeR in the dark (blue) and under illumination at λ = 540 ± 10 nm (green). Most of the retinal (96%) bound to TaHeR adopts an all-trans configuration in the dark (n = 3). When the retinal was extracted after illumination, the proportion of the 13-cis form increased to 39% (n = 3). The representative data are shown, f, Deprotonation of the retinal Schiff base of TaHeR at an alkaline pH. Difference absorption spectra (left) and absorption change at λ = 542 nm (right, red solid circles) of TaHeR upon a pH change from 8.4 to 11.7 and higher values. The deprotonated form of the retinal Schiff base showed the absorption at λ = 392 nm. The acid dissociation constant (pKa) value in the right panel indicates mean ± s.d., which is fitted with the Henderson–Hasselbalch equation (grey dashed line). This experiment was performed once. g, Red-shift of the UV–visible absorption spectrum of TaHeR, and protonation of the counterion. The UV–visible absorption spectra (left) and the λmax (right, red solid circles) of TaHeR at pH 1.6–7.0. When the pH is lowered, a red-shift of the absorption is observed, and this is commonly reported for many type-1 rhodopsins and reflects the protonation of counterions. Thus, the red-shift of TaHeR originates from the protonation of Glu108, which is fitted with the Henderson–Hasselbalch equation (grey dashed line), and the pKa of the counterion (Glu108) is estimated to be 3.6. At pH values less than 2.1, a large blue-shift to 450 nm is observed, presumably due to the acid denaturation of the protein. The pKa value in the right panel indicates mean ± s.d. on fitting. This experiment was performed once.

Extended Data Fig. 2 Photocycle and sequence conservation.

a, Transient absorption spectra of TaHeR in E. coli membrane excited at λpump = 532 nm. b, Time evolutions of transient absorption changes at specific wavelengths of TaHeR in E. coli membrane. Each wavelength probes a different photochemical species: 409 nm for the M intermediate; 549 nm for the bleached dark state; and 605 nm for the K and O intermediates. ΔOD, change in optical density. c, Reconstructed absorption spectra obtained by the multi-exponential fitting for the time evolution of the transient absorption spectra shown in a. d, The photocycle of TaHeR, determined by the multi-exponential fitting for the time evolution of the transient absorption changes shown in a and b. In the measurement of the transient absorption spectra at t > 200 ms, a notch filter was placed in front of the ICCD detector, to minimize the scattered excitation pulse, and thus the absorption change could not be observed at λ = 523–540 nm in a and c (see details in Methods). These spectroscopy experiments were performed twice, and the representative data are shown. e, Maximum conserved residues and their conservation ratios in the HeR family members. The corresponding residues of TaHeR are also indicated with their residue numbers.

Extended Data Fig. 3 Inverted topology.

a, Crystals of the HeR protein. The crystallization was reproduced more than three times. b, Snake plot of the HeR structure. c, Expression of Flag-eGFP-HeR-Myc in cultured ND7/23 cells, with TaHeR bearing the Myc epitope tag at the C terminus. eGFP fluorescence (left, green), probed with a Myc antibody under unpermeabilized conditions for immunofluorescent staining with Alexa Fluor 594 (middle, magenta) and merge (right). Fluorescence from Alexa Fluor 594 was observed under unpermeabilized conditions, indicating that the C-terminal Myc-epitope tag was localized on the extracellular side. d, Expression of Myc-eGFP-HeR-Flag in cultured ND7/23 cells, with HeR bearing the Myc epitope tag at the N terminus. eGFP fluorescence (left, green), probed with a Myc antibody under unpermeabilized conditions for immunofluorescent staining with Alexa Fluor 594 (middle, magenta) and merge (right). Fluorescence from Alexa Fluor 594 was not observed under unpermeabilized conditions, indicating that the N-terminal Myc-epitope tag was not localized on the extracellular side. e, Expression of Myc-eGFP-HeR-Flag in cultured ND7/23 cells. eGFP fluorescence (left, green), probed with a Myc antibody under permeabilized conditions for immunofluorescent staining with Alexa Fluor 594 (middle, magenta) and merge (right). Fluorescence from Alexa Fluor 594 was observed under permeabilized conditions, indicating that the N-terminal Myc-epitope tag was localized on the intracellular side. Scale bar, 20 μm. These immunofluorescent staining experiments were performed twice, and the representative data are shown. f, g, Structures of HeR (f) and BR (g), aligned in the direction of the membrane plane. The rhodopsins and retinal chromophores are shown as ribbon representations and sticks, respectively.

Extended Data Fig. 4 Dimer interface.

a, SEC–MALLS analysis of HeR, using Shimazu HPLC and Superdex 200 increase 10/300 column equilibrated with buffer, containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.03% DDM. The three chromatograms show the readings from the UV absorption, refractive index, and light-scattering detectors. The traces were normalized to the peak maxima. The cyan and red curves in the light-scattering chromatogram indicate the calculated molecular masses of the protein–detergent complex (Mc) and the protein (Mp), respectively. The UV extinction coefficient of the protein was calculated by PROTPARAM and assumed to be 1.624. The refractive index increments (dn/dc) of the protein and the detergent were assumed to be 0.185 and 0.144, respectively. This experiment was performed once. b, Molecular mass values determined by the SEC–MALLS experiment with the standard errors on fitting or calculated from the amino acid sequence. The experimental protein mass was determined to be about 61.75 kDa, corresponding to the theoretical mass of the His-tagged HeR dimer, 59.7 kDa. c, d, UV–visible CD (blue) and absorption (green) spectra of HeR in a lipid membrane (POPE/POPG) (c) and in DDM (d). These experiments were performed once. e, f, In-cell thrombin-digestion of extracellular loops. Schematic diagram of the thrombin-digestion sites in the thrombin-digestion mutants of the extracellular loops (e). Immunoblotting analysis of the mutants (f). Protein digestion patterns were detected by the S-tag antibody. These results revealed that the thrombin digested only ECL1, and not ECL2 and ECL3. This pattern indicated that the first loop blocked the access of thrombin to ECL2 and ECL3, consistent with the crystal structure and also with the inverted topology as compared to the type-1 rhodopsins. This experiment was performed twice, and the representative data are shown. g, The four-helix bundle is expanded and shown in detail, with the interacting residues within 4.2 Å depicted as stick models. h, Tomographic representation along the dimer interface viewed from the extracellular side, showing the high surface complementarity within the four-helix bundle interface. This interface consists of an extensive network of interactions involving 16 residues in TM4 and TM5. i, List of rhodopsin oligomerization states.

Extended Data Fig. 5 Validation of dimer formation.

a, Gel filtration chromatogram of the purified HeR, using AKTA purifier and Superdex 200 increase 10/300 column equilibrated with buffer, containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.03% DDM. Black line indicates the absorbance at 280 nm. Dashed line indicates the peak fractions used in the SDS–PAGE analysis in b. This experiment was performed twice, and the representative data are shown. b, SDS–PAGE analysis of the gel filtration fractions in a. The left and right panels show the images of the Coomassie brilliant blue-stained and unstained gels, respectively. The band corresponding to the HeR with colour indicates that stable dimer formation without the dissociation of the retinal even in the SDS–PAGE. Both bands corresponding to the monomer and dimer are double, The HeR structure contains all the residues of HeR except for the N-terminal His6 tag and the residues 1–3, denying the proteolysis of the purified HeR. We suppose that the double bands reflect the difference in denaturation by SDS. c, E. coli cells expressing the wild-type and deletion mutant of HeR. This experiment was performed twice, and the representative data are shown. d, Absorption spectra of the wild-type and deletion mutant of HeR solubilized with DDM from E. coli membrane, in the presence of 500 mM hydroxylamine. Black and coloured spectra were measured under dark and light conditions, respectively. These experiments were performed twice, and the representative data are shown. e, Dark-minus-light difference spectra of the wild-type and deletion mutant of HeR in d. The orange spectrum is multiplied by 5.0. f, Conservation of the surface residues of the HeR structure. The sequence conservation among 531 HeRs was calculated using the ConSurf server (http://consurf.tau.ac.il), and is coloured from cyan (low) to maroon (high). The residues constituting the dimer interface, especially those in ECL1, show low sequence conservation among the HeRs. g, Sequence difference in the residues constituting the dimer interface. The conserved and not-conserved residues between TaHeR and HeR 48C12 are coloured in dark turquoise and orange, respectively. 23 of the 30 residues constituting the interface are different between TaHeR and HeR 48C12. h, A typical HS-AFM image of HeR 48C12 dimers in a lipid membrane. The HS-AFM experiments were performed four times and similar images were obtained. Representative data are shown. i, An averaged image (50 frames) of HeR 48C12 dimers. Despite the sequence difference between TaHeR and HeR 48C12, the HS-AFM images revealed that HeR 48C12 also forms a dimer in the membrane.

Extended Data Fig. 6 Conservation of the retinal-binding site.

a, Maximum conserved residues around retinal and their conservation ratios in HeR family members, with their residue numbers in HeR and BR. The variations of the amino acid species in TaHeR, HeR 48C12, and representative type-1 rhodopsins are shown in the lower part. b, Light-induced difference absorption spectra of wild-type (black curve) and 17 mutants (red curve) of HeR solubilized with DDM from E. coli membrane, in the presence of 500 mM hydroxylamine (HA). Positive and negative signals show the spectra before and after illuminations, corresponding to those of the rhodopsin and the retinal oxime, respectively. Mutant spectra were normalized to a positive peak at 542 nm in the case of the wild type, whereas the mutant spectra were multiplied by factors of 0.8–3.1, as shown in each graph. These experiments were performed once. c, The results of the mutagenesis effects mapped on the retinal-binding site.

Extended Data Fig. 7 UV–visible and FTIR spectroscopic analyses.

a, UV–visible difference spectra of K (magenta curve) and M (blue curve) intermediates were obtained by illumination with 540 nm light at 77 K and with λ > 520 nm light at 230 K, respectively. The K and M intermediates were completely reverted to the original state by illumination with 590 nm and 400 nm light, respectively (dotted curves). One division of the y axis is 0.03. b, The FTIR difference spectra of the K and M intermediates were obtained by the same illumination conditions used for UV–visible spectroscopy (upper panel). The FTIR difference spectra of the O intermediate were reconstructed from the time-resolved FTIR spectroscopy at 298 K (lower panel). The black and red spectra were measured under H2O and D2O conditions, respectively. One division of the y axes is 0.0005. c, The time traces at 1,694 cm−1 (red curve) and 1,673  cm−1 (blue curve) were obtained by time-resolved FTIR spectroscopy at 298 K. The solid and dotted curves were measured under H2O and D2O conditions, respectively. One division of the y axis is 0.0004. d, The FTIR difference spectra upon the formation of the K intermediate are shown in the X–D stretching vibration region. The red and blue spectra were measured under D2O and D218O conditions. The green-tagged bands are assigned to the O–D stretching vibrations of water molecules. These spectroscopy experiments were performed once.

Extended Data Fig. 8 ConSurf analysis and transient absorption changes of HeR mutants.

a, b, Conservation of the residues of HeR. The sequence conservation among 531 HeRs was calculated using the ConSurf server (http://consurf.tau.ac.il) and is coloured from cyan (low) to maroon (high). b, A focus on the residues constituting the lateral fenestration. c, Fo − Fc omit maps for waters around the Schiff base, contoured at 3.0σ. The water molecules are shown as red spheres. di, Time evolutions of transient absorption changes at specific wavelengths for wild-type HeR in E. coli membranes (d), and wild-type HeR (e), HeR(E227Q) (f), HeR(H23F) (g), HeR(H82F) (h) and HeR(H23F/H82F) (i) in nanodiscs. ΔOD, change in optical density, using laser flash photolysis. These spectroscopy experiments were performed twice, and representative data are shown.

Extended Data Fig. 9 Comparison of lateral fenestrations in microbial rhodopsins.

ad, Surface representations of HeR (a), BR (b), XR (c, PDB code: 3DDL), and KR2 (d, PDB code: 3X3B). The side chains above the β-ionone ring are shown as sticks. e, f, Surface representations and cross sections of HeR (e) and metarhodopsin II intermediate (f, PDB code: 3PXO). Black arrows show two openings of the seven-transmembrane region. gi, The difference UV–visible absorption spectra of wild-type HeR (i), HeR(G171W) (h) and GR (i) upon bleaching by 500 mM HA at different times in 20 mM HEPES–NaOH, pH 7.0, 100 mM NaCl and 0.1% DDM. j, Time evolution of the bleaching of the visible absorptions shown in gi. The bleaching rate of the retinal chromophore in HeR is much faster than those in GR, suggesting that the retinal binding in HeR is not as tight as that in the type-1 rhodopsins. This observation is consistent with the fact that the residues constituting the retinal-binding site in HeR are smaller than those in the type-1 rhodopsins. These experiments were performed twice, and representative data are shown.

Extended Data Fig. 10 Characterization of HeRs in other domains of life and investigation of scrambling activity.

a, Phylogenetic tree of HeRs. The four HeRs investigated spectroscopically are shown in red. b, Alignment of the amino acid sequences of TaHeR, HeR 48C12, McHeR and EhvHeR. Secondary structure elements for α-helices and β-strands are indicated by cylinders and arrows, respectively. Conservation of the residues is indicated as follows: red panels for completely conserved; red letters for partially conserved; and black letters for not conserved. This alignment was done using ESPript3. c, d, The absorption spectra (c) and time evolution of the transient absorption changes (d) of HeR 48C12, TaHeR, McHeR, and EhvHeR, using laser flash photolysis. The absorption spectra were measured in 0.1% DDM for HeR 48C12, TaHeR and EhvHeR, and in 0.03% DDM for McHeR. McHeR and EhvHeR showed blue-shifted absorptions, as compared with those of HeR 48C12 and TaHeR. Transient absorption changes were measured with HeR 48C12 and TaHeR in the E. coli membrane fraction and McHeR and EhvHeR in 0.03% and 0.1% DDM, respectively. The pH values were 8.5, 8.0, 8.0 and 7.5 for HeR 48C12, TaHeR, McHeR and EhvHeR, respectively. These spectroscopy experiments were performed once. e, Schematic representation of the fluorescent NBD-labelled scramblase activity assay. f, Fluorescence traces of scrambling by protein-free (black), bovine opsin (red), bovine rhodopsin (pink), opsin of TaHeR (light green) and TaHeR (dark green), reconstituted in POPE:POPG (3:1) phospholipid liposomes. g, Quantification of the scrambling activities of bovine opsin, bovine rhodopsin, opsin of TaHeR and TaHeR. The data are the mean ± s.d. of three measurements. h, Model diagram representing the scramblase activities of HeR and bovine opsin (PDB code: 3CAP).

Extended Data Table 1 Data collection and refinement statistics

Supplementary information

Supplementary Information

This file contains Supplementary Text and Supplementary References.

Reporting Summary

Supplementary Data 1

Sequence alignment of the representative type-1 rhodopsins and heliorhodopsins.

Supplementary Data 2

Phylogenetic tree of the representative type-1 rhodopsins and heliorhodopsins.

Video 1

: HS-AFM video of TaHeR An HS-AFM video of TaHeR embedded in an asolectin lipid bilayer. Scan size: 40 × 31 nm2 (120 × 80 pixels2). Imaging rate: 100 ms/frame. The HS-AFM experiments were performed four times and similar images were obtained. Representative data are shown.

Video 2

: HS-AFM video of HeR 48C12 An HS-AFM video of HeR 48C12 embedded in an asolectin lipid bilayer. Four dimers, indicated by magenta ellipses on the first frame, are observed in the lipid-bilayer island. The blob encircled by the cyan circle probably corresponds to an unstructured monomer, due to the instability of HeR 48C12. Scan size: 50 × 35 nm2 (120 × 84 pixels2). Imaging rate: 150 ms/frame. The HS-AFM experiments were performed four times and similar images were obtained. Representative data are shown.

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Shihoya, W., Inoue, K., Singh, M. et al. Crystal structure of heliorhodopsin. Nature 574, 132–136 (2019). https://doi.org/10.1038/s41586-019-1604-6

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