A distinct abundant group of microbial rhodopsins discovered using functional metagenomics

Abstract

Many organisms capture or sense sunlight using rhodopsin pigments^1,2, which are integral membrane proteins that bind retinal chromophores. Rhodopsins comprise two distinct protein families ¹ , type-1 (microbial rhodopsins) and type-2 (animal rhodopsins). The two families share similar topologies and contain seven transmembrane helices that form a pocket in which retinal is linked covalently as a protonated Schiff base to a lysine at the seventh transmembrane helix^2,3. Type-1 and type-2 rhodopsins show little or no sequence similarity to each other, as a consequence of extensive divergence from a common ancestor or convergent evolution of similar structures ¹ . Here we report a previously unknown and diverse family of rhodopsins-which we term the heliorhodopsins-that we identified using functional metagenomics and that are distantly related to type-1 rhodopsins. Heliorhodopsins are embedded in the membrane with their N termini facing the cell cytoplasm, an orientation that is opposite to that of type-1 or type-2 rhodopsins. Heliorhodopsins show photocycles that are longer than one second, which is suggestive of light-sensory activity. Heliorhodopsin photocycles accompany retinal isomerization and proton transfer, as in type-1 and type-2 rhodopsins, but protons are never released from the protein, even transiently. Heliorhodopsins are abundant and distributed globally; we detected them in Archaea, Bacteria, Eukarya and their viruses. Our findings reveal a previously unknown family of light-sensing rhodopsins that are widespread in the microbial world.

Extended Data Fig. 1 |. Full alignment of heliorhodopsin and type-1 rhodopsin.

a, Multiple amino acid alignment of different heliorhodopsins. Positions 23, 80, 104 and 107 (heliorhodopsin 48C12 numbering) are marked with black arrows, as well as position 237 (grey arrow) and lysine in position 241 (red arrow). b, Multiple amino acid alignment of heliorhodopsin (48C12) with green absorbing proteorhodopsin (GPR), bacteriorhodopsin (BR), sensory rhodopsin I from Halobacterium salinarum (HsSRI) and Salinibacter ruber (SrSRI), sensory rhodopsin II from H. salinarum (HsSRII) and Natronomonas pharaonis (NpSRII), Anabaena sensory rhodopsin (ASR), xenorhodopsin from Parvularcula oceani (PoXeR), halorhodopsin from H. salinarum (HsHR), eubacterial chloride-pump rhodopsin from Nonlabens marinus (NmClR), sodium-pump rhodopsin from Krokinobacter eikastus (KR2) (K. eikastus is also known as Dokdonia eikasta), anion channelrhodopsin from Guillardia theta (GtACR1) and cation channelrhodopsin2 from Chlamydomonas reinhardtii (C-terminal side omitted, ChR2 ΔC-term). Positions 85, 89 and 96 (bacteriorhodopsin numbering) are marked with black arrows, as well as position 212 (grey arrow) and lysine in position 216 (red arrow).

Extended Data Fig. 2 |. Relationship and depth distribution of heliorhodopsins and type-1 rhodopsins.

a, Clustering analysis separates heliorhodopsins (purple leaves in dendrogram tree) from type-1 rhodopsins (grey leaves in dendrogram tree) based on per cent identity obtained using protein–protein blast v.2.7.1. The hierarchical clustering was performed using the clustermap function from the Python package seaborn, with default parameters for metric and linkage. The code used to generate the figure is available as a Jupyter notebook at https://github.com/BejaLab/heliorhodopsin. b, Detailed phylogenetic relationships within the heliorhodopsin family. White circles represent bootstrap values of >80%. The scale bar indicates the average number of amino acid substitutions per site. Coloured circles indicate heliorhodopsins expressed in this study. c, Depth profiles of the relative abundance of type-1 rhodopsins and heliorhodopsins from marine metagenomes collected during the Tara Oceans expedition.

Extended Data Fig. 3 |. Heliorhodopsin membrane topology.

a, Predictions of membrane topology for type-1 rhodopsins, type-2 rhodopsins and heliorhodopsin. Upper panel, suggested schematic topologies for type-1 rhodopsins, type-2 rhodopsins and heliorhodopsins. Positively charged amino acids are labelled. N, amino-terminal tail, C, carboxy-terminal tail. Middle panel, membrane topology predictions by TMHMM, Phobius, Philius and SPOCTOPUS. Lower panel, the rhodopsin sequences used for membrane topology predictions. b, Arrangement of heliorhodopsin 48C12 protein across the E. coli membrane. The heliorhodopsin–β-lactamase fusion sites resulting in ampicillin resistance (amp^r) and ampicillin sensitivity (amp^s) are indicated. This experiment was repeated twice.

Extended Data Fig. 4 |. Ion-transport activity assay of heliorhodopsins.

a, Ion-transport activity assay of six kinds of heliorhodopsin (48C12, and contigs 172728, 2376895, 381806, 1205911 and 161490), and a comparison with a type-1 rhodopsin (a light-driven proton pump; green-absorbing proteorhodopsin, GPR) in the absence and presence of the protonophore CCCP. Light was present for the time region indicated by the orange bars. b, Patch-clamp assay of heliorhodopsin 48C12. The photocurrent was measured at 60, 0 or −60 mV by whole-cell mode. Left, 48C12-expressing cells were illuminated with 550-nm light (2.6 mW/mm²), indicated by a yellow bar (n = 10 cells). Right, cells expressing GtCCR4 were illuminated with 520-nm light (2.4 mW/mm²), indicated by a green bar.

Extended Data Fig. 5 |. UV-visible absorption of heliorhodopsin 48C12 at different pH values.

a, Deprotonation of the retinal Schiff base of heliorhodopsin 48C12 at alkaline pH. Difference absorption spectra (left) and absorption change at λ = 553 nm (right, orange solid circles) of heliorhodopsin 48C12 upon pH change from 8.5 to higher values. The deprotonated form of retinal Schiff base showed the absorption at λ = 373 nm. b, Red-shift of UV-visible absorption spectrum of heliorhodopsin 48C12, and protonation of the counterion. UV-visible absorption spectra (left) and the λ_max (right, orange solid circles) of heliorhodopsin 48C12 at pH 2.8–8.4. When pH is lowered a red-shift of the absorption is observed, which is commonly reported for many type-1 rhodopsins and reflects the protonation of counterions. Thus, the red-shift of heliorhodopsin 48C12 originates from protonation of E107, which is fitted with the Henderson–Hasselbalch equation (blue dashed line), and the pK_a of counterion (E107) is estimated to be 3.7. At pH values of less than 2.8, a large blue-shift to 443 nm is observed, presumably owing to the acid denaturation of the protein. The pK_a values in right panels indicate mean ± s.d.

Extended Data Fig. 6 |. HPLC pattern of retinal extracted from heliorhodopsin 48C12.

HPLC pattern of retinal extracted from heliorhodopsin 48C12 in the dark (blue) and under illumination at λ = 540 ± 10 nm (green). Most of the retinal (>97%) bound to heliorhodopsin 48C12 adopts an all-trans configuration in the dark (n = 4). When the retinal is extracted after illumination (λ > 500 nm), the proportion of the 13-cis form increased to 59 ± 5% (mean ± s.d., n = 4).

Extended Data Fig. 7 |. Photocycle of heliorhodopsins.

a, The photocycle of heliorhodopsin 48C12 determined by the multi-exponential fitting for the time evolution of transient absorption change shown in Fig. 4a, b. The lifetimes of the intermediates are indicated as mean ± s.d. b, c, Time evolution of transient absorption change of photo-excited contigs 381806 (b) and 172728 (c).

Extended Data Fig. 8 |. Light-induced FTIR difference spectra of heliorhodopsin 48C12.

a, Light-induced FTIR spectra of heliorhodopsin 48C12 at 77 K (upper spectrum), 240 K (middle spectrum) and 277 K (lower spectrum) in the 1,800−900 cm⁻¹, in which the intermediate produced is the K, M and O intermediate, respectively. Spectra are measured in H₂O (black) and D₂O (red). The amide-I vibration of the peptide backbone and the C=N stretch vibration of the Schiff base appear at 1,700−1,600 cm⁻¹. Peaks at 1,659(−) and 1,629(+) cm⁻¹ in H₂O, and at 1,637(−) and 1,613(+) cm⁻¹ in D₂O at 77 K probably originate from a C=N stretch of the Schiff base, and small changes in amide-I suggest that the primary K intermediate does not undergo a major conformational change. This is also the case for the M intermediate, for which negative peaks at 1,656 cm⁻¹ in H₂O and at 1,638 cm⁻¹ in D₂O originate from the C=N stretch of the Schiff base, and changes in amide-I vibrations are small. By contrast, the appearance of strong peaks at 1,694 and 1,675 cm⁻¹ in the O intermediate indicates extensive conformational changes. b, Light-induced difference FTIR absorption spectra of the K intermediate of heliorhodopsin 48C12 and bacteriorhodopsin. Light-induced difference FTIR absorption spectra of heliorhodopsin 48C12 (upper spectrum) and bacteriorhodopsin (lower spectrum) at 77 K in the 1,230−1,150 cm⁻¹ region, which were measured in H₂O. The bands at 1,200(−) and 1,188(+) cm⁻¹ were observed at 77 K, similar to the type-1 rhodopsin bacteriorhodopsin, which suggests that the primary photochemical reaction of heliorhodopsin is the all-trans to 13-cis isomerization.

Extended Data Fig. 9 |. Transient absorption change of heliorhodopsin 48C12 mutants.

a–c, Transient absorption spectra of photo-excited heliorhodopsin 48C12 E107Q (a), H23F (b) and H80F (c) mutants. The M intermediate is formed for all these mutants, which suggests that none of these residues is the proton acceptor of the Schiff base. In the case of the H80F mutation, the O intermediate is not formed. d–f, Time evolution of transient absorption change of photo-excited heliorhodopsin 48C12 D36N (d), E62D (e) and E107D (f) mutants. The fact that the M intermediate forms in these mutants excludes the possibility that one of these residues acts as the proton acceptor for the Schiff base.

Extended Data Fig. 10 |. Proton release and uptake observed with cresol red.

a, UV-visible absorption spectra of cresol red (left) and its absorbance at 429 nm (blue circles) and 573 nm (green circles) (right), at different pH values in 100 mM NaCl and 6-mix buffer (10 mM citrate, 10 mM MES, 10 mM HEPES, 10 mM MOPS, 10 mM CHES and 10 mM CAPS). Global fitting for absorption at different pH values with the Henderson–Hasselbalch equation showed the pK_a of cresol red to be 8.123 ± 0.004 (mean ± s.d.). b, c, Time evolutions of transient absorption change of GPR (b) and heliorhodopsin 48C12 (c) in unbuffered 100 mM NaCl and 0.1% DDM. The pH value was adjusted to be approximately 8.1 by addition of NaOH. The transient absorption change of cresol red was calculated by the subtracting transient absorption changes obtained with cresol red from those without cresol red at 429 and 573 nm (corresponding to accumulations of protonated and deprotonated state of cresol red, respectively).

Fig. 1 |. Heliorhodopsin 48C12.

a, Fosmid clones from Lake Kinneret from 96-well plate number 48 in the presence of all-trans retinal. Clone KIN48C12 is circled. b, Membranes from E. coli containing fosmid KIN48C12 with (+) and without (−) 10 μM all-trans retinal. c, Predicted secondary structure of heliorhodopsin. The transmembrane helices in heliorhodopsin are marked I to VII to distinguish them from the seven transmembrane helices of type-1 and type-2 rhodopsins (which are routinely referred to as helices A-F and helices 1−7, respectively). Negatively charged residues are circled in red and positively charged residues are circled in blue (the proton-accepting groups H23 and H80 are marked with double blue circles), and the conserved lysine in TMVII is marked in solid blue. d, Schematic of membrane topologies of type-1 rhodopsin, type-2 rhodopsin and heliorhodopsins. The presentation of type-1 rhodopsin is based on proteorhodopsin, and the type-2 rhodopsin is based on bovine rhodopsin. N, amino-terminal tail, C, carboxy-terminal tail. Retinal shown for type-1 and heliorhodopsins is all-trans retinal, and for type-2 it is 11-cis retinal. Positively charged amino acids are marked.

Fig. 2 |. Microbial rhodopsins.

a, Unrooted phylogenetic tree for representative proteins from heliorhodopsins and type-1 rhodopsins. Heliorhodopsin 48C12 (HeR 48C12) is indicated in purple. BR, bacteriorhodopsin; PR, proteorhodopsin; SRI, sensory rhodopsin I; SRII, sensory rhodopsin II; NaR, sodium-transporting rhodopsin; NR, Neorospora rhodopsin; ClR, chloride-transporting rhodopsin; HR, halorhodopsin; ActR, actinobacteria rhodopsin; XeR, xenorhodopsin; XR, xanthorhodopsin; VR, viral rhodopsin; CCR, cation channelrhodopsin; enzyme R, enzyme rhodopsin; ACR, anion channelrhodopsin. Coloured circles indicate heliorhodopsins expressed in this study. White circles represent bootstrap values of >80%. The scale bar indicates the average number of amino acid substitutions per site. See Supplementary Data 2 for alignment and Supplementary Data 3 for the tree. b, The relative abundance of type-1 rhodopsins and heliorhodopsins (depicted in grey and purple, respectively), presented in reads per kilobase per million (RPKM), was measured in marine metagenomes that correspond to bacterial, giant virus and viral fractions collected during the Tara Oceans expedition (n = 382 metagenomes in total) and selected non-marine metagenomes (n = 33). Distribution of the abundances are shown as box plots, whiskers include observations within 1.5× interquartile range of the upper and lower quartiles. If the number of samples was less than five per group, individual dots are presented. MS, Mediterranean Sea; RS, Red Sea; IO, Indian Ocean; SAO, South Atlantic Ocean; NAO, North Atlantic Ocean; SPO, South Pacific Ocean; NPO, North Pacific Ocean; SO, Southern Ocean; AMZ, Amazon lakes; ISL, Iranian Saline Lakes; XXR, Xiangxi River; QRE, Queensland river estuaries.

Fig. 3 |. Molecular properties of heliorhodopsin 48C12.

a, Ion transport activity assay of heliorhodopsin 48C12 by observing the pH changes of E. coli cell suspensions, and comparison with type-1 rhodopsin (a light-driven proton-pump proteorhodopsin, green-absorbing proteorhodopsin GPR) in the absence and presence of the protonophore (carbonyl cyanide m-chlorophenyl hydrazone (CCCP)). Light was present for the time region indicated by the orange bars. b, Electrophysiological measurements of heliorhodopsin 48C12 by observing photocurrents on ND7/23 cells, and a comparison with type-1 rhodopsin (a channelrhodopsin from Guillardia theta (Gt)CCR4). The membrane potential was held at −60 mV and light was present for the time region indicated by the orange or green bar (550 nm for heliorhodopsin 48C12 and 520 nm for CCR4, n = 10 cells). c, E. coli cells expressing heliorhodopsin 48C12 wild type, as well as the K241A and K241Q mutants. d, Absorption spectra of heliorhodopsin 48C12 and the E107Q mutant.

Fig. 4 |. Photocycle of heliorhodopsin 48C12.

a, Transient absorption spectra of heliorhodopsin 48C12 excited at λ_pump = 532 nm. OD, optical density. b, Time evolutions of transient absorption change at specific wavelengths. Each of the wavelengths probes different photochemical species: 400 nm for the M intermediate; 540 nm for the L and N intermediates, and bleached dark state; and 625 nm for the K and O intermediates. c, Light-induced difference FTIR absorption spectra of heliorhodopsin 48C12 at 240 K (top spectrum) and 277 K (middle spectrum), and bacteriorhodopsin at 250 K (bottom spectrum) in the region of 1,790−1,710 cm⁻¹, in which the intermediates produced are the M and O intermediates of heliorhodopsin 48C12 and the M intermediate of bacteriorhodopsin, respectively. Spectra are measured in H₂O (black) and D₂O (red). d, Time evolutions of transient absorption change at specific wavelengths for heliorhodopsin 48C12 E107Q (top), H23F (middle) and H80F (bottom) mutants. e, Light-induced difference FTIR absorption spectra of heliorhodopsin 48C12 at 77 K (top spectrum), 240 K (middle spectrum) and 277 K (bottom spectrum) in the region of 1,720−1,600 cm⁻¹. Spectra are measured in H₂O (black) and D₂O (red). f, Photoreaction cycle of heliorhodopsin 48C12. The chromophore retinal isomerizes from all-trans to 13-cis form upon illumination (dark-to-K). Then, the proton of the retinal Schiff base is transferred to the proton-accepting group (PAG), which is probably composed of several amino acid residues that include H23 and H80 (K-to-M). The proton returns to the Schiff base and a large conformational change of protein moiety occurs on the O (M-to-O). The conformational change is expected to generate an inter-protein signal to an unidentified counterpart protein. Finally, the protein returns to the dark state and deactivation occurs by retinal thermal isomerization (O-to-dark).