Omega Rhodopsins: A Versatile Class of Microbial Rhodopsins
1Division of Life Science, Gyeongsang National University, 501 Jinju-daero, Jinju, Gyeongsangnam-do 52828, Republic of Korea
2Electron Microscopy Research Center, Republic of Korea
3Department of Systems Biology, Division of Life Sciences, and Institute for Life Science and Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea
J. Microbiol. Biotechnol. 2020; 30(5): 633-641
Published May 28, 2020
Copyright © The Korean Society for Microbiology and Biotechnology.
Microbial rhodopsins, termed as ‘type I rhodopsins’ to distinguish them from the G-protein coupled receptor superfamily (type II rhodopsins) that comprises several opsin families, are photoactive proteins consisting of seven transmembrane (TM) domains. These proteins mainly act as light-driven ion pumps or sensors with the configurational change in incorporated all-
Key milestones in microbial rhodopsin research.Rhodopsins that function as ion pumps or channels are shown in black circles; sensory rhodopsins are in red. Omega rhodopsins are indicated Ω.
The first discovered and most widely understood rhodopsin is bacteriorhodopsin (BR), which was found from Archaea in the 1970s [5, 6], and its ability to translocate the proton ion that results from ATP biosynthesis was further elucidated [5, 6]. The subsequent discovery of an inward chloride ion pump, halorhodopsin (HR), and sensory rhodopsin (SR), which is responsible for phototactic responses, have broadened the spectra of haloarchaeal rhodopsins [7, 8]. Additionally, channel rhodopsin (ChR), a light-gated cation channel, was discovered in eukaryotic green algae [9, 10]. Members of ChR are adopted as successful tools for optogenetics, wherein they react to light and allow the influx of positive ions to act as an ‘on’ switch. In contrast, HR, which translocates negatively charged chloride ion, is widely used for inactivation in neuroscience.
Research on microbial rhodopsins has markedly expanded along with the discovery of proteorhodopsin (PR) from Proteobacteria through a marine metagenomic analysis . PR shares structural and functional similarities with the archaeal BR; however, its presence in Bacteria led to the expectation that rhodopsin may have a greater impact on the marine ecosystem. Subsequently, three haloarchaeal-type rhodopsin genes including those of an HR and two SRs were detected in the genome of
In the 2010s, wing to mind-boggling developments in genomics, new clades of bacterial rhodopsins had been continuously discovered. A sodium-pumping rhodopsin (NaR) with the NDQ motif in its active site and a chloride-pumping rhodopsin (ClR) with the NTQ motif were reported in turn from marine flavobacteria [15-17]. Xenorhodopsin, a natural inward proton pump, was also characterized, which is unique since other previously reported unidirectional cation pumps are all outward . As the newest members of the bandwagon, viral-type proton-pumping rhodopsins were retrieved from giant viruses of eukaryotic algae through single-cell metagenomics . More recently, heliorhodopsin with its speculated function of light-sensing activity was found to be widespread globally across the three domains of life and algal viruses . All of these efforts have profoundly expanded the spectrum of type I microbial rhodopsins and phylogenetically related rhodopsins.
Among the microbial rhodopsins, this review focuses on the ion-translocating rhodopsins that comprise the ‘3 omega motif ’ that forms a stack of three non-consecutive aromatic amino acids arising from the TM helices A and B, and the B–C loop. We refer to the class of microbial rhodopsins containing this structural motif as ‘omega rhodopsins.’ We review recent developments in elucidating the functions and characteristics of this rhodopsin group. A thorough understanding of this rhodopsin class will provide insights into the evolution of 7-TM proteins with consideration to their phylogenetic distribution and functional or ecological diversification.
Recognition of the ‘3 Omega Motif’
We noticed the presence of the 3 omega motif during the structural analysis of a ClR from
Characteristic features of type I microbial rhodopsins with the ‘3 omega motif.’ Sequence alignment in the active sites and 3 omega motif.The representative amino-acid sequences of each rhodopsin adopted from Halobacterium salinarumNRC-1 (BR), Dokdoniasp. MED134 (PR), Actinobacterium_MWH_Uga1 (ActR), Salinibacter ruberDSM 13855 (XR), Gleobacter violaceusPCC7421 (GR), Thermus thermophilusJL-18 (TR), Dokdonia erikastaNBTC 100814 (NaR), Nonlabens marinusS1-08 (ClR), Halobacterium salinarumNRC-1 (HR).
Three-dimensional structures of the 3 omega motifs.NDQ motif-containing sodium-pump rhodopsin, NaR (cyan, PDB ID: 3X3B); NTQ motif-containing chloride-pump rhodopsin, ClR (green, PDB ID: 5G28); xanthorhodopsin, XR (magenta, PDB ID: 3DDL); Gleobacterrhodopsin, GR (orange, PDB ID: 6NWD); thermophilic rhodopsin, TR (blue, PDB ID: 5AZD).
ActR is a group of green light-dependent, outward proton-pumping rhodopsins and is phylogenetically close to XR (Fig. 4). Although the protein structure has not yet been elucidated for any of the members, alignment of protein sequences revealed that the amino acids with aromatic rings are conserved in TM A and TM B, but not in the B-C loop (Fig. 2). Even without the presence of the third aromatic functional group, the stacking of two aromatic rings in TMs A and B could be sufficient to contribute to the stability of the two helices and the whole protein. Thus, we classify ActR as an omega rhodopsin in this review.
Phylogenetic relationships between omega rhodopsins and other microbial rhodopsin families.The maximum-likelihood method using the Jones-Taylor-Thornton matrix-based model of amino acid substitution rates with empirical amino acid frequencies and the gamma model was used. Representative microbial rhodopsins were aligned by MUSCLE. Evolutionary analyses were conducted in MEGA X. Blue-colored circles on the tip of some branches indicate the rhodopsins listed in Fig. 2. NDQ motif-containing sodium-pump rhodopsin, NaR; NTQ motif-containing chloride-pump rhodopsin, ClR; xanthorhodopsin, XR; Gleobacterrhodopsin, GR; thermophilic rhodopsin, TR; actinorhodopsin, ActR; proteorhodopsin, PR; bacteriorhodopsin, BR; halorhodopsin, HR; channel rhodopsin, ChR; sensory rhodopsin, SR; xenorhodopsin, XeR; heliorhodopsin, HeR; viral rhodopsin, ViR.
The function of the 3 omega motif is yet to be clearly elucidated. The ﬂuorescent thermal stability assay of mutants, wherein the aromatic amino acids were substituted with alanine, revealed that mutants were slightly unstable compared with the wild-type ClR . Moreover, Morizumi
We hypothesize that the structural rigidity and stability of TM helices A and B generated by stacked aromatic rings could support the diagonal flexibility and freedom of other transmembrane columns, resulting in functional innovation with respect to the extended ion specificity and absorption spectrum during the evolution of rhodopsins in this class.
Functional Diversity of Omega Rhodopsins
Omega rhodopsins have a wide functional versatility in terms of ion specificity and absorption spectrum. Fig. 4 presents the phylogenetic relationships within or between omega rhodopsins and other type I rhodopsins. Phylogenetic analysis indicates that this class of microbial rhodopsins is evolutionally distinct from the others, thus constituting a unique phylogenetic clade. We will summarize the brief history and recent studies on omega rhodopsins according to their phylogenetic affiliation and translocated ions, namely H+, Na+, and Cl- ions.
The XR Family of Proton Pump Rhodopsins
The most representative family of proton pumps among the omega rhodopsins is the XRs. The photocycle and functional amino acids in the ion transfer pathway in XR remain similar to the other proton pumps, such as PR or BR, but their structure differs considerably. In addition to retinal, XR is associated with a particular carotenoid antenna molecule, salinixanthin, whose function is light-harvesting and acting as an energy provider for the acceptor, retinal . The structure of the XR initially found in
A proton pump was found in the hyperthermophilic bacterium
GR is a proton pump found in
Actinorhodopsin Proton Pumps
ActRs are most closely related to XR in the phylogenetic tree (Fig. 4). This group of rhodopsins was initially found by mining the metagenomic data of non-marine samples [14, 35]. Genes that are present in the metagenomic assemblies in this rhodopsin clade are linked to the sequenced actinobacterial genomes, and therefore it is termed ‘ActR’ . ActRs have been excavated from numerous mixed cultures containing Actinobacteria and environmental DNA samples from various freshwater environments, thus revealing that these genes are dispersed globally.
ActRs in freshwater comprise the three phylogenetic groups LG1, LG2, and PCL1, and these sequences were also reported from freshwater lakes, estuaries, and hypersaline lagoon ecosystems . LG1 group is split into subgroups, and LG1-A groups are encoded by acI, which is the most abundant actinobacterial lineage among the freshwater inhabitants, whereas genes of the LG1-B group are carried by the Luna lineage [36, 37]. The ability of ActR to pump proton in the native cells was confirmed by supplementing with exogenous retinal  or innate retinal . ActR in
NDQ Motif-Containing Sodium Pump Rhodopsins
In 2013, a novel class of microbial rhodopsins possessing Asn and Gln at the positions of the proton acceptor and donor residues of BR was found in
Although NaR is optimized for sodium ion pumping, it can also pump lithium and even convert to a proton pump in the absence of sodium or lithium ions. However, its proton-pumping photocycle is slower than that during sodium pumping, and its efficiency is considerably lower than that of the typical proton pumps . Extensive studies on ion selectivity using KR2 revealed that the rate constant of ion uptake is dependent on ion concentration, and NaR pumps sodium under physiological conditions in which the sodium concentration is significantly greater than that of the proton [43-45]. The cation tuning exhibited by NaR is unique among microbial rhodopsins. In the KCl solution, NaR acts as a proton pump, whereas it functions as a sodium pump in the NaCl solution. Its cation selectivity is based on the different helical movements in the presence of K+ or Na+ . Switching mechanism from sodium pumping to proton pumping at acidic pH was proposed by Kovalev
The crystal structures of NaR under neutral and acidic conditions, and three different acid pHs in the monomeric state or pentameric states, were reported . Along with detailed biochemical characterization and mutagenesis, both studies successfully figured out the sodium-translocation mechanism. In particular, the NaR structure provides the first clue how positively charged sodium ion, which is unable to bind covalently to the Schiff base, achieves transport across the conduit. Unlike proton pumps, NaR contains distinguishable structures including N-terminal helix capping the ion-release cavity and loop B-C over a hydrophilic cavity to be part of the sodium release region. The structures also revealed the pentamerization of KR2 and the binding of sodium ions at the interface [49, 50].
NaR holds significant value for application as a next-generation optogenetics tool with its variants that preferentially pump potassium, another ion that triggers the neuronal responses. Potassium preferential pumps were achieved by manipulating Gly263 or Asn61 residues in the ion-uptake cavity that affect the selectivity of the pump. Furthermore, Kato
Additionally, the mechanism for the rapid photoreaction of KR2 was elucidated via femtosecond time-resolved absorption study or femto- to submillisecond transient stimulated Raman spectroscopy [53, 54]. Moreover, extensive classical and quantum molecular dynamics simulations of transient photocycle states revealed the molecular mechanism for sodium pumping by presenting the electrostatic transitions by internal conformational dynamics and proton transfer reactions . These intensive studies on the molecular mechanism of NaR may provide a basis for the rational design of NaR with optogenetic applications.
NTQ Motif-Containing Chloride Pump Rhodopsins
Chloride-pumping rhodopsins are found in Archaea and Bacteria living in highly saline habitats, and the presence of these pumps is reported in various marine bacteria inhabiting a broad range of environments . In proton pumps, the proton acceptor and donor amino acids (amino acid residues 85 and 96 in BR) that protonate the retinal Schiff base are conserved as carboxylic amino acids, Asp or Glu [2, 57]. These two residues along with Thr 89 form a highly conserved motif, DTD or DTE, in BR or PR, respectively. HR has the TSA motif at the corresponding position. The single amino acid change in BR (D85T) resulted in the conversion of a proton pump to a chloride pump . The TSD motif was found in cyanobacterial HR, and the single amino acid replacement (T74D) successfully turned the inward chloride pump into an outward proton pump .
Notably, ClR shares higher sequence identity with NaRs than HRs. Structural studies of ClR indicate that its architecture is similar to that of NaR than that of HR despite the opposite transport directions and charges of the ion molecules transported [21, 62]. The retinal conformation of ClR around the Schiff base is different from that of HR and more similar to those of NaR and XR. The amino-acid residues comprising the internal cavity are highly conserved between ClR and NaR, and they share the 3 omega motif .
Interchangeability of Omega Rhodopsins and Implications in Evolutionary Engineering
Moreover, artificial evolution studies to extend ion or light specificity have been conducted using omega rhodopsins. Successful NaR variants obtained by manipulating residues in the ion-uptake cavity that translocate potassium ions were previously described [49, 50]. Similarly, tuning the same amino acids, Asn61 and Gly263 at the cytoplasmic surface in NaR, turned its variant to permeate Cs+ as well as other monovalent cations . Recently, a 40-nm red-shift in the absorption wavelength of NaR was achieved by controlling the polarity of amino-acid residues (P219T/S254A) around the retinal chromophore without impairing its sodium-transport efficiency. Collectively, with the tuning approach, a natural sodium pump with red-shifted absorption, which lacks Pro at the position of P219, was identified from a bacterium living in a solar saltern. Rhodopsins with longer-wavelength light will see expanded use as optogenetics tools due to their low phototoxicity and high tissue penetration .
Evolutionary Relationship Between Omega Rhodopsins and Other Rhodopsins
Although type I and type II rhodopsins share the noticeable structural similarities of possessing the seven-transmembrane α-helical architecture and an internal pocket in which the chromophore retinal is bound, these two types of rhodopsins differ in their sequences and structural details. Moreover, they are also taxonomically distinct and found in evolutionarily distant organisms. Type I rhodopsins that often function as light-driven ion pumps have been found in both prokaryotes and eukaryotes so far, whereas type II rhodopsins are found only in higher animals and classified into the G-protein coupled receptor (GPCR) superfamily since they transduce the light signal to linked G protein [1, 67]. Whether these two protein superfamilies diverge from an ancient common ancestor or converge on the same protein fold and the covalent linkage to the retinal from independent origins remains under debate [67, 68]. Mackin
After solving the high-resolution structure of NaR, Shalaeva and colleagues revealed that NaR and sodium-dependent GPCRs share the striking similarity of their sodium-binding sites through structural superposition . Based on the phylogenetic position of NaR between other type I rhodopsins and class A GPCRs, they speculated its proximity to the common ancestor of both superfamilies, which apparently contained a sodium-binding site and presumably was a light-driven sodium export pump. A single aromatic residue (Trp215 in NaR) in the 6th helix turned out to be highly conserved in visual rhodopsins and most type I rhodopsins. It functions in rotation or tilting of the helix, and it might be involved in signal transduction via interacting with ligand in type II rhodopsins and ion translocation by opening a conduit in type I rhodopsins, respectively. Shalaeva
The biosynthetic pathway for retinal and carotenoids could be associated with rhodopsin evolution as the key cofactors of rhodopsins. For example, compared to the proteobacterial strains with PR possessing a minimum set of genes for retinal biosynthesis, the flavobacterial strains having NaR or ClR encode an additional
Ecophysiological Roles of Omega Rhodopsins
Mechanistic evidence on omega rhodopsins has been accumulating rapidly via biochemical studies on photochemical properties and ion specificities and their successful structural analyses, whereas their physiological functions and roles in cellular processes and ecological consequences remain largely unexplored.
It is widely accepted that proton pumps, particularly PR, which is the most abundant phototrophic protein in oceanic surface waters, play a significant role in providing alternative energy generation and metabolic strategies under oligotrophic conditions . The roles of bacterial sodium and chloride pumps in marine microbial ecology are to be determined. Since sodium and chloride are the two most predominant dissolved ions in seawater, their pumps may help in maintaining osmotic balance . They can be more productive if they function in generating energy via a chloride or sodium chemiosmotic potential. Some extremophilic archaea and marine bacteria use the sodium-motive force to drive energy transmission . A respiration-dependent primary sodium pump directly couples Na+ translocation to a chemical reaction, or a Na+/H+ antiporter transforms the H+ gradient generated by primary proton pumps into a Na+ gradient .
In numerous genomes, sodium or chloride pumps coexist with the PR family of proton pumps, and some photo-responsible studies on cell growth or gene expression have been conducted.
Microbes with omega rhodopsins have been isolated from various environments including freshwater, seawater, halophilic lakes, and salterns. Recruitment of metagenomic reads revealed that the occurrence of NQ rhodopsin-carrying prokaryotes reaches about 17% in a hypersaline microbial mat sample comprising approximately 90 practical salinity units . The metagenomics analysis also verified the existence of XR- and NaR-family omega rhodopsins in Antarctic desert hypolithic communities. It was the first observation of rhodopsins from the terrestrial environment, and they are presumed to exist in ubiquitous species in hypolithons and to adapt well to this environment .
The ecological roles of ActR in actinobacteria have been characterized mainly in a ubiquitous freshwater actinobacterial lineage, acI. All retinal biosynthesis pathway genes were transcribed, and ActR was most highly transcribed in a eutrophic lake . A metatranscriptomic study of acI also reported the high expression of ActR and some transport proteins . Notably, the expression level of ActR from the acI lineage in freshwater is not directly light-driven, and it is rather constitutive circadian-minimized at dusk and maximized at dawn .
In conclusion, microbial rhodopsins are distributed among microbes belonging to various taxa, spanning a wide range of different characteristics . Among these, we reviewed the studies on rhodopsins with the 3 omega motif in terms of their functional diversity, physiology in the cell, and evolutionary relationship. The reason for the existence of this motif was not clearly elucidated, but we speculate that it may contribute to the structural stability and arrangement of rhodopsins. The presence of this motif may have contributed to the functional diversification of rhodopsins of this class including the ion specificity and the absorption spectrum. Further biochemical research on the 3 omega motif and physiological studies will be required to reveal the importance of omega rhodopsins in evolutionary relationships among rhodopsin (super)families.
This work was financially supported by grants from the National Research Foundation of Korea (2020R1C1C1004778, NRF-2017R1C1B2009025, and NRF-2016R1E1A1A01943552).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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