Multiple Roles of a Conserved Glutamate Residue for Unique Biophysical Properties in a New Group of Microbial Rhodopsins Homologous to TAT Rhodopsin.

TAT rhodopsin, a microbial rhodopsin found in the marine SAR11 bacterium HIMB114, uniquely possesses a Thr-Ala-Thr (TAT) motif in the third transmembrane helix. Because of a low pKa value of the retinal Schiff base (RSB), TAT rhodopsin exhibits both a visible light-absorbing state with the protonated RSB and a UV-absorbing state with the deprotonated RSB at a neutral pH. The UV-absorbing state, in contrast to the visible light-absorbing one, converts to a long-lived photointermediate upon light absorption, implying that TAT rhodopsin functions as a pH-dependent light sensor. Despite detailed biophysical characterization and mechanistic studies on the TAT rhodopsin, it has been unknown whether other proteins with similarly unusual features exist. Here, we identified several new rhodopsin genes homologous to the TAT rhodopsin of HIMB114 (TATHIMB) from metagenomic data. Based on the absorption spectra of expressed proteins from these genes with visible and UV peaks similar to that of TATHIMB, they were classified as Twin-peaked Rhodopsin (TwR) family. TwR genes form a gene cluster with a set of 13 ORFs conserved in subclade IIIa of SAR11 bacteria. A glutamic acid in the second transmembrane helix, Glu54, is conserved in all of the TwRs. We investigated E54Q mutants of two TwRs and revealed that Glu54 plays critical roles in regulating the RSB pKa, oligomer formation, and the efficient photoreaction of the UV-absorbing state. The discovery of novel TwRs enables us to study the universality and individuality of the characteristics revealed so far in the original TATHIMB and contributes to further studies on mechanisms of unique properties of TwRs.

Effects of the Unique Chromophore-Protein Interactions on the Primary Photoreaction of Schizorhodopsin.

Schizorhodopsin (SzR) is a newly discovered microbial rhodopsin subfamily, functioning as an unusual inward-proton (H+) pump upon absorbing light. Two major protein structural differences around the chromophore have been found, resulting in unique chromophore-protein interactions that may be responsible for its unusual function. Therefore, it is important to elucidate how such a difference affects the primary photoreaction dynamics. We study the primary dynamics of SzR and its C75S mutant by femtosecond time-resolved absorption (TA) spectroscopy. The obtained TA data revealed that the photoisomerization in SzR proceeds more slowly and less efficiently than typical outward H+-pumping rhodopsins and that it further slows in the C75S mutant. We performed impulsive stimulated Raman measurements to clarify the effect of the cysteine residue on the retinal chromophore and found that interactions with Cys75 flatten the retinal chromophore of wild-type SzR. We discuss the effect of the unique chromophore-cysteine interaction on the retinal isomerization dynamics and structure of SzR.

Structural basis for ion selectivity in potassium-selective channelrhodopsins.

KCR channelrhodopsins (K+-selective light-gated ion channels) have received attention as potential inhibitory optogenetic tools but more broadly pose a fundamental mystery regarding how their K+ selectivity is achieved. Here, we present 2.5-2.7 Å cryo-electron microscopy structures of HcKCR1 and HcKCR2 and of a structure-guided mutant with enhanced K+ selectivity. Structural, electrophysiological, computational, spectroscopic, and biochemical analyses reveal a distinctive mechanism for K+ selectivity; rather than forming the symmetrical filter of canonical K+ channels achieving both selectivity and dehydration, instead, three extracellular-vestibule residues within each monomer form a flexible asymmetric selectivity gate, while a distinct dehydration pathway extends intracellularly. Structural comparisons reveal a retinal-binding pocket that induces retinal rotation (accounting for HcKCR1/HcKCR2 spectral differences), and design of corresponding KCR variants with increased K+ selectivity (KALI-1/KALI-2) provides key advantages for optogenetic inhibition in vitro and in vivo. Thus, discovery of a mechanism for ion-channel K+ selectivity also provides a framework for next-generation optogenetics.

A light-gated cation channel with high reactivity to weak light.

The cryptophyte algae, Guillardia theta, possesses 46 genes that are homologous to microbial rhodopsins. Five of them are functionally light-gated cation channelrhodopsins (GtCCR1-5) that are phylogenetically distinct from chlorophyte channelrhodopsins (ChRs) such as ChR2 from Chlamydomonas reinhardtii. In this study, we report the ion channel properties of these five CCRs and compared them with ChR2 and other ChRs widely used in optogenetics. We revealed that light sensitivity varied among GtCCR1-5, in which GtCCR1-3 exhibited an apparent EC50 of 0.21-1.16 mW/mm2, similar to that of ChR2, whereas GtCCR4 and GtCCR5 possess two EC50s, one of which is significantly small (0.025 and 0.032 mW/mm2). GtCCR4 is able to trigger action potentials in high temporal resolution, similar to ChR2, but requires lower light power, when expressed in cortical neurons. Moreover, a high light-sensitive response was observed when GtCCR4 was introduced into blind retina ganglion cells of rd1, a mouse model of retinitis pigmentosa. Thus, GtCCR4 provides optogenetic neuronal activation with high light sensitivity and temporal precision.

Time-resolved detection of light-induced conformational changes of heliorhodopsin.

Heliorhodopsins (HeRs) are a new category of rhodopsins. They exist as a dimer and exhibit a characteristic inverted topology. HeRs bind all-trans-retinal as a chromophore in the dark, and its isomerization to the 13-cis form by light illumination leads to a photocyclic reaction involving several photo-intermediates: K, L, M, and O. In this study, the kinetics of conformational changes of HeR from Thermoplasmatales archaeon SG8-52-1 (TaHeR) were studied by the transient grating (TG) and circular dichroism (CD) methods. The TG method reveals that the diffusion coefficient (D) does not change until the O formation suggesting no significant conformation change at the surface of the protein during the early steps of the reaction. Subsequently, D decreases upon the O formation. Although two time constants (202 µs and 2.6 ms) are observed for the conversion from the M to O by the absorption detection, D decreases only at the first step (202 µs). Light-induced unfolding of helical structure is detected by the CD method. To examine the contribution of a characteristic helix in the intracellular loop 1 (ICL1 helix), Tyr93 on the ICL1 helix was replaced by Gly (Y93G), and the reaction of this mutant was also investigated. It was found that this replacement partially suppresses the D-change, although the CD-change is almost the same as that of the wild type. These results are interpreted in terms of different sensitivities of TG and CD methods, that is, D is sensitive to the structure of the solvent-exposed surface and selectively observes the conformational change in the ICL1 region. It is suggested that the structure of hydrophilic residues in the ICL1 helix is changed during this process.

Converting a Natural-Light-Driven Outward Proton Pump Rhodopsin into an Artificial Inward Proton Pump.

Microbial rhodopsins are a large family of photoreceptive membrane proteins with diverse light-regulated functions. While the most ubiquitous microbial rhodopsins are light-driven outward proton (H+) pumps, new subfamilies of microbial rhodopsins transporting H+ inwardly, i.e., light-driven inward H+ pumps, have been discovered recently. Although structural and spectroscopic studies provide insights into their ion transport mechanisms, the minimum key element(s) that determine the direction of H+ transport have not yet been clarified. Here, we conducted the first functional conversion study by substituting key amino acids in a natural outward H+-pumping rhodopsin (PspR) with those in inward H+-pumping rhodopsins. Consequently, an artificial inward H+ pump was constructed by mutating only three residues of PspR. This result indicates that these residues govern the key processes that discriminate between outward and inward H+ pumps. Spectroscopic studies revealed the presence of an inward H+-accepting residue in the H+ transport pathway and direct H+ uptake from the extracellular solvent. This finding of the simple element for determining H+ transport would provide a new basis for understanding the concept of ion transport not only by microbial rhodopsins but also by other ion-pumping proteins.

Phototrophy by antenna-containing rhodopsin pumps in aquatic environments.

Energy transfer from light-harvesting ketocarotenoids to the light-driven proton pump xanthorhodopsins has been previously demonstrated in two unique cases: an extreme halophilic bacterium1 and a terrestrial cyanobacterium2. Attempts to find carotenoids that bind and transfer energy to abundant rhodopsin proton pumps3 from marine photoheterotrophs have thus far failed4-6. Here we detected light energy transfer from the widespread hydroxylated carotenoids zeaxanthin and lutein to the retinal moiety of xanthorhodopsins and proteorhodopsins using functional metagenomics combined with chromophore extraction from the environment. The light-harvesting carotenoids transfer up to 42% of the harvested energy in the violet- or blue-light range to the green-light absorbing retinal chromophore. Our data suggest that these antennas may have a substantial effect on rhodopsin phototrophy in the world's lakes, seas and oceans. However, the functional implications of our findings are yet to be discovered.

Biophysical characterization of microbial rhodopsins with DSE motif.

Microbial rhodopsins are photoreceptive transmembrane proteins that transport ions or regulate other intracellular biological processes. Recent genomic and metagenomic analyses found many microbial rhodopsins with unique sequences distinct from known ones. Functional characterization of these new types of microbial rhodopsins is expected to expand our understanding of their physiological roles. Here, we found microbial rhodopsins having a DSE motif in the third transmembrane helix from members of the Actinobacteria. Although the expressed proteins exhibited blue-green light absorption, either no or extremely small outward H+ pump activity was observed. The turnover rate of the photocycle reaction of the purified proteins was extremely slow compared to typical H+ pumps, suggesting these rhodopsins would work as photosensors or H+ pumps whose activities are enhanced by an unknown regulatory system in the hosts. The discovery of this rhodopsin group with the unique motif and functionality expands our understanding of the biological role of microbial rhodopsins.

Proton-transporting heliorhodopsins from marine giant viruses.

Rhodopsins convert light into signals and energy in animals and microbes. Heliorhodopsins (HeRs), a recently discovered new rhodopsin family, are widely present in archaea, bacteria, unicellular eukaryotes, and giant viruses, but their function remains unknown. Here, we report that a viral HeR from Emiliania huxleyi virus 202 (V2HeR3) is a light-activated proton transporter. V2HeR3 absorbs blue-green light, and the active intermediate contains the deprotonated retinal Schiff base. Site-directed mutagenesis study revealed that E191 in TM6 constitutes the gate together with the retinal Schiff base. E205 and E215 form a PAG of the Schiff base, and mutations at these positions converted the protein into an outward proton pump. Three environmental viral HeRs from the same group as well as a more distantly related HeR exhibited similar proton-transport activity, indicating that HeR functions might be diverse similarly to type-1 microbial rhodopsins. Some strains of E. huxleyi contain one HeR that is related to the viral HeRs, while its viruses EhV-201 and EhV-202 contain two and three HeRs, respectively. Except for V2HeR3 from EhV-202, none of these proteins exhibit ion transport activity. Thus, when expressed in the E. huxleyi cell membranes, only V2HeR3 has the potential to depolarize the host cells by light, possibly to overcome the host defense mechanisms or to prevent superinfection. The neuronal activity generated by V2HeR3 suggests that it can potentially be used as an optogenetic tool, similarly to type-1 microbial rhodopsins.

Kinetic study on the molecular mechanism of light-driven inward proton transport by schizorhodopsins.

Schizorhodopsins (SzRs) are light-driven inward proton pumping membrane proteins. A H+ is released to the cytoplasmic solvent from the chromophore, retinal Schiff base (RSB), after light absorption, and then another H+ is bound to the RSB at the end of photocyclic reaction. However, the mechanistic detail of H+ transfers in SzR is almost unknown. Here we studied the deuterium isotope effect and the temperature dependence of the reaction rate constants of elementary steps in the photocycles of SzRs. The former indicated that deprotonation and reprotonation of RSB is mainly accomplished by H+ hopping between heavy atoms with similar H+ affinity. Furthermore, the temperature dependence of the rate constants revealed that most of H+ transfer events have a high entropy barrier. In contrast, the activation enthalpy and entropy of extremely thermostable SzR (MsSzR) are significantly higher than other types of SzRs (SzR1 and MtSzR) suggesting that its highly thermostable structure is optimized with at the cost of slower reaction rates at ambient temperatures.

Rhodopsin-bestrophin fusion proteins from unicellular algae form gigantic pentameric ion channels.

Many organisms sense light using rhodopsins, photoreceptive proteins containing a retinal chromophore. Here we report the discovery, structure and biophysical characterization of bestrhodopsins, a microbial rhodopsin subfamily from marine unicellular algae, in which one rhodopsin domain of eight transmembrane helices or, more often, two such domains in tandem, are C-terminally fused to a bestrophin channel. Cryo-EM analysis of a rhodopsin-rhodopsin-bestrophin fusion revealed that it forms a pentameric megacomplex (~700 kDa) with five rhodopsin pseudodimers surrounding the channel in the center. Bestrhodopsins are metastable and undergo photoconversion between red- and green-absorbing or green- and UVA-absorbing forms in the different variants. The retinal chromophore, in a unique binding pocket, photoisomerizes from all-trans to 11-cis form. Heterologously expressed bestrhodopsin behaves as a light-modulated anion channel.

Saccharibacteria harness light energy using type-1 rhodopsins that may rely on retinal sourced from microbial hosts.

Microbial rhodopsins are a family of photoreceptive membrane proteins with a wide distribution across the Tree of Life. Within the candidate phyla radiation (CPR), a diverse group of putatively episymbiotic bacteria, the genetic potential to produce rhodopsins appears to be confined to a small clade of organisms from sunlit environments. Here, we characterize the metabolic context and biophysical features of Saccharibacteria Type-1 rhodopsin sequences derived from metagenomic surveys and show that these proteins function as outward proton pumps. This provides one of the only known mechanisms by which CPR can generate a proton gradient for ATP synthesis. These Saccharibacteria do not encode the genetic machinery to produce all-trans-retinal, the chromophore essential for rhodopsin function, but their rhodopsins are able to rapidly uptake this cofactor when provided in experimental assays. We found consistent evidence for the capacity to produce retinal from ß-carotene in microorganisms co-occurring with Saccharibacteria, and this genetic potential was dominated by members of the Actinobacteria, which are known hosts of Saccharibacteria in other habitats. If Actinobacteria serve as hosts for Saccharibacteria in freshwater environments, exchange of retinal for use by rhodopsin may be a feature of their associations.

Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine.

ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.

Structural characterization of proton-pumping rhodopsin lacking a cytoplasmic proton donor residue by X-ray crystallography.

DTG/DTS rhodopsin, which was named based on a three-residue motif (DTG or DTS) that is important for its function, is a light-driven proton-pumping microbial rhodopsin using a retinal chromophore. In contrast to other light-driven ion-pumping rhodopsins, DTG/DTS rhodopsin does not have a cytoplasmic proton donor residue, such as Asp, Glu, or Lys. Because of the lack of cytoplasmic proton donor residue, proton directly binds to the retinal chromophore from the cytoplasmic solvent. However, mutational experiments that showed the complicated effects of mutations were not able to clarify the roles played by each residue, and the detail of proton uptake pathway is unclear because of the lack of structural information. To understand the proton transport mechanism of DTG/DTS rhodopsin, here we report the three-dimensional structure of one of the DTG/DTS rhodopsins, PspR from Pseudomonas putida, by X-ray crystallography. We show that the structure of the cytoplasmic side of the protein is significantly different from that of bacteriorhodopsin, the best-characterized proton-pumping rhodopsin, and large cytoplasmic cavities were observed. We propose that these hydrophilic cytoplasmic cavities enable direct proton uptake from the cytoplasmic solvent without the need for a specialized cytoplasmic donor residue. The introduction of carboxylic residues homologous to the cytoplasmic donors in other proton-pumping rhodopsins resulted in higher pumping activity with less pH dependence, suggesting that DTG/DTS rhodopsins are advantageous for producing energy and avoiding intracellular alkalization in soil and plant-associated bacteria.

Ion Transport Activity Assay for Microbial Rhodopsin Expressed in Escherichia coli Cells.

Microbial rhodopsins have diverse functions, including roles as light-driven ion pumps, light-gated ion channels, photosensors, and light-regulated enzymes. As the number of rhodopsin-like genes identified has increased in recent years, so has the requirement for rapid identification of their functions. The patch-clamp method is often used to investigate the ion transport mechanism of microbial rhodopsins in mammalian cells; however, this requires a dedicated system and advanced techniques. The ion transport assay using the Escherichia coli expression system described here evaluates the ion transport capacity by monitoring the pH change in E. coli suspensions; if the target rhodopsin has a light-dependent ion transport activity, a light-dependent pH change is observed. The pH increase or decrease corresponds to proton release from the cell or proton uptake into the cell, respectively. This method can be used to evaluate ion transport capacity in a high-throughput manner using a combination of general-purpose equipment and common techniques. Graphic abstract: Schematic diagram of the ion transport assay in rhodopsin-expressing E. coli cells.

Crystal structure of schizorhodopsin reveals mechanism of inward proton pumping.

Schizorhodopsins (SzRs), a new rhodopsin family identified in Asgard archaea, are phylogenetically located at an intermediate position between type-1 microbial rhodopsins and heliorhodopsins. SzRs work as light-driven inward H+ pumps as xenorhodopsins in bacteria. Although E81 plays an essential role in inward H+ release, the H+ is not metastably trapped in such a putative H+ acceptor, unlike the other H+ pumps. It remains elusive why SzR exhibits different kinetic behaviors in H+ release. Here, we report the crystal structure of SzR AM_5_00977 at 2.1 Å resolution. The SzR structure superimposes well on that of bacteriorhodopsin rather than heliorhodopsin, suggesting that SzRs are classified with type-1 rhodopsins. The structure-based mutagenesis study demonstrated that the residues N100 and V103 around the ß-ionone ring are essential for color tuning in SzRs. The cytoplasmic parts of transmembrane helices 2, 6, and 7 are shorter than those in the other microbial rhodopsins, and thus E81 is located near the cytosol and easily exposed to the solvent by light-induced structural change. We propose a model of untrapped inward H+ release; H+ is released through the water-mediated transport network from the retinal Schiff base to the cytosol by the side of E81. Moreover, most residues on the H+ transport pathway are not conserved between SzRs and xenorhodopsins, suggesting that they have entirely different inward H+ release mechanisms.

Exploration of natural red-shifted rhodopsins using a machine learning-based Bayesian experimental design.

Microbial rhodopsins are photoreceptive membrane proteins, which are used as molecular tools in optogenetics. Here, a machine learning (ML)-based experimental design method is introduced for screening rhodopsins that are likely to be red-shifted from representative rhodopsins in the same subfamily. Among 3,022 ion-pumping rhodopsins that were suggested by a protein BLAST search in several protein databases, the ML-based method selected 65 candidate rhodopsins. The wavelengths of 39 of them were able to be experimentally determined by expressing proteins with the Escherichia coli system, and 32 (82%, p = 7.025 × 10-5) actually showed red-shift gains. In addition, four showed red-shift gains >20 nm, and two were found to have desirable ion-transporting properties, indicating that they would be potentially useful in optogenetics. These findings suggest that data-driven ML-based approaches play effective roles in the experimental design of rhodopsin and other photobiological studies. (141/150 words).

Expression analysis of microbial rhodopsin-like genes in Guillardia theta.

The Cryptomonad Guillardia theta has 42 genes encoding microbial rhodopsin-like proteins in their genomes. Light-driven ion-pump activity has been reported for some rhodopsins based on heterologous E. coli or mammalian cell expression systems. However, neither their physiological roles nor the expression of those genes in native cells are known. To reveal their physiological roles, we investigated the expression patterns of these genes under various growth conditions. Nitrogen (N) deficiency induced color change in exponentially growing G. theta cells from brown to green. The 29 rhodopsin-like genes were expressed in native cells. We found that the expression of 6 genes was induced under N depletion, while that of another 6 genes was reduced under N depletion.

Schizorhodopsins: A family of rhodopsins from Asgard archaea that function as light-driven inward H+ pumps.

Schizorhodopsins (SzRs), a rhodopsin family first identified in Asgard archaea, the archaeal group closest to eukaryotes, are present at a phylogenetically intermediate position between typical microbial rhodopsins and heliorhodopsins. However, the biological function and molecular properties of SzRs have not been reported. Here, SzRs from Asgardarchaeota and from a yet unknown microorganism are expressed in Escherichia coli and mammalian cells, and ion transport assays and patch clamp analyses are used to demonstrate SzR as a novel type of light-driven inward H+ pump. The mutation of a cytoplasmic glutamate inhibited inward H+ transport, suggesting that it functions as a cytoplasmic H+ acceptor. The function, trimeric structure, and H+ transport mechanism of SzR are similar to that of xenorhodopsin (XeR), a light-driven inward H+ pumping microbial rhodopsins, implying that they evolved convergently. The inward H+ pump function of SzR provides new insight into the photobiological life cycle of the Asgardarchaeota.