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.

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.

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.

High-performance optical control of GPCR signaling by bistable animal opsins MosOpn3 and LamPP in a molecular property-dependent manner.

Optical control of G protein-coupled receptor (GPCR) signaling is a highly valuable approach for comprehensive understanding of GPCR-based physiologies and controlling them precisely. However, optogenetics for GPCR signaling is still developing and requires effective and versatile tools with performance evaluation from their molecular properties. Here, we systematically investigated performance of two bistable opsins that activate Gi/Go-type G protein (mosquito Opn3 (MosOpn3) and lamprey parapinopsin (LamPP)) in optical control in vivo using Caenorhabditis elegans. Transgenic worms expressing MosOpn3, which binds 13-cis retinal to form photopigments, in nociceptor neurons showed light-induced avoidance responses in the presence of all-trans retinal, a retinal isomer ubiquitously present in every tissue, like microbial rhodopsins and unlike canonical vertebrate opsins. Remarkably, transgenic worms expressing MosOpn3 were ~7,000 times more sensitive to light than transgenic worms expressing ChR2 in this light-induced behavior, demonstrating the advantage of MosOpn3 as a light switch. LamPP is a UV-sensitive bistable opsin having complete photoregenerative ability by green light. Accordingly, transgenic worms expressing LamPP in cholinergic motor neurons stopped moving upon violet light illumination and restored coordinate movement upon green light illumination, demonstrating color-dependent control of behavior using LamPP. Furthermore, we applied molecular engineering to produce MosOpn3-based tools enabling light-dependent upregulation of cAMP or Ca2+ levels and LamPP-based tool enabling clamping cAMP levels color dependently and context independently, extending their usability. These findings define the capacity of two bistable opsins with similar retinal requirement as ChR2, providing numerous strategies for optical control of various GPCR-based physiologies as well as GPCR signaling itself.

Direct inhibition of human APOBEC3 deaminases by HIV-1 Vif independent of the proteolysis pathway.

HIV-1 Vif is known to counteract the antiviral activity of human apolipoprotein B mRNA-editing catalytic polypeptide-like (A3), a cytidine deaminase, in various ways. However, the precise mechanism behind this interaction has remained elusive. Within infected cells, Vif forms a complex called VßBCC, comprising CBFß and the components of E3 ubiquitin ligase, Elongin B, Elongin C, and Cullin5. Together with the ubiquitin-conjugating enzyme, VßBCC induces ubiquitination-mediated proteasomal degradation of A3. However, Vif exhibits additional counteractive effects. In this study, we elucidate that VßBCC inhibits deamination by A3G, A3F, and A3B independently of proteasomal degradation. Surprisingly, we discovered that this inhibition for A3G is directly attributed to the interaction between VßBCC and the C-terminal domain of A3G. Previously, it was believed that Vif did not interact with the C-terminal domain. Our findings suggest that inhibiting the interaction between VßBCC and the C-terminal domain, as well as the N-terminal domain known to be targeted for ubiquitination, of A3G may be needed to prevent counteraction by Vif.

Structural Insights into the Interaction between the C-Terminal-Deleted BH3-like Motif Peptide of Hepatitis B Virus X Protein and Bcl-xL.

Hepatitis B virus X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) infection. The full-length HBx protein interacts with Bcl-xL and is involved in the HBV replication and cell death processes. The three hydrophobic residues Trp120, Leu123, and Ile127 of the HBx BH3-like motif are essential for the Bcl-xL-binding. On the other hand, various lengths of C-terminal-truncated HBx mutants are frequently detected in HCC tissues, and these mutants, rather than the full-length HBx, appear to be responsible for HCC development. Notably, the region spanning residues 1-120 of HBx [HBx(1 and 120)] has been strongly associated with an increased risk of HCC development. However, the mode of interaction between HBx(1-120) and Bcl-xL remains unclear. HBx(1-120) possesses only Trp120 among the three hydrophobic residues essential for the Bcl-xL-binding. To elucidate this interaction mode, we employed a C-terminal-deleted HBx BH3-like motif peptide composed of residues 101-120. Here, we present the NMR complex structure of Bcl-xL and HBx(101-120). Our results demonstrate that HBx(101-120) binds to Bcl-xL in a weaker manner. Considering the high expression of Bcl-xL in HCC cells, this weak interaction, in conjunction with the overexpression of Bcl-xL in HCC cells, may potentially contribute to HCC development through the interaction between C-terminal-truncated HBx and Bcl-xL.

Functional characterization of four opsins and two G alpha subtypes co-expressed in the molluscan rhabdomeric photoreceptor.

BACKGROUND: Rhabdomeric photoreceptors of eyes in the terrestrial slug Limax are the typical invertebrate-type but unique in that three visual opsins (Gq-coupled rhodopsin, xenopsin, Opn5A) and one retinochrome, all belonging to different groups, are co-expressed. However, molecular properties including spectral sensitivity and G protein selectivity of any of them are not determined, which prevents us from understanding an advantage of multiplicity of opsin properties in a single rhabdomeric photoreceptor. To gain insight into the functional role of the co-expression of multiple opsin species in a photoreceptor, we investigated the molecular properties of the visual opsins in the present study. RESULTS: First, we found that the fourth member of visual opsins, Opn5B, is also co-expressed in the rhabdomere of the photoreceptor together with previously identified three opsins. The photoreceptors were also demonstrated to express Gq and Go alpha subunits. We then determined the spectral sensitivity of the four visual opsins using biochemical and spectroscopic methods. Gq-coupled rhodopsin and xenopsin exhibit maximum sensitivity at ~ 456 and 475 nm, respectively, and Opn5A and Opn5B exhibit maximum sensitivity at ~ 500 and 470 nm, respectively, with significant UV sensitivity. Notably, in vitro experiments revealed that Go alpha was activated by all four visual opsins, in contrast to the specific activation of Gq alpha by Gq-coupled rhodopsin, suggesting that the eye photoreceptor of Limax uses complex G protein signaling pathways. CONCLUSIONS: The eye photoreceptor in Limax expresses as many as four different visual opsin species belonging to three distinct classes. The combination of opsins with different spectral sensitivities and G protein selectivities may underlie physiological properties of the ocular photoreception, such as a shift in spectral sensitivity between dark- and light-adapted states. This may be allowed by adjustment of the relative contribution of the four opsins without neural networks, enabling a simple strategy for fine-tuning of vision.

Tropical Diseases in Okinawa: Overview and Emerging Infectious Diseases.

Okinawa prefecture is at risk for emerging infectious diseases due to its subtropical climate and its location within the Indo-Pacific region. Understanding the existing vectors and infectious agents contextualizes current threats, guides treatment, and informs prevention, and may be of unique concern in the setting of complex emergencies.

Marine Envenomation in Okinawa: Overview and Treatment Concept.

Okinawa prefecture is a popular tourist destination due to its beaches and reefs. The reefs host a large variety of animals, including a number of venomous species. Because of the popularity of the reefs and marine activities, people are frequently in close contact with dangerous venomous species and, thus, are exposed to potential envenomation. Commonly encountered venomous animals throughout Okinawa include the invertebrate cone snail, sea urchin, crown-of-thorns starfish, blue-ringed octopus, box jellyfish, and fire coral. The vertebrates include the stonefish, lionfish, sea snake, and moray eel. Treatment for marine envenomation can involve first aid, hot water immersion, antivenom, supportive care, regional anesthesia, and pharmaceutical administration. Information on venomous animals, their toxins, and treatment should be well understood by prehospital care providers and physicians practicing in the prefecture.

Reversible Photoreaction of a Retinal Photoisomerase, Retinal G-Protein-Coupled Receptor RGR.

Retinal G-protein-coupled receptor (RGR) plays a crucial role in the visual system of vertebrates as a retinal photoisomerase, which isomerizes all-trans-retinal to 11-cis-retinal to maintain the photosensitivity of visual rhodopsins. Despite the previous characterization of bovine RGR, little is known about the spectral properties of RGR from other species. In addition, photoreactivity of the 11-cis-retinal-binding form remains unclear. In this study, we revealed that human and chicken RGRs form blue-absorbing pigments similar to bovine RGR. Furthermore, the spectroscopic and biochemical analyses revealed that bovine and chicken RGRs are bistable rhodopsins displaying a reversible photoreaction. These findings provide insight into the behavior of RGR as a retinal photoisomerase and aid in understanding its role in the visual system.

Difference FTIR Spectroscopy of Jumping Spider Rhodopsin-1 at 77 K.

Animal visual rhodopsins can be classified into monostable and bistable rhodopsins, which are typically found in vertebrates and invertebrates, respectively. The former example is bovine rhodopsin (BovRh), whose structures and functions have been extensively studied. On the other hand, those of bistable rhodopsins are less known, despite their importance in optogenetics. Here, low-temperature Fourier-transform infrared (FTIR) spectroscopy was applied to jumping spider rhodopsin-1 (SpiRh1) at 77 K, and the obtained light-induced spectral changes were compared with those of squid rhodopsin (SquRh) and BovRh. Although chromophore distortion of the resting state monitored by HOOP vibrations is not distinctive between invertebrate and vertebrate rhodopsins, distortion of the all-trans chromophore after photoisomerization is unique for BovRh, and the distortion was localized at the center of the chromophore in SpiRh1 and SquRh. Highly conserved aspartate (D83 in BovRh) does not change the hydrogen-bonding environment in invertebrate rhodopsins. Thus, present FTIR analysis provides specific structural changes, leading to activation of invertebrate and vertebrate rhodopsins. On the other hand, the analysis of O-D stretching vibrations in D2O revealed unique features of protein-bound water molecules. Numbers of water bands in SpiRh1 and SquRh were less and more than those in BovRh. The X-ray crystal structure of SpiRh1 observed a bridged water molecule between the protonated Schiff base and its counterion (E194), but strongly hydrogen-bonded water molecules were never detected in SpiRh1, as well as SquRh and BovRh. Thus, absence of strongly hydrogen-bonded water molecules is substantial for animal rhodopsins, which is distinctive from microbial rhodopsins.

Rhodopsins at a glance.

Rhodopsins are photoreceptive membrane proteins consisting of a common heptahelical transmembrane architecture that contains a retinal chromophore. Rhodopsin was first discovered in the animal retina in 1876, but a different type of rhodopsin, bacteriorhodopsin, was reported to be present in the cell membrane of an extreme halophilic archaeon, Halobacterium salinarum, 95 years later. Although these findings were made by physiological observation of pigmented tissue and cell bodies, recent progress in genomic and metagenomic analyses has revealed that there are more than 10,000 microbial rhodopsins and 9000 animal rhodopsins with large diversity and tremendous new functionality. In this Cell Science at a Glance article and accompanying poster, we provide an overview of the diversity of functions, structures, color discrimination mechanisms and optogenetic applications of these two rhodopsin families, and will also highlight the third distinctive rhodopsin family, heliorhodopsin.

NMR characterization of the structure of the intrinsically disordered region of human origin recognition complex subunit 1, hORC1, and of its interaction with G-quadruplex DNAs.

Human origin recognition complex (hORC) binds to the DNA replication origin and then initiates DNA replication. However, hORC does not exhibit DNA sequence-specificity and how hORC recognizes the replication origin on genomic DNA remains elusive. Previously, we found that hORC recognizes G-quadruplex structures potentially formed near the replication origin. Then, we showed that hORC subunit 1 (hORC1) preferentially binds to G-quadruplex DNAs using a hORC1 construct comprising residues 413 to 511 (hORC1413-511). Here, we investigate the structural characteristics of hORC1413-511 in its free and complex forms with G-quadruplex DNAs. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopic studies indicated that hORC1413-511 is disordered except for a short α-helical region in both the free and complex forms. NMR chemical shift perturbation (CSP) analysis suggested that basic residues, arginines and lysines, and polar residues, serines and threonines, are involved in the G-quadruplex DNA binding. Then, this was confirmed by mutation analysis. Interestingly, CSP analysis indicated that hORC1413-511 binds to both parallel- and (3 + 1)-type G-quadruplex DNAs using the same residues, and thereby in the same manner. Our study suggests that hORC1 uses its intrinsically disordered G-quadruplex binding region to recognize parallel-type and (3 + 1)-type G-quadruplex structures at replication origin.

Complex Formation of an RNA Aptamer with a Part of HIV-1 Tat through Induction of Base Triples in Living Human Cells Proven by In-Cell NMR.

An RNA aptamer that strongly binds to a target molecule has the potential to be a nucleic acid drug inside living human cells. To investigate and improve this potential, it is critical to elucidate the structure and interaction of RNA aptamers inside living cells. We examined an RNA aptamer for HIV-1 Tat (TA), which had been found to trap Tat and repress its function in living human cells. We first used in vitro NMR to examine the interaction between TA and a part of Tat containing the binding site for trans-activation response element (TAR). It was revealed that two U-A∗U base triples are formed in TA upon binding of Tat. This was assumed to be critical for strong binding. Then, TA in complex with a part of Tat was incorporated into living human cells. The presence of two U-A∗U base triples was also revealed for the complex in living human cells by in-cell NMR. Thus, the activity of TA in living human cells was rationally elucidated by in-cell NMR.

Diverse heliorhodopsins detected via functional metagenomics in freshwater Actinobacteria, Chloroflexi and Archaea.

The recently discovered rhodopsin family of heliorhodopsins (HeRs) is abundant in diverse microbial environments. So far, the functional and biological roles of HeRs remain unknown. To tackle this issue, we combined experimental and computational screens to gain some novel insights. Here, 10 readily expressed HeR genes were found using functional metagenomics on samples from two freshwater environments. These HeRs originated from diverse prokaryotic groups: Actinobacteria, Chloroflexi and Archaea. Heterologously expressed HeRs absorbed light in the green and yellow wavelengths (543-562 nm) and their photocycles exhibited diverse kinetic characteristics. To approach the physiological function of the HeRs, we used our environmental clones along with thousands of microbial genomes to analyze genes neighbouring HeRs. The strongest association was found with the DegV family involved in activation of fatty acids, which allowed us to hypothesize that HeRs might be involved in light-induced membrane lipid modifications.

Proton conduction in hydronium solvate ionic liquids affected by ligand shape.

We investigated the ligand dependence of the proton conduction of hydronium solvate ionic liquids (ILs), consisting of a hydronium ion (H3O+), polyether ligands, and a bis[(trifluoromethyl)sulfonyl]amide anion (Tf2N-; Tf = CF3SO2). The ligands were changed from previously reported 18-crown-6 (18C6) to other cyclic or acyclic polyethers, namely, dicyclohexano-18-crown-6 (Dh18C6), benzo-18-crown-6 (B18C6) and pentaethylene glycol dimethyl ether (G5). Pulsed-field gradient spin echo nuclear magnetic resonance results revealed that the protons of H3O+ move faster than those of cyclic 18C6-based ligands but as fast as those of acyclic G5 ligands. Based on these results and density functional theory calculations, we propose that the coordination of a cyclic ether ligand to the H3O+ ion is essential for fast proton conduction in hydronium solvate ILs. Our results attract special interest for many electro- and bio-chemical applications such as electrolyte systems for fuel cells and artificial ion channels for biological cells.

Optogenetic Potentials of Diverse Animal Opsins: Parapinopsin, Peropsin, LWS Bistable Opsin.

Animal opsin-based pigments are light-activated G-protein-coupled receptors (GPCRs), which drive signal transduction cascades via G-proteins. Thousands of animal opsins have been identified, and molecular phylogenetic and biochemical analyses have revealed the unexpected diversity in selectivity of G-protein activation and photochemical property. Here we discuss the optogenetic potentials of diverse animal opsins, particularly recently well-characterized three non-canonical opsins, parapinopsin, peropsin, and LWS bistable opsin. Unlike canonical opsins such as vertebrate visual opsins that have been conventionally used for optogenetic applications, these opsins are bistable; opsin-based pigments do not release the chromophore retinal after light absorption, and the stable photoproducts revert to their original dark states upon subsequent light absorption. Parapinopsins have a "complete photoregeneration ability," which allows a clear color-dependent regulation of signal transductions. On the other hand, peropsins serve as a "dark-active and light-inactivated" GPCR to regulate signal transductions in the opposite way compared with usual opsins. In addition, an LWS bistable opsin from a butterfly was revealed to be the longest wavelength-sensitive animal opsin with its absorption maximum at ~570 nm. The property-dependent optical regulations of signal transductions were demonstrated in mammalian cultured cells, showing potentials of new optogenetic tools.

Color opponency with a single kind of bistable opsin in the zebrafish pineal organ.

Lower vertebrate pineal organs discriminate UV and visible light. Such color discrimination is typically considered to arise from antagonism between two or more spectrally distinct opsins, as, e.g., human cone-based color vision relies on antagonistic relationships between signals produced by red-, green-, and blue-cone opsins. Photosensitive pineal organs contain a bistable opsin (parapinopsin) that forms a signaling-active photoproduct upon UV exposure that may itself be returned to the signaling-inactive "dark" state by longer-wavelength light. Here we show the spectrally distinct parapinopsin states (with antagonistic impacts on signaling) allow this opsin alone to provide the color sensitivity of this organ. By using calcium imaging, we show that single zebrafish pineal photoreceptors held under a background light show responses of opposite signs to UV and visible light. Both such responses are deficient in zebrafish lacking parapinopsin. Expressing a UV-sensitive cone opsin in place of parapinopsin recovers UV responses but not color opponency. Changes in the spectral composition of white light toward enhanced UV or visible wavelengths respectively increased vs. decreased calcium signal in parapinopsin-sufficient but not parapinopsin-deficient photoreceptors. These data reveal color opponency from a single kind of bistable opsin establishing an equilibrium-like mixture of the two states with different signaling abilities whose fractional concentrations are defined by the spectral composition of incident light. As vertebrate visual color opsins evolved from a bistable opsin, these findings suggest that color opponency involving a single kind of bistable opsin might have been a prototype of vertebrate color opponency.

Convergent evolution of tertiary structure in rhodopsin visual proteins from vertebrates and box jellyfish.

Box jellyfish and vertebrates are separated by >500 million years of evolution yet have structurally analogous lens eyes that employ rhodopsin photopigments for vision. All opsins possess a negatively charged residue-the counterion-to maintain visible-light sensitivity and facilitate photoisomerization of their retinaldehyde chromophore. In vertebrate rhodopsins, the molecular evolution of the counterion position-from a highly conserved distal location in the second extracellular loop (E181) to a proximal location in the third transmembrane helix (E113)-is established as a key driver of higher fidelity photoreception. Here, we use computational biology and heterologous action spectroscopy to determine whether the appearance of the advanced visual apparatus in box jellyfish was also accompanied by changes in the opsin tertiary structure. We found that the counterion in an opsin from the lens eye of the box jellyfish Carybdea rastonii (JellyOp) has also moved to a unique proximal location within the transmembrane bundle-E94 in TM2. Furthermore, we reveal that this Schiff base/counterion system includes an additional positive charge-R186-that has coevolved with E94 to functionally separate E94 and E181 in the chromophore-binding pocket of JellyOp. By engineering this pocket-neutralizing R186 and E94, or swapping E94 with the vertebrate counterion E113-we can recreate versions of the invertebrate and vertebrate counterion systems, respectively, supporting a relatively similar overall architecture in this region of animal opsins. In summary, our data establish the third only counterion site in animal opsins and reveal convergent evolution of tertiary structure in opsins from distantly related species with advanced visual systems.

Investigation of the Interaction of Human Origin Recognition Complex Subunit 1 with G-Quadruplex DNAs of Human c-myc Promoter and Telomere Regions.

Origin recognition complex (ORC) binds to replication origins in eukaryotic DNAs and plays an important role in replication. Although yeast ORC is known to sequence-specifically bind to a replication origin, how human ORC recognizes a replication origin remains unknown. Previous genome-wide studies revealed that guanine (G)-rich sequences, potentially forming G-quadruplex (G4) structures, are present in most replication origins in human cells. We previously suggested that the region comprising residues 413-511 of human ORC subunit 1, hORC1413-511, binds preferentially to G-rich DNAs, which form a G4 structure in the absence of hORC1413-511. Here, we investigated the interaction of hORC1413-511 with various G-rich DNAs derived from human c-myc promoter and telomere regions. Fluorescence anisotropy revealed that hORC1413-511 binds preferentially to DNAs that have G4 structures over ones having double-stranded structures. Importantly, circular dichroism (CD) and nuclear magnetic resonance (NMR) showed that those G-rich DNAs retain the G4 structures even after binding with hORC1413-511. NMR chemical shift perturbation analyses revealed that the external G-tetrad planes of the G4 structures are the primary binding sites for hORC1413-511. The present study suggests that human ORC1 may recognize replication origins through the G4 structure.