Identification of Phosphorylation Codes for Arrestin Recruitment by G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) mediate diverse signaling in part through interaction with arrestins, whose binding promotes receptor internalization and signaling through G protein-independent pathways. High-affinity arrestin binding requires receptor phosphorylation, often at the receptor's C-terminal tail. Here, we report an X-ray free electron laser (XFEL) crystal structure of the rhodopsin-arrestin complex, in which the phosphorylated C terminus of rhodopsin forms an extended intermolecular ß sheet with the N-terminal ß strands of arrestin. Phosphorylation was detected at rhodopsin C-terminal tail residues T336 and S338. These two phospho-residues, together with E341, form an extensive network of electrostatic interactions with three positively charged pockets in arrestin in a mode that resembles binding of the phosphorylated vasopressin-2 receptor tail to ß-arrestin-1. Based on these observations, we derived and validated a set of phosphorylation codes that serve as a common mechanism for phosphorylation-dependent recruitment of arrestins by GPCRs.

Unconventional Roles of Opsins.

Rhodopsin is the classical light sensor. Although rhodopsin has long been known to be important for image formation in the eye, the requirements for opsins in non-image formation and in extraocular light sensation were revealed much later. Most recent is the demonstration that an opsin in the fruit fly, Drosophila melanogaster, is expressed in pacemaker neurons in the brain and functions in light entrainment of circadian rhythms. However, the biggest surprise is that opsins have light-independent roles, countering more than a century of dogma that they function exclusively as light sensors. Through studies in Drosophila, light-independent roles of opsins have emerged in temperature sensation and hearing. Although these findings have been uncovered in the fruit fly, there are hints that opsins have light-independent roles in a wide array of animals, including mammals. Thus, despite the decades of focus on opsins as light detectors, they represent an important new class of polymodal sensory receptor.

Structural view of G protein-coupled receptor signaling in the retinal rod outer segment.

Visual phototransduction is the most extensively studied G protein-coupled receptor (GPCR) signaling pathway because of its quantifiable stimulus, non-redundancy of genes, and immense importance in vision. We summarize recent discoveries that have advanced our understanding of rod outer segment (ROS) morphology and the pathological basis of retinal diseases. We have combined recently published cryo-electron tomography (cryo-ET) data on the ROS with structural knowledge on individual proteins to define the precise spatial limitations under which phototransduction occurs. Although hypothetical, the reconstruction of the rod phototransduction system highlights the potential roles of phosphodiesterase 6 (PDE6) and guanylate cyclases (GCs) in maintaining the spacing between ROS discs, suggesting a plausible mechanism by which intrinsic optical signals are generated in the retina.

Functional opsin patterning for Drosophila color vision is established through signaling pathways in adjacent object-detection neurons.

Vision is mainly based on two different tasks, object detection and color discrimination, carried out by photoreceptor (PR) cells. The Drosophila compound eye consists of ∼800 ommatidia. Every ommatidium contains eight PR cells, six outer cells (R1-R6) and two inner cells (R7 and R8), by which object detection and color vision are achieved, respectively. Expression of opsin genes in R7 and R8 is highly coordinated through the instructive signal from R7 to R8, and two major ommatidial subtypes are distributed stochastically; pale type expresses Rh3/Rh5 and yellow type expresses Rh4/Rh6 in R7/R8. The homeodomain protein Defective proventriculus (Dve) is expressed in yellow-type R7 and in six outer PRs, and it is involved in Rh3 repression to specify the yellow-type R7. dve mutant eyes exhibited atypical coupling, Rh3/Rh6 and Rh4/Rh5, indicating that Dve activity is required for proper opsin coupling. Surprisingly, Dve activity in R1 is required for the instructive signal, whereas activity in R6 and R7 blocks the signal. Our results indicate that functional coupling of two different neurons is established through signaling pathways from adjacent neurons that are functionally different.

Structures of the Rhodopsin-Transducin Complex: Insights into G-Protein Activation.

Rhodopsin (Rho), a prototypical G-protein-coupled receptor (GPCR) in vertebrate vision, activates the G-protein transducin (GT) by catalyzing GDP-GTP exchange on its α subunit (GαT). To elucidate the determinants of GT coupling and activation, we obtained cryo-EM structures of a fully functional, light-activated Rho-GT complex in the presence and absence of a G-protein-stabilizing nanobody. The structures illustrate how GT overcomes its low basal activity by engaging activated Rho in a conformation distinct from other GPCR-G-protein complexes. Moreover, the nanobody-free structures reveal native conformations of G-protein components and capture three distinct conformers showing the GαT helical domain (αHD) contacting the Gßγ subunits. These findings uncover the molecular underpinnings of G-protein activation by visual rhodopsin and shed new light on the role played by Gßγ during receptor-catalyzed nucleotide exchange.

Engineering bionanoreactor in bacteria for efficient hydrogen production.

Hydrogen production through water splitting is a vital strategy for renewable and sustainable clean energy. In this study, we developed an approach integrating nanomaterial engineering and synthetic biology to establish a bionanoreactor system for efficient hydrogen production. The periplasmic space (20 to 30 nm) of an electroactive bacterium, Shewanella oneidensis MR-1, was engineered to serve as a bionanoreactor to enhance the interaction between electrons and protons, catalyzed by hydrogenases for hydrogen generation. To optimize electron transfer, we used the microbially reduced graphene oxide (rGO) to coat the electrode, which improved the electron transfer from the electrode to the cells. Native MtrCAB protein complex on S. oneidensis and self-assembled iron sulfide (FeS) nanoparticles acted in tandem to facilitate electron transfer from an electrode to the periplasm. To enhance proton transport, S. oneidensis MR-1 was engineered to express Gloeobacter rhodopsin (GR) and the light-harvesting antenna canthaxanthin. This led to efficient proton pumping when exposed to light, resulting in a 35.6% increase in the rate of hydrogen production. The overexpression of native [FeFe]-hydrogenase further improved the hydrogen production rate by 56.8%. The bionanoreactor engineered in S. oneidensis MR-1 achieved a hydrogen yield of 80.4 µmol/mg protein/day with a Faraday efficiency of 80% at a potential of -0.75 V. This periplasmic bionanoreactor combines the strengths of both nanomaterial and biological components, providing an efficient approach for microbial electrosynthesis.

Dark continuous noise from mutant G90D-rhodopsin predominantly underlies congenital stationary night blindness.

Congenital stationary night blindness (CSNB) is an inherited retinal disease that causes a profound loss of rod sensitivity without severe retinal degeneration. One well-studied rhodopsin point mutant, G90D-Rho, is thought to cause CSNB because of its constitutive activity in darkness causing rod desensitization. However, the nature of this constitutive activity and its precise molecular source have not been resolved for almost 30 y. In this study, we made a knock-in (KI) mouse line with a very low expression of G90D-Rho (equal in amount to ~0.1% of normal rhodopsin, WT-Rho, in WT rods), with the remaining WT-Rho replaced by REY-Rho, a mutant with a very low efficiency of activating transducin due to a charge reversal of the highly conserved ERY motif to REY. We observed two kinds of constitutive noise: one being spontaneous isomerization (R*) of G90D-Rho at a molecular rate (R* s-1) 175-fold higher than WT-Rho and the other being G90D-Rho-generated dark continuous noise comprising low-amplitude unitary events occurring at a very high molecular rate equivalent in effect to ~40,000-fold of R* s-1 from WT-Rho. Neither noise type originated from G90D-Opsin because exogenous 11-cis-retinal had no effect. Extrapolating the above observations at low (0.1%) expression of G90D-Rho to normal disease exhibited by a KI mouse model with RhoG90D/WTand RhoG90D/G90D genotypes predicts the disease condition very well quantitatively. Overall, the continuous noise from G90D-Rho therefore predominates, constituting the major equivalent background light causing rod desensitization in CSNB.

Multiple retinal isomerizations during the early phase of the bestrhodopsin photoreaction.

Bestrhodopsins constitute a class of light-regulated pentameric ion channels that consist of one or two rhodopsins in tandem fused with bestrophin ion channel domains. Here, we report on the isomerization dynamics in the rhodopsin tandem domains of Phaeocystis antarctica bestrhodopsin, which binds all-trans retinal Schiff-base (RSB) absorbing at 661 nm and, upon illumination, converts to the meta-stable P540 state with an unusual 11-cis RSB. The primary photoproduct P682 corresponds to a mixture of highly distorted 11-cis and 13-cis RSB directly formed from the excited state in 1.4 ps. P673 evolves from P682 in 500 ps and contains highly distorted 13-cis RSB, indicating that the 11-cis fraction in P682 converts to 13-cis. Next, P673 establishes an equilibrium with P595 in 1.2 µs, during which RSB converts to 11-cis and then further proceeds to P560 in 48 µs and P540 in 1.0 ms while remaining 11-cis. Hence, extensive isomeric switching occurs on the early ground state potential energy surface (PES) on the hundreds of ps to µs timescale before finally settling on a metastable 11-cis photoproduct. We propose that P682 and P673 are trapped high up on the ground-state PES after passing through either of two closely located conical intersections that result in 11-cis and 13-cis RSB. Co-rotation of C11=C12 and C13=C14 bonds results in a constricted conformational landscape that allows thermal switching between 11-cis and 13-cis species of highly strained RSB chromophores. Protein relaxation may release RSB strain, allowing it to evolve to a stable 11-cis isomeric configuration in microseconds.

Stress resilience-enhancing drugs preserve tissue structure and function in degenerating retina via phosphodiesterase inhibition.

Chronic, progressive retinal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa, arise from genetic and environmental perturbations of cellular and tissue homeostasis. These disruptions accumulate with repeated exposures to stress over time, leading to progressive visual impairment and, in many cases, legal blindness. Despite decades of research, therapeutic options for the millions of patients suffering from these disorders remain severely limited, especially for treating earlier stages of pathogenesis when the opportunity to preserve the retinal structure and visual function is greatest. To address this urgent, unmet medical need, we employed a systems pharmacology platform for therapeutic development. Through integrative single-cell transcriptomics, proteomics, and phosphoproteomics, we identified universal molecular mechanisms across distinct models of age-related and inherited retinal degenerations, characterized by impaired physiological resilience to stress. Here, we report that selective, targeted pharmacological inhibition of cyclic nucleotide phosphodiesterases (PDEs), which serve as critical regulatory nodes that modulate intracellular second messenger signaling pathways, stabilized the transcriptome, proteome, and phosphoproteome through downstream activation of protective mechanisms coupled with synergistic inhibition of degenerative processes. This therapeutic intervention enhanced resilience to acute and chronic forms of stress in the degenerating retina, thus preserving tissue structure and function across various models of age-related and inherited retinal disease. Taken together, these findings exemplify a systems pharmacology approach to drug discovery and development, revealing a new class of therapeutics with potential clinical utility in the treatment or prevention of the most common causes of blindness.

Whale shark rhodopsin adapted to deep-sea lifestyle by a substitution associated with human disease.

Spectral tuning of visual pigments often facilitates adaptation to new environments, and it is intriguing to study the visual ecology of pelagic sharks with secondarily expanded habitats. The whale shark, which dives into the deep sea of nearly 2,000 meters besides near-surface filter feeding, was previously shown to possess the 'blue-shifted' rhodopsin (RHO), which is a signature of deep-sea adaptation. In this study, our spectroscopy of recombinant whale shark RHO mutants revealed that this blue shift is caused dominantly by an unprecedented spectral tuning site 94. In humans, the mutation at the site causes congenital stationary night blindness (CSNB) by reducing the thermal stability of RHO. Similarly, the RHO of deep-diving whale shark has reduced thermal stability, which was experimentally shown to be achieved by site 178 and 94. RHOs having the natural substitution at site 94 are also found in some Antarctic fishes, suggesting that the blue shift by the substitution at the CSNB site associated with the reduction in thermal stability might be allowed in cold-water deep-sea habitats.

Widespread use of proton-pumping rhodopsin in Antarctic phytoplankton.

Photosynthetic carbon (C) fixation by phytoplankton in the Southern Ocean (SO) plays a critical role in regulating air-sea exchange of carbon dioxide and thus global climate. In the SO, photosynthesis (PS) is often constrained by low iron, low temperatures, and low but highly variable light intensities. Recently, proton-pumping rhodopsins (PPRs) were identified in marine phytoplankton, providing an alternate iron-free, light-driven source of cellular energy. These proteins pump protons across cellular membranes through light absorption by the chromophore retinal, and the resulting pH energy gradient can then be used for active membrane transport or for synthesis of adenosine triphosphate. Here, we show that PPR is pervasive in Antarctic phytoplankton, especially in iron-limited regions. In a model SO diatom, we found that it was localized to the vacuolar membrane, making the vacuole a putative alternative phototrophic organelle for light-driven production of cellular energy. Unlike photosynthetic C fixation, which decreases substantially at colder temperatures, the proton transport activity of PPR was unaffected by decreasing temperature. Cellular PPR levels in cultured SO diatoms increased with decreasing iron concentrations and energy production from PPR photochemistry could substantially augment that of PS, especially under high light intensities, where PS is often photoinhibited. PPR gene expression and high retinal concentrations in phytoplankton in SO waters support its widespread use in polar environments. PPRs are an important adaptation of SO phytoplankton to growth and survival in their cold, iron-limited, and variable light environment.

Ultrafast spectra and kinetics of human green-cone visual pigment at room temperature.

Rhodopsin is the pigment that enables night vision, whereas cone opsins are the pigments responsible for color vision in bright-light conditions. Despite their importance for vision, cone opsins are poorly characterized at the molecular level compared to rhodopsin. Spectra and kinetics of the intermediate states of human green-cone visual pigment (mid-wavelength sensitive, or MWS opsin) were measured and compared with the intermediates and kinetics of bovine rhodopsin. All the major intermediates of the MWS opsin were recorded in the picosecond to millisecond time range. Several intermediates in MWS opsin appear to have characteristics similar to the intermediates of bovine rhodopsin; however, there are some marked differences. One of the most striking differences is in their kinetics, where the kinetics of the MWS opsin intermediates are slower compared to those of the bovine rhodopsin intermediates.

Deconstructing the transmembrane core of class A G protein-coupled receptors.

Class A G protein-coupled receptors have evolved to recognize ligands ranging from small-molecule odorants to proteins. Although they are among the most diverse membrane receptors in eukaryotic organisms, they possess a highly conserved core within their seven-transmembrane helix framework. The conservation of the transmembrane core has led to the idea of a common mechanism by which ligand binding is coupled to the outward rotation of helix H6, the hallmark of an active receptor. Nevertheless, there is still no consensus on the mechanism of coupling or on the roles of specific residues within the core. Recent insights from crystallography and NMR spectroscopy provide a way to decompose the core into its essential structural and functional elements that shed new light on this important region.

Eyeless razor clam Sinonovacula constricta discriminates light spectra through opsins to guide Ca2+ and cAMP signaling pathways.

Phototransduction is based on opsins that drive distinct types of Gα cascades. Although nonvisual photosensitivity has long been known in marine bivalves, the underlying molecular basis and phototransduction mechanism are poorly understood. Here, we introduced the eyeless razor clam Sinonovacula constricta as a model to clarify this issue. First, we showed that S. constricta was highly diverse in opsin family members, with a significant expansion in xenopsins. Second, the expression of putative S. constricta opsins was highly temporal-spatio specific, indicating their potential roles in S. constricta development and its peripheral photosensitivity. Third, by cloning four S. constricta opsins with relatively higher expression (Sc_opsin1, 5, 7, and 12), we found that they exhibited different expression levels in response to different light environments. Moreover, we demonstrated that these opsins (excluding Sc_opsin7) couple with Gαq and Gαi cascades to mediate the light-dependent Ca2+ (Sc_opsin1 and 5) and cAMP (Sc_opsin12) signaling pathways. The results indicated that Sc_opsin1 and 5 belonged to Gq-opsins, Sc_opsin12 belonged to Gi-opsins, while Sc_opsin7 might act as a photo-isomerase. Furthermore, we found that the phototransduction function of S. constricta Gq-opsins was dependent on the lysine at the seventh transmembrane domain, and greatly influenced by the external light spectra in a complementary way. Thus, a synergistic photosensitive system mediated by opsins might exist in S. constricta to rapidly respond to the transient or subtle changes of the external light environment. Collectively, our findings provide valuable insights into the evolution of opsins in marine bivalves and their potential functions in nonvisual photosensitivity.

Convergent mechanism underlying the acquisition of vertebrate scotopic vision.

High sensitivity of scotopic vision (vision in dim light conditions) is achieved by the rods' low background noise, which is attributed to a much lower thermal activation rate (kth) of rhodopsin compared with cone pigments. Frogs and nocturnal geckos uniquely possess atypical rods containing noncanonical cone pigments that exhibit low kth, mimicking rhodopsin. Here, we investigated the convergent mechanism underlying the low kth of rhodopsins and noncanonical cone pigments. Our biochemical analysis revealed that the kth of canonical cone pigments depends on their absorption maximum (λmax). However, rhodopsin and noncanonical cone pigments showed a substantially lower kth than predicted from the λmax dependency. Given that the λmax is inversely proportional to the activation energy of the pigments in the Hinshelwood distribution-based model, our findings suggest that rhodopsin and noncanonical cone pigments have convergently acquired low frequency of spontaneous-activation attempts, including thermal fluctuations of the protein moiety, in the molecular evolutionary processes from canonical cone pigments, which contributes to highly sensitive scotopic vision.

A cholesterol switch controls phospholipid scrambling by G protein-coupled receptors.

Class A G protein-coupled receptors (GPCRs), a superfamily of cell membrane signaling receptors, moonlight as constitutively active phospholipid scramblases. The plasma membrane of metazoan cells is replete with GPCRs yet has a strong resting trans-bilayer phospholipid asymmetry, with the signaling lipid phosphatidylserine confined to the cytoplasmic leaflet. To account for the persistence of this lipid asymmetry in the presence of GPCR scramblases, we hypothesized that GPCR-mediated lipid scrambling is regulated by cholesterol, a major constituent of the plasma membrane. We now present a technique whereby synthetic vesicles reconstituted with GPCRs can be supplemented with cholesterol to a level similar to that of the plasma membrane and show that the scramblase activity of two prototypical GPCRs, opsin and the ß1-adrenergic receptor, is impaired upon cholesterol loading. Our data suggest that cholesterol acts as a switch, inhibiting scrambling above a receptor-specific threshold concentration to disable GPCR scramblases at the plasma membrane.

A short story on how chromophore is hydrolyzed from rhodopsin for recycling.

The photocycle of visual opsins is essential to maintain the light sensitivity of the retina. The early physical observations of the rhodopsin photocycle by Böll and Kühne in the 1870s inspired over a century's worth of investigations on rhodopsin biochemistry. A single photon isomerizes the Schiff-base linked 11-cis-retinylidene chromophore of rhodopsin, converting it to the all-trans agonist to elicit phototransduction through photoactivated rhodopsin (Rho*). Schiff base hydrolysis of the agonist is a key step in the photocycle, not only diminishing ongoing phototransduction but also allowing for entry and binding of fresh 11-cis chromophore to regenerate the rhodopsin pigment and maintain light sensitivity. Many challenges have been encountered in measuring the rate of this hydrolysis, but recent advancements have facilitated studies of the hydrolysis within the native membrane environment of rhodopsin. These techniques can now be applied to study hydrolysis of agonist in other opsin proteins that mediate phototransduction or chromophore turnover. In this review, we discuss the progress that has been made in characterizing the rhodopsin photocycle and the journey to characterize the hydrolysis of its all-trans-retinylidene agonist.

Chromophore hydrolysis and release from photoactivated rhodopsin in native membranes.

For sustained vision, photoactivated rhodopsin (Rho*) must undergo hydrolysis and release of all-trans-retinal, producing substrate for the visual cycle and apo-opsin available for regeneration with 11-cis-retinal. The kinetics of this hydrolysis has yet to be described for rhodopsin in its native membrane environment. We developed a method consisting of simultaneous denaturation and chromophore trapping by isopropanol/borohydride, followed by exhaustive protein digestion, complete extraction, and liquid chromatography-mass spectrometry. Using our method, we tracked Rho* hydrolysis, the subsequent formation of N-retinylidene-phosphatidylethanolamine (N-ret-PE) adducts with the released all-trans-retinal, and the reduction of all-trans-retinal to all-trans-retinol. We found that hydrolysis occurred faster in native membranes than in detergent micelles typically used to study membrane proteins. The activation energy of the hydrolysis in native membranes was determined to be 17.7 ± 2.4 kcal/mol. Our data support the interpretation that metarhodopsin II, the signaling state of rhodopsin, is the primary species undergoing hydrolysis and release of its all-trans-retinal. In the absence of NADPH, free all-trans-retinal reacts with phosphatidylethanolamine (PE), forming a substantial amount of N-ret-PE (∼40% of total all-trans-retinal at physiological pH), at a rate that is an order of magnitude faster than Rho* hydrolysis. However, N-ret-PE formation was highly attenuated by NADPH-dependent reduction of all-trans-retinal to all-trans-retinol. Neither N-ret-PE formation nor all-trans-retinal reduction affected the rate of hydrolysis of Rho*. Our study provides a comprehensive picture of the hydrolysis of Rho* and the release of all-trans-retinal and its reentry into the visual cycle, a process in which alteration can lead to severe retinopathies.

Hydration-mediated G-protein-coupled receptor activation.

The Rhodopsin family of G-protein­coupled receptors (GPCRs) comprises the targets of nearly a third of all pharmaceuticals. Despite structural water present in GPCR X-ray structures, the physiological relevance of these solvent molecules to rhodopsin signaling remains unknown. Here, we show experimental results consistent with the idea that rhodopsin activation in lipid membranes is coupled to bulk water movements into the protein. To quantify hydration changes, we measured reversible shifting of the metarhodopsin equilibrium due to osmotic stress using an extensive series of polyethylene glycol (PEG) osmolytes. We discovered clear evidence that light activation entails a large influx of bulk water (∼80­100 molecules) into the protein, giving insight into GPCR activation mechanisms. Various size polymer osmolytes directly control rhodopsin activation, in which large solutes are excluded from rhodopsin and dehydrate the protein, favoring the inactive state. In contrast, small osmolytes initially forward shift the activation equilibrium until a quantifiable saturation point is reached, similar to gain-of-function protein mutations. For the limit of increasing osmolyte size, a universal response of rhodopsin to osmotic stress is observed, suggesting it adopts a dynamic, hydrated sponge-like state upon photoactivation. Our results demand a rethinking of the role of water dynamics in modulating various intermediates in the GPCR energy landscape. We propose that besides bound water, an influx of bulk water plays a necessary role in establishing the active GPCR conformation that mediates signaling.

Conformational alterations in unidirectional ion transport of a light-driven chloride pump revealed using X-ray free electron lasers.

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10-µs and 1-ms time points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied by a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.