Crystal structure of metarhodopsin II

G-protein-coupled receptors (GPCRs) are seven transmembrane helix (TM) proteins that transduce signals into living cells by binding extracellular ligands and coupling to intracellular heterotrimeric G proteins (Gαβγ). The photoreceptor rhodopsin couples to transducin and bears its ligand 11-cis-retinal covalently bound via a protonated Schiff base to the opsin apoprotein. Absorption of a photon causes retinal cis/trans isomerization and generates the agonist all-trans-retinal in situ. After early photoproducts, the active G-protein-binding intermediate metarhodopsin II (Meta?II) is formed, in which the retinal Schiff base is still intact but deprotonated. Dissociation of the proton from the Schiff base breaks a major constraint in the protein and enables further activating steps, including an outward tilt of TM6 and formation of a large cytoplasmic crevice for uptake of the interacting C terminus of the Gα subunit. Owing to Schiff base hydrolysis, Meta?II is short-lived and notoriously difficult to crystallize. We therefore soaked opsin crystals with all-trans-retinal to form Meta?II, presuming that the crystal's high concentration of opsin in an active conformation (Ops*) may facilitate all-trans-retinal uptake and Schiff base formation. Here we present the 3.0?? and 2.85?? crystal structures, respectively, of Meta?II alone or in complex with an 11-amino-acid C-terminal fragment derived from Gα (GαCT2). GαCT2 binds in a large crevice at the cytoplasmic side, akin to the binding of a similar Gα-derived peptide to Ops* (ref. 7). In the Meta?II structures, the electron density from the retinal ligand seamlessly continues into the Lys?296 side chain, reflecting proper formation of the Schiff base linkage. The retinal is in a relaxed conformation and almost undistorted compared with pure crystalline all-trans-retinal. By comparison with early photoproducts we propose how retinal translocation and rotation induce the gross conformational changes characteristic for Meta?II. The structures can now serve as models for the large GPCR family.     

Effect of bleached rhodopsin on signal amplification in rod visual receptors

Bleaching of rhodopsin markedly desensitizes the vertebrate visual system during a subsequent period of dark adaptation. Previous studies have indicated an origin of bleaching desensitization in the visual pigment itself, but have not identified the mechanism of action. A candidate for the site at which densensitization is initially expressed is the activation of transducin (formation of T*) on the rod disk membranes; this reaction directly involves rhodopsin in its photoactivated (R*) form and mediates initial amplification of the visual signal (reviewed in refs 7-9). We have analysed the effect of bleaching on the sensitivity of a flash-induced light-scattering signal known to monitor the disk-based amplifier, and which has been established as specifically monitoring transducin activation. We have recorded this signal from functioning retinal rods in situ ('ATR' signal) and find that bleaches inducing a pronounced, sustained loss in rod electrophysiological sensitivity do not alter the sensitivity of the ATR response after correction for reduced quantum catch. Our results indicate that the biochemical gain of the R*----T* transduction stage remains unchanged in the presence of bleached pigment and implicate a subsequent reaction as the first to show a sustained, bleaching-dependent gain reduction.     

Light-dependent phosphorylation of rhodopsin by beta-adrenergic receptor kinase

The structural components involved in transduction of extracellular signals as diverse as a photon of light impinging on the retina or a hormone molecule impinging on a cell have been highly conserved. These components include a recognition unit or receptor (for example, the beta-adrenergic receptor (beta AR) for catecholamines or the 'light receptor' rhodopsin), a guanine nucleotide regulatory or transducing protein, and an effector enzyme (for example, adenylate cyclase or cyclic GMP phosphodiesterase). Molecular cloning has revealed that the beta AR shares significant sequence and three-dimensional homology with rhodopsin. The function of the beta AR is diminished by exposure to stimulatory agonists, leading to desensitization. Similarly, 'light adaptation' involves decreased coupling of photoactivated rhodopsin to cGMP phosphodiesterase activation. Both forms of desensitization involve receptor phosphorylation. The latter is mediated by a unique protein kinase, rhodopsin kinase, which phosphorylates only the light-bleached form of rhodopsin. An analogous enzyme (termed beta AR kinase or beta ARK) phosphorylates only the agonist-occupied beta AR. We report here that beta ARK is also capable of phosphorylating rhodopsin in a totally light-dependent fashion. Moreover, rhodopsin kinase can phosphorylate the agonist-occupied beta AR. Thus the mechanisms which regulate the function of these disparate signalling systems also appear to be similar.     

The structural basis of agonist-induced activation in constitutively active rhodopsin

G-protein-coupled receptors (GPCRs) comprise the largest family of membrane proteins in the human genome and mediate cellular responses to an extensive array of hormones, neurotransmitters and sensory stimuli. Although some crystal structures have been determined for GPCRs, most are for modified forms, showing little basal activity, and are bound to inverse agonists or antagonists. Consequently, these structures correspond to receptors in their inactive states. The visual pigment rhodopsin is the only GPCR for which structures exist that are thought to be in the active state. However, these structures are for the apoprotein, or opsin, form that does not contain the agonist all-trans retinal. Here we present a crystal structure at a resolution of 3 ? for the constitutively active rhodopsin mutant Glu 113 Gln in complex with a peptide derived from the carboxy terminus of the α-subunit of the G protein transducin. The protein is in an active conformation that retains retinal in the binding pocket after photoactivation. Comparison with the structure of ground-state rhodopsin suggests how translocation of the retinal β-ionone ring leads to a rotation of transmembrane helix 6, which is the critical conformational change on activation. A key feature of this conformational change is a reorganization of water-mediated hydrogen-bond networks between the retinal-binding pocket and three of the most conserved GPCR sequence motifs. We thus show how an agonist ligand can activate its GPCR.     

Observations of light-induced structural changes of retinal within rhodopsin

Photo-isomerization of the 11-cis retinal chromophore activates the mammalian light-receptor rhodopsin, a representative member of a major superfamily of transmembrane G-protein-coupled receptor proteins (GPCRs) responsible for many cell signal communication pathways. Although low-resolution (5 A) electron microscopy studies confirm a seven transmembrane helix bundle as a principal structural component of rhodopsin, the structure of the retinal within this helical bundle is not known in detail. Such information is essential for any theoretical or functional understanding of one of the fastest occurring photoactivation processes in nature, as well as the general mechanism behind GPCR activation. Here we determine the three-dimensional structure of 11-cis retinal bound to bovine rhodopsin in the ground state at atomic level using a new high-resolution solid-state NMR method. Significant structural changes are observed in the retinal following activation by light to the photo-activated M(I) state of rhodopsin giving the all-trans isomer of the chromophore. These changes are linked directly to the activation of the receptor, providing an insight into the activation mechanism of this class of receptors at a molecular level.     

Bicycle-pedal model for the first step in the vision process.

Computer simulation of the molecular dynamics of retinal during its photoisomerisation inside a restrictive active site gives a detailed model for the sequence of events in the first step of the vision process. It is proposed that the prelumirhodopsin intermediate contains a strained all-trans retinal molecule produced directly and rapidly from the 11-cis, 12-s-trans conformation in rhodopsin by a bicycle-pedal isomerisation. The model reproduces the main experimental observations and explains how the protein makes the photoisomerisation path unique.     

Membrane protein diffusion sets the speed of rod phototransduction

Retinal rods signal the activation of a single receptor molecule by a photon. To ensure efficient photon capture, rods maintain about 109 copies of rhodopsin densely packed into membranous disks. But a high packing density of rhodopsin may impede other steps in phototransduction that take place on the disk membrane, by restricting the lateral movement of, and hence the rate of encounters between, the molecules involved. Although it has been suggested that lateral diffusion of proteins on the membrane sets the rate of onset of the photoresponse, it was later argued that the subsequent processing of the complexes was the main determinant of this rate. The effects of protein density on response shut-off have not been reported. Here we show that a roughly 50% reduction in protein crowding achieved by the hemizygous knockout of rhodopsin in transgenic mice accelerates the rising phases and recoveries of flash responses by about 1.7-fold in vivo. Thus, in rods the rates of both response onset and recovery are set by the diffusional encounter frequency between proteins on the disk membrane.     

A rhodopsin is the functional photoreceptor for phototaxis in the unicellular eukaryote Chlamydomonas

Rhodopsin is a visual pigment ubiquitous in multicellular animals. If visual pigments have a common ancient origin, as is believed, then some unicellular organisms might also use a rhodopsin photoreceptor. We show here that the unicellular alga Chlamydomonas does indeed use a rhodopsin photoreceptor. We incorporated analogues of its retinal chromophore into a blind mutant; normal photobehaviour was restored and the colour of maximum sensitivity was shifted in a manner consistent with the nature of the retinal analogue added. The data suggest that 11-cis-retinal is the natural chromophore and that the protein environment of this retinal is similar to that found in bovine rhodopsin, suggesting homology with the rhodopsins of higher organisms. This is the first demonstration of a rhodopsin photoreceptor in an alga or eukaryotic protist and also the first report of behavioural spectral shifts caused by exogenous synthetic retinals in a eukaryote. A survey of the morphology and action spectra of other protists suggests that rhodopsins may be common photoreceptors of chlorophycean, prasinophycean and dinophycean algae. Thus, Chlamydomonas represents a useful new model for studying photoreceptor cells.     

Crystal structure of squid rhodopsin

Invertebrate phototransduction uses an inositol-1,4,5-trisphosphate signalling cascade in which photoactivated rhodopsin stimulates a G(q)-type G protein, that is, a class of G protein that stimulates membrane-bound phospholipase Cbeta. The same cascade is used by many G-protein-coupled receptors, indicating that invertebrate rhodopsin is a prototypical member. Here we report the crystal structure of squid (Todarodes pacificus) rhodopsin at 2.5 A resolution. Among seven transmembrane alpha-helices, helices V and VI extend into the cytoplasmic medium and, together with two cytoplasmic helices, they form a rigid protrusion from the membrane surface. This peculiar structure, which is not seen in bovine rhodopsin, seems to be crucial for the recognition of G(q)-type G proteins. The retinal Schiff base forms a hydrogen bond to Asn 87 or Tyr 111; it is far from the putative counterion Glu 180. In the crystal, a tight association is formed between the amino-terminal polypeptides of neighbouring monomers; this intermembrane dimerization may be responsible for the organization of hexagonally packed microvillar membranes in the photoreceptor rhabdom.     

Development of the light response in neonatal mammalian rods

The sensitivity to light is low in many neonatal mammals when compared with that in the adult. In human infants at one month of age, for example, the dark-adapted sensitivity for detection of large stimuli is 50 times lower than in the adult, and in rats the overall sensitivity of the neonatal retina is also low compared with the adult. This low sensitivity in the neonate has been attributed to a number of factors, but the possibility that the photoreceptors themselves might be an important limitation on the overall visual sensitivity has not so far been clearly established. Here we record the light response of single neonatal rat rods and find that the sensitivity is considerably lower than in the adult. The response to a single photoisomerization is normal in the neonate, and the sensitivity deficit can therefore be attributed to a low level of functional rhodopsin. Opsin, the protein component of rhodopsin, must be present in normal amounts, as the sensitivity can be restored to adult levels by treating the retina with 9-cis retinal, an active homologue of the native chromophore 11-cis retinal. The low sensitivity of photoreceptors in the neonate can therefore be attributed mainly to a low concentration of 11-cis retinal in the developing retina.     

Crystal structure of the ligand-free G-protein-coupled receptor opsin

In the G-protein-coupled receptor (GPCR) rhodopsin, the inactivating ligand 11-cis-retinal is bound in the seven-transmembrane helix (TM) bundle and is cis/trans isomerized by light to form active metarhodopsin II. With metarhodopsin II decay, all-trans-retinal is released, and opsin is reloaded with new 11-cis-retinal. Here we present the crystal structure of ligand-free native opsin from bovine retinal rod cells at 2.9 ?ngstr?m (A) resolution. Compared to rhodopsin, opsin shows prominent structural changes in the conserved E(D)RY and NPxxY(x)(5,6)F regions and in TM5-TM7. At the cytoplasmic side, TM6 is tilted outwards by 6-7 A, whereas the helix structure of TM5 is more elongated and close to TM6. These structural changes, some of which were attributed to an active GPCR state, reorganize the empty retinal-binding pocket to disclose two openings that may serve the entry and exit of retinal. The opsin structure sheds new light on ligand binding to GPCRs and on GPCR activation.     

A semiconductor source of triggered entangled photon pairs

Entangled photon pairs are an important resource in quantum optics, and are essential for quantum information applications such as quantum key distribution and controlled quantum logic operations. The radiative decay of biexcitons-that is, states consisting of two bound electron-hole pairs-in a quantum dot has been proposed as a source of triggered polarization-entangled photon pairs. To date, however, experiments have indicated that a splitting of the intermediate exciton energy yields only classically correlated emission. Here we demonstrate triggered photon pair emission from single quantum dots suggestive of polarization entanglement. We achieve this by tuning the splitting to zero, through either application of an in-plane magnetic field or careful control of growth conditions. Entangled photon pairs generated 'on demand' have significant fundamental advantages over other schemes, which can suffer from multiple pair emission, or require post-selection techniques or the use of photon-number discriminating detectors. Furthermore, control over the pair generation time is essential for scaling many quantum information schemes beyond a few gates. Our results suggest that a triggered entangled photon pair source could be implemented by a simple semiconductor light-emitting diode.     

Low retinal noise in animals with low body temperature allows high visual sensitivity

The weakest pulse of light a human can detect sends about 100 photons through the pupil and produces 10-20 rhodopsin isomerizations in a small retinal area. It has been postulated that we cannot see single photons because of a retinal noise arising from randomly occurring thermal isomerizations. Direct recordings have since demonstrated the existence of electrical 'dark' rod events indistinguishable from photoisomerization signals. Their mean rate of occurrence is roughly consistent with the 'dark light' in psychophysical threshold experiments, and their thermal parameters justify an identification with thermal isomerizations. In the retina of amphibians, a small proportion of sensitive ganglion cells have a performance-limiting noise that is low enough to be well accounted for by these events. Here we study the performance of dark-adapted toads and frogs and show that the performance limit of visually guided behaviour is also set by thermal isomerizations. As visual sensitivity limited by thermal events should rise when the temperature falls, poikilothermous vertebrates living at low temperatures should then reach light sensitivities unattainable by mammals and birds with optical factors equal. Comparison of different species at different temperatures shows a correlation between absolute threshold intensities and estimated thermal isomerization rates in the retina.     

Subsecond deactivation of transducin by endogenous GTP hydrolysis

The response of a retinal rod cell to a weak flash of light is mediated by a receptor/GTP-binding protein (rhodopsin/transducin) signal transduction system and terminates within a second. The T alpha subunit of transducin (composed of subunits T alpha, T beta and T gamma) is triggered by photoexcited rhodopsin (R*) to release GDP and bind GTP. The binding of GTP causes release of the T alpha unit from T beta gamma and allows it to modulate the activity of an enzyme that generates a second messenger. Termination of the response requires the hydrolysis of the GTP by intrinsic GTPase. As with other G proteins, the GTPase activity of transducin seems to be slow. Reported in vitro turnover rates of a few molecules of GTP hydrolysed per molecule of transducin per minute imply a T alpha-GTP deactivation time of many seconds. But this time might be only a small fraction of that of the GTPase cycle. We have now used time-resolved microcalorimetry in bovine rod outer segments (ROS) to monitor the heat release due to the hydrolysis of GTP by a transducin population that had been quickly activated by flash illumination of rhodopsin. The enthalpy of GTP hydrolysis is released within 1 s at 23 degrees C. This deactivation time seems to be independent of any diffusible factor in the preparation and concurs with the termination kinetics of the rod's response. Thereafter, transducin seems unable to reload GTP for many seconds. This refractory 'resetting' time may account for the low steady-state GTPase rates in vitro.     

Development of the signal in sensory rhodopsin and its transfer to the cognate transducer

The microbial phototaxis receptor sensory rhodopsin II (NpSRII, also named phoborhodopsin) mediates the photophobic response of the haloarchaeon Natronomonas pharaonis by modulating the swimming behaviour of the bacterium. After excitation by blue-green light NpSRII triggers, by means of a tightly bound transducer protein (NpHtrII), a signal transduction chain homologous with the two-component system of eubacterial chemotaxis. Two molecules of NpSRII and two molecules of NpHtrII form a 2:2 complex in membranes as shown by electron paramagnetic resonance and X-ray structure analysis. Here we present X-ray structures of the photocycle intermediates K and late M (M2) explaining the evolution of the signal in the receptor after retinal isomerization and the transfer of the signal to the transducer in the complex. The formation of late M has been correlated with the formation of the signalling state. The observed structural rearrangements allow us to propose the following mechanism for the light-induced activation of the signalling complex. On excitation by light, retinal isomerization leads in the K state to a rearrangement of a water cluster that partly disconnects two helices of the receptor. In the transition to late M the changes in the hydrogen bond network proceed further. Thus, in late M state an altered tertiary structure establishes the signalling state of the receptor. The transducer responds to the activation of the receptor by a clockwise rotation of about 15 degrees of helix TM2 and a displacement of this helix by 0.9 A at the cytoplasmic surface.     

Measurement of thermal contribution to photoreceptor sensitivity

Activation of a visual pigment molecule to initiate phototransduction requires a minimum energy, Ea, that need not be wholly derived from a photon, but may be supplemented by heat. Theory predicts that absorbance at very long wavelengths declines with the fraction of molecules that have a sufficient complement of thermal energy, and that Ea is inversely related to the wavelength of maximum absorbance (lambda(max)) of the pigment. Consistent with the first of these predictions, warming increases relative visual sensitivity to long wavelengths. Here we measure this effect in amphibian photoreceptors with different pigments to estimate Ea (refs 2, 5-7) and test experimentally the predictions of an inverse relation between Ea and lambda(max). For rods and 'red' cones in the adult frog retina, we find no significant difference in Ea between the two pigments involved, although their lambda(max) values are very different. We also determined Ea for the rhodopsin in toad retinal rods--spectrally similar to frog rhodopsin but differing in amino-acid sequence--and found that it was significantly higher. In addition, we estimated Ea for two pigments whose lambda(max) difference was due only to a chromophore difference (A1 and A2 pigment, in adult and larval frog cones). Here Ea for A2 was lower than for A1. Our results refute the idea of a necessary relation between lambda(max) and Ea, but show that the A1 --> A2 chromophore substitution decreases Ea.     

myo-Inositol polyphosphate may be a messenger for visual excitation in Limulus photoreceptors

Photoreceptor excitation begins with the absorption of a photon by rhodopsin and proceeds through an unknown sequence of steps that leads to changes in specific ionic conductances. These conductance changes produce the receptor potential. It has been proposed that hydrolysis of phosphoinositides is involved in the control of a variety of physiological processes. Recent studies have implicated inositol 1,4,5-trisphosphate as an intracellular messenger in the cascade mediating hormone-stimulated secretion. We propose that one of the steps in the excitatory cascade in Limulus ventral photoreceptors may be an increase in intracellular concentration of myo-inositol polyphosphates, derived from hydrolysis of the membrane component phosphatidylinositol bisphosphate by a phospholipase. Here we present biochemical and electrophysiological evidence that an inositol polyphosphate may be an intracellular messenger in the cascade mediating excitation, based on the following criteria: the cells possess the synthetic and degradative metabolism for the messenger; the natural stimulus leads to a change in the concentration of the messenger within the cells; and intracellular injection of exogenous messenger mimics naturally occurring electrophysiological events.     

Millisecond activation of transducin in the cyclic nucleotide cascade of vision

Cyclic GMP has been implicated as a messenger molecule involved in visual transduction. Photoexcited rhodopsin (R*) binds to a multisubunit membrane protein called transducin (T) and stimulates the exchange of a bound GDP molecule for GTP. This leads to the release of the alpha-subunit of T with bound GTP (T alpha-GTP), which activates a cyclic GMP phosphodiesterase. The question arises as to whether the hydrolysis of cyclic GMP that results from activation of the phosphodiesterase is sufficiently rapid to be involved in visual excitation, which occurs on a time scale of approximately 2 s in the single-photon limit. Previous studies have suggested that the cyclic GMP phosphodiesterase is activated in less than 100 ms at moderate light levels. We report here light scattering studies of magnetically orientated frog rod outer segments which show that a molecule of R* catalyses the activation of a molecule of T in about 1 ms. Thus, hundreds of molecules can be activated within the response time of vision in the single-photon limit, and the formation of T alpha-GTP is fast enough for it to be a key step in visual transduction.     

Photoreceptor-specific degeneration caused by tunicamycin

The antibiotic tunicamycin inhibits the biosynthesis of N-acetylglucosaminylpyrophosphoryl polyisoprenol, a key intermediate in the formation of the asparagine-linked oligosaccharides of glycoproteins. The effects of tunicamycin have been studied in various biological systems, primarily with the aim of elucidating the role of the carbohydrate moieties in the cellular function of glycoproteins. Rhodopsin, the visual pigment of retinal rod photoreceptor cells, is a membrane glycoprotein which consists of a single polypeptide chain (opsin) to which a chromophoric prosthetic group (II-cis-retinaldehyde) and two asparagine-linked oligosaccharide chains are covalently attached. The glycosylation of opsin can be blocked with tunicamycin in vitro in conditions where polypeptide synthesis is only slightly decreased. We have reported that tunicamycin can disrupt the normal assembly of rod outer segment membranes in vitro without significantly inhibiting the biosynthesis or intracellular transport of opsin. Here we report that intraocular injection of tunicamycin produces a photoreceptor-specific degeneration characterized by progressive shortening of rod outer segment, decreased membrane assembly, and eventual photoreceptor cell death.