Rhodopsin photoproducts in 2D crystals

The published electron microscope and X-ray structures of rhodopsin have made available a detailed picture of the inactive dark state of rhodopsin. Yet, the photointermediates of rhodopsin that ultimately lead to the activated receptor species still await a similar analysis. Such an analysis first requires the generation and characterization of the photoproducts that can be obtained in crystals of rhodopsin. We therefore studied with Fourier-transform infrared (FTIR) difference spectroscopy the photoproducts in 2D crystals of bovine rhodopsin in a p22(1)2(1) crystal form. The spectra obtained by cryotrapping revealed that in this crystal form the still inactive early intermediates batho, lumi, and meta I are similar to those obtained from rhodopsin in native disk membranes, although the transition from lumi to meta I is shifted to a higher temperature. However, at room temperature, the formation of the active state, meta II, is blocked in the crystalline environment. Instead, an intermediate state is formed that bears some features of meta II but lacks the specific conformational changes required for activity. Despite being unable to activate its cognate G protein, transducin, to a significant extent, this intermediate state is capable of interacting with functional transducin-derived peptides to a limited extent. Therefore, while unable to support formation of rhodopsin's active state meta II, 2D p22(1)2(1) crystals proved to be very suitable for determining 3D structures of its still inactive precursors, batho, lumi, and meta I. In future studies, FTIR spectroscopy may serve as a sensitive assay to screen crystals grown under altered conditions for potential formation of the active state, meta II.     

Formation of Meta III during the decay of activated rhodopsin proceeds via Meta I and not via Meta II

Thermal isomerization of the retinal Schiff base C=N double bond is known to trigger the decay of rhodopsin's Meta I/Meta II photoproduct equilibrium to the inactive Meta III state [Vogel, R., Siebert, F., Mathias, G., Tavan, P., Fan, G., and Sheves, M. (2003) Biochemistry 42, 9863-9874]. Previous studies have indicated that the transition to Meta III does not occur under conditions that strongly favor the active state Meta II but requires a residual amount of Meta I in the initial photoproduct equilibrium. In this study we show that the triggering event, the thermal isomerization of the protonated Schiff base, is independent of the presence of Meta II and occurs even under conditions where the transition to Meta II is completely prevented. We have examined two examples in which the transitions from Lumi to Meta I or from Meta I to Meta II are blocked. This was achieved using dry films of rhodopsin and rhodopsin reconstituted into rather rigid lipid bilayers. In both cases, the resulting fully inactive room temperature photoproducts decay specifically by thermal isomerization of the protonated Schiff base C=N double bond to an all-trans 15-syn chromophore isomer, corresponding to that of Meta III. This thermal isomerization becomes less efficient as the conformation of the respective photoproduct approaches that of Meta II and is fully absent in a pure Meta II state. These results indicate that the decay of the Meta I/Meta II photoproduct equilibrium to Meta III proceeds via Meta I and not via Meta II.     

Structural impact of the E113Q counterion mutation on the activation and deactivation pathways of the G protein-coupled receptor rhodopsin

Disruption of an interhelical salt bridge between the retinal protonated Schiff base linked to H7 and Glu113 on H3 is one of the decisive steps during activation of rhodopsin. Using previously established stabilization strategies, we engineered a stabilized E113Q counterion mutant that converted rhodopsin to a UV-absorbing photoreceptor with deprotonated Schiff base and allowed reconstitution into native-like lipid membranes. Fourier-transform infrared difference spectroscopy reveals a deprotonated Schiff base in the photoproducts of the mutant up to the active state Meta II, the absence of the classical pH-dependent Meta I/Meta II conformational equilibrium in favor of Meta II, and an anticipation of active state features under conditions that stabilize inactive photoproduct states in wildtype rhodopsin. Glu181 on extracellular loop 2, is found to be unable to maintain a counterion function to the Schiff base on the activation pathway of rhodopsin in the absence of the primary counterion, Glu113. The Schiff base becomes protonated in the transition to Meta III. This protonation is, however, not associated with a deactivation of the receptor, in contrast to wildtype rhodopsin. Glu181 is suggested to be the counterion in the Meta III state of the mutant and appears to be capable of stabilizing a protonated Schiff base in Meta III, but not of constraining the receptor in an inactive conformation.     

Conformation and stability of alpha-helical membrane proteins. 1. Influence of salts on conformational equilibria between active and Inactive states of rhodopsin

We studied the influence of salts on the pH-dependent conformational equilibria between the active and the inactive photoproduct states of rhodopsin, Meta II and Meta I, respectively, and between the active and inactive conformations of the apoprotein opsin. In both equilibria, the active species is favored in the presence of medium to high concentration of salt. The ion selectivity for the Meta I/Meta II equilibrium is particularly pronounced for the anions and follows the series trichloroacetate > thiocyanate > iodide > bromide > sulfate > chloride > acetate. The Hill coefficient of this salt-induced transition is close to 2.0. Both ion selectivity and Hill coefficient suggest that the transition is mainly regulated by ion binding to two specific charged binding sites in the protein with smaller contributions being due to the Hofmeister effect. We propose that these putative ion binding sites are identical to those sites that are titrated in the corresponding pH-dependent conformational transition. They presumably function as ionic locks, which keep the receptor in an inactive conformation, and which may be disrupted either by pH-dependent protonation or by salt-dependent ion binding.     

A beta-arrestin binding determinant common to the second intracellular loops of rhodopsin family G protein-coupled receptors

beta-Arrestins have been shown to inhibit competitively G protein-dependent signaling and to mediate endocytosis for many of the hundreds of nonvisual rhodopsin family G protein-coupled receptors (GPCR). An open question of fundamental importance concerning the regulation of signal transduction of several hundred rhodopsin-like GPCRs is how these receptors of limited sequence homology, when considered in toto, can all recruit and activate the two highly conserved beta-arrestin proteins as part of their signaling/desensitization process. Although the serine and threonine residues that form GPCR kinase phosphorylation sites are common beta-arrestin-associated receptor determinants regulating receptor desensitization and internalization, the agonist-activated conformation of a GPCR probably reveals the most fundamental determinant mediating the GPCR and arrestin interaction. Here we identified a beta-arrestin binding determinant common to the rhodopsin family GPCRs formed from the proximal 10 residues of the second intracellular loop. We demonstrated by both gain and loss of function studies for the serotonin 2C, beta2-adrenergic, alpha2a)adrenergic, and neuropeptide Y type 2 receptors that the highly conserved amino acids, proline and alanine, naturally occurring in rhodopsin family receptors six residues distal to the highly conserved second loop DRY motif regulate beta-arrestin binding and beta-arrestin-mediated internalization. In particular, as demonstrated for the beta2 AR, this occurs independently of changes in GPCR kinase phosphorylation. These results suggest that a GPCR conformation directed by the second intracellular loop, likely using the loop itself as a binding patch, may function as a switch for transitioning beta-arrestin from its inactive form to its active receptor-binding state.     

Engineering a "steric doorstop" in rhodopsin: converting an inverse agonist to an agonist

The crystal structures of rhodopsin depict the inactive conformation of rhodopsin in the dark. The 11-cis retinoid chromophore, the inverse agonist holding rhodopsin inactive, is well-resolved. Thr118 in helix 3 is the closest amino acid residue next to the 9-methyl group of the chromophore. The 9-methyl group of retinal facilitates the transition from an inactive metarhodopsin I to the active metarhodopsin II intermediate. In this study, a site-specific mutation of Thr118 to the bulkier Trp was made with the idea to induce an active conformation of the protein. The data indicate that such a mutation does indeed result in an active protein that depends on the presence of the ligand, specifically the 9-methyl group. As a result of this mutation, 11-cis retinal has been converted to an agonist. The apoprotein form of this mutant is no more active than the wild-type apoprotein. However, unlike wild-type rhodopsin, the covalent linkage of the ligand can be attacked by hydroxylamine in the dark. The combination of the Thr118Trp mutation and the 9-methyl group of the chromophore behaves as a "steric doorstop" holding the protein in an open and active conformation.     

Toward the active conformations of rhodopsin and the beta2-adrenergic receptor

Using sets of experimental distance restraints, which characterize active or inactive receptor conformations, and the X-ray crystal structure of the inactive form of bovine rhodopsin as a starting point, we have constructed models of both the active and inactive forms of rhodopsin and the beta2-adrenergic G-protein coupled receptors (GPCRs). The distance restraints were obtained from published data for site-directed crosslinking, engineered zinc binding, site-directed spin-labeling, IR spectroscopy, and cysteine accessibility studies conducted on class A GPCRs. Molecular dynamics simulations in the presence of either "active" or "inactive" restraints were used to generate two distinguishable receptor models. The process for generating the inactive and active models was validated by the hit rates, yields, and enrichment factors determined for the selection of antagonists in the inactive model and for the selection of agonists in the active model from a set of nonadrenergic GPCR drug-like ligands in a virtual screen using ligand docking software. The simulation results provide new insights into the relationships observed between selected biochemical data, the crystal structure of rhodopsin, and the structural rearrangements that occur during activation.     

Signaling states of rhodopsin. Retinal provides a scaffold for activating proton transfer switches

The G-protein-coupled receptor rhodopsin is activated by photoconversion of its covalently bound ligand 11-cis-retinal to the agonist all-trans-retinal. After light-induced isomerization and early photointermediates, the receptor reaches a G-protein-dependent equilibrium between active and inactive conformations distinguished by the protonation of key opsin residues. In this report, we study the role of the 9-methyl group of retinal, one of the crucial steric determinants of light activation. We find that when this group is removed, the protonation equilibrium is strongly shifted to the inactive conformation. The residually formed active species is very similar to the active form of normal rhodopsin, metarhodopsin II. It has a deprotonated Schiff base, binds to the retinal G-protein transducin, and is favored at acidic pH. Our data show that the normal proton transfer reactions are inhibited in 9-demethyl rhodopsin but are still mandatory for receptor activation. We propose that retinal and its 9-methyl group act as a scaffold for opsin to adjust key proton donor and acceptor side chains for the proton transfer reactions that stabilize the active conformation. The mechanism may also be applicable to related receptors and may thus explain the partial agonism of certain ligands.     

A purified agonist-activated G-protein coupled receptor: truncated octopus Acid Metarhodopsin

G-protein coupled receptors (GPCRs) mediate responses to many types of extracellular signals. So far, bovine rhodopsin, the inactive form of a GPCR, is the only member of the family whose three dimensional structure has been determined. It would be desirable to determine the structure of the active form of a GPCR. In this paper, we report the large scale preparation of a stable, homogenous species, truncated octopus rhodopsin (t-rhodopsin) in which proteolysis has removed the proline-rich C-terminal; this species retains the spectral properties and the ability for light-induced G-protein activation of unproteolyzed octopus rhodopsin. Moreover, starting from this species we can prepare a pure, active form of pigment, octopus t-Acid Metarhodopsin which has an all-trans-retinal as its agonist. Photoisomerization of t-Acid Metarhodopsin leads back to the inactive form, t-rhodopsin with the inverse agonist 11-cis-retinal. Octopus t-Acid Metarhodopsin can activate an endogenous octopus G-protein in the dark and this activity is reduced by irradiation with orange light which photoregenerates t-Acid Metarhodopsin back to the initial species, t-rhodopsin.     

Active state of sensory rhodopsin II: structural determinants for signal transfer and proton pumping

The molecular mechanism of transmembrane signal transduction is still a pertinent question in cellular biology. Generally, a receptor can transfer an external signal via its cytoplasmic surface, as found for G-protein-coupled receptors such as rhodopsin, or via the membrane domain, such as that in sensory rhodopsin II (SRII) in complex with its transducer, HtrII. In the absence of HtrII, SRII functions as a proton pump. Here, we report on the crystal structure of the active state of uncomplexed SRII from Natronomonas pharaonis, NpSRII. The problem with a dramatic loss of diffraction quality upon loading of the active state was overcome by growing better crystals and by reducing the occupancy of the state. The conformational changes in the region comprising helices F and G are similar to those observed for the NpSRII-transducer complex but are much more pronounced. The meaning of these differences for the understanding of proton pumping and signal transduction by NpSRII is discussed.     

Partial agonism in a G Protein-coupled receptor: role of the retinal ring structure in rhodopsin activation

The visual process in rod cells is initiated by absorption of a photon in the rhodopsin retinal chromophore and consequent retinal cis/trans-isomerization. The ring structure of retinal is thought to be needed to transmit the photonic energy into conformational changes culminating in the active metarhodopsin II (Meta II) intermediate. Here, we demonstrate that cis-acyclic retinals, lacking four carbon atoms of the ring, can activate rhodopsin. Detailed analysis of the activation pathway showed that, although the photoproduct pathway is more complex, Meta II formed with almost normal kinetics. However, lack of the ring structure resulted in a low amount of Meta II and a fast decay of activity. We conclude that the main role of the ring structure is to maintain the active state, thus specifying a mechanism of activation by a partial agonist of the G protein-coupled receptor rhodopsin.     

Conformational changes of G protein-coupled receptors during their activation by agonist binding

The superfamily of G protein-coupled receptors (GPCRs) is the largest and most diverse group of transmembrane proteins involved in signal transduction. Many of the over 1000 human GPCRs represent important pharmaceutical targets. However, despite high interest in this receptor family, no high-resolution structure of a human GPCR has been resolved yet. This is mainly due to difficulties in obtaining large quantities of pure and active protein. Until now, only a high-resolution x-ray structure of an inactive state of bovine rhodopsin is available. Since no structure of an active state has been solved, information of the GPCR activation process can be gained only by biophysical techniques. In this review, we first describe what is known about the ground state of GPCRs to then address questions about the nature of the conformational changes taking place during receptor activation and the mechanism controlling the transition from the resting to the active state. Finally, we will also address the question to what extent information about the three-dimensional GPCR structure can be included into pharmaceutical drug design programs.     

Equilibrium between metarhodopsin-I and metarhodopsin-II is dependent on the conformation of the third cytoplasmic loop

Rhodopsin is a G-protein-coupled receptor (GPCR) that is the light detector in the rod cells of the eye. Rhodopsin is the best understood member of the large GPCR superfamily and is the only GPCR for which atomic resolution structures have been determined. However, these structures are for the inactive, dark-adapted form. Characterization of the conformational changes in rhodopsin caused by light-induced activation is of wide importance, because the metarhodopsin-II photoproduct is analogous to the agonist-occupied conformation of other GPCRs, and metarhodopsin-I may be similar to antagonist-occupied GPCR conformations. In this work we characterize the interaction of antibody K42-41L with the metarhodopsin photoproducts. K42-41L is shown to inhibit formation of metarhodopsin-II while it stabilizes the metarhodopsin-I state. Thus, K42-41L recognizes an epitope accessible in dark-adapted rhodopsin and metarhodopsin-I that is lost upon formation of metarhodopsin-II. Previous work has shown that the peptide TGALQERSK is able to mimic the K42-41L epitope, and we have now determined the structure of the K42-41L-peptide complex. The structure demonstrates a central role for elements of the rhodopsin C3 loop, particularly Gln238 and Glu239, in the interaction with K42-41L. Geometric constraints taken from the antibody-bound peptide were used to model the epitope on the rhodopsin surface. The resulting model suggests that K42-41L locks the C3 loop into an extended conformation that is intermediate between two compact conformations seen in crystal structures of dark-adapted rhodopsin. Together, the structural and functional data strongly suggest that the equilibrium between metarhodopsin-I and metarhodopsin-II is dependent upon the conformation of the C3 loop. The biological implications of this model and its possible relations to dimeric and multimeric complexes of rhodopsin are discussed.     

Conformational Changes of G Protein‐Coupled Receptors During Their Activation by Agonist Binding

Abstract

The superfamily of G protein‐coupled receptors (GPCRs) is the largest and most diverse group of transmembrane proteins involved in signal transduction. Many of the over 1000 human GPCRs represent important pharmaceutical targets. However, despite high interest in this receptor family, no high‐resolution structure of a human GPCR has been resolved yet. This is mainly due to difficulties in obtaining large quantities of pure and active protein. Until now, only a high‐resolution x‐ray structure of an inactive state of bovine rhodopsin is available. Since no structure of an active state has been solved, information of the GPCR activation process can be gained only by biophysical techniques. In this review, we first describe what is known about the ground state of GPCRs to then address questions about the nature of the conformational changes taking place during receptor activation and the mechanism controlling the transition from the resting to the active state. Finally, we will also address the question to what extent information about the three‐dimensional GPCR structure can be included into pharmaceutical drug design programs.     

Single-Molecule Observation of the Ligand-Induced Population Shift of Rhodopsin,A G-Protein-Coupled Receptor

Rhodopsin is a G-protein-coupled receptor, in which retinal chromophore acts as inverse-agonist or agonist depending on its configuration and protonation state. Photostimulation of rhodopsin results in a pH-dependent equilibrium between the active state (Meta-II) and its inactive precursor (Meta-I). Here, we monitored conformational changes of rhodopsin using a fluorescent probe Alexa594 at the cytoplasmic surface, which shows fluorescence increase upon the generation of active state, by single-molecule measurements. The fluorescence intensity of a single photoactivated rhodopsin molecule alternated between two states. Interestingly, such a fluorescence alternation was also observed for ligand-free rhodopsin (opsin), but not for dark-state rhodopsin. In addition, the pH-dependences of Meta-I/Meta-II equilibrium estimated by fluorescence measurements deviated notably from estimates based on absorption spectra, indicating that both Meta-I and Meta-II are mixtures of two conformers. Our observations indicate that rhodopsin molecules intrinsically adopt both active and inactive conformations, and the ligand retinal shifts the conformational equilibrium. These findings provide dynamical insights into the activation mechanisms of G-protein-coupled receptors.     

Single-Molecule Observation of the Ligand-Induced Population Shift of Rhodopsin,A G-Protein-Coupled Receptor

Rhodopsin is a G-protein-coupled receptor, in which retinal chromophore acts as inverse-agonist or agonist depending on its configuration and protonation state. Photostimulation of rhodopsin results in a pH-dependent equilibrium between the active state (Meta-II) and its inactive precursor (Meta-I). Here, we monitored conformational changes of rhodopsin using a fluorescent probe Alexa594 at the cytoplasmic surface, which shows fluorescence increase upon the generation of active state, by single-molecule measurements. The fluorescence intensity of a single photoactivated rhodopsin molecule alternated between two states. Interestingly, such a fluorescence alternation was also observed for ligand-free rhodopsin (opsin), but not for dark-state rhodopsin. In addition, the pH-dependences of Meta-I/Meta-II equilibrium estimated by fluorescence measurements deviated notably from estimates based on absorption spectra, indicating that both Meta-I and Meta-II are mixtures of two conformers. Our observations indicate that rhodopsin molecules intrinsically adopt both active and inactive conformations, and the ligand retinal shifts the conformational equilibrium. These findings provide dynamical insights into the activation mechanisms of G-protein-coupled receptors.     

Function of extracellular loop 2 in rhodopsin: glutamic acid 181 modulates stability and absorption wavelength of metarhodopsin II

The second extracellular loop of rhodopsin folds back into the membrane-embedded domain of the receptor to form part of the binding pocket for the 11-cis-retinylidene chromophore. A carboxylic acid side chain from this loop, Glu181, points toward the center of the retinal polyene chain. We studied the role of Glu181 in bovine rhodopsin by characterizing a set of site-directed mutants. Sixteen of the 19 single-site mutants expressed and bound 11-cis-retinal to form pigments. The lambda(max) value of mutant pigment E181Q showed a significant spectral red shift to 508 nm only in the absence of NaCl. Other substitutions did not significantly affect the spectral features of the mutant pigments in the dark. Thus, Glu181 does not contribute significantly to spectral tuning of the ground state of rhodopsin. The most likely interpretation of these data is that Glu181 is protonated and uncharged in the dark state of rhodopsin. The Glu181 mutants displayed significantly increased reactivity toward hydroxylamine in the dark. The mutants formed metarhodopsin II-like photoproducts upon illumination but many of the photoproducts displayed shifted lambda(max) values. In addition, the metarhodopsin II-like photoproducts of the mutant pigments had significant alterations in their decay rates. The increased reactivity of the mutants to hydroxylamine supports the notion that the second extracellular loop prevents solvent access to the chromophore-binding pocket. In addition, Glu181 strongly affects the environment of the retinylidene Schiff base in the active metarhodopsin II photoproduct.     

β-arrestin functionally regulates the non-bleaching pigment parapinopsin in lamprey pineal

The light response of vertebrate visual cells is achieved by light-sensing proteins such as opsin-based pigments as well as signal transduction proteins, including visual arrestin. Previous studies have indicated that the pineal pigment parapinopsin has evolutionally and physiologically important characteristics. Parapinopsin is phylogenetically related to vertebrate visual pigments. However, unlike the photoproduct of the visual pigment rhodopsin, which is unstable, dissociating from its chromophore and bleaching, the parapinopsin photoproduct is stable and does not release its chromophore. Here, we investigated arrestin, which regulates parapinopsin signaling, in the lamprey pineal organ, where parapinopsin and rhodopsin are localized to distinct photoreceptor cells. We found that beta-arrestin, which binds to stimulated G protein-coupled receptors (GPCRs) other than opsin-based pigments, was localized to parapinopsin-containing cells. This result stands in contrast to the localization of visual arrestin in rhodopsin-containing cells. Beta-arrestin bound to cultured cell membranes containing parapinopsin light-dependently and translocated to the outer segments of pineal parapinopsin-containing cells, suggesting that beta-arrestin binds to parapinopsin to arrest parapinopsin signaling. Interestingly, beta-arrestin colocalized with parapinopsin in the granules of the parapinopsin-expressing cell bodies under light illumination. Because beta-arrestin, which is a mediator of clathrin-mediated GPCR internalization, also served as a mediator of parapinopsin internalization in cultured cells, these results suggest that the granules were generated light-dependently by beta-arrestin-mediated internalization of parapinopsins from the outer segments. Therefore, our findings imply that beta-arrestin-mediated internalization is responsible for eliminating the stable photoproduct and restoring cell conditions to the original dark state. Taken together with a previous finding that the bleaching pigment evolved from a non-bleaching pigment, vertebrate visual arrestin may have evolved from a "beta-like" arrestin by losing its clathrin-binding domain and its function as an internalization mediator. Such changes would have followed the evolution of vertebrate visual pigments, which generate unstable photoproducts that independently decay by chromophore dissociation.     

From Induced Fit to Conformational Selection: A Continuum of Binding Mechanism Controlled by the Timescale of Conformational Transitions

In receptor-ligand binding, a question that generated considerable interest is whether the mechanism is induced fit or conformational selection. This question is addressed here by a solvable model, in which a receptor undergoes transitions between active and inactive forms. The inactive form is favored while unbound but the active form is favored while a ligand is loosely bound. As the active-inactive transition rates increase, the binding mechanism gradually shifts from conformational selection to induced fit. The timescale of conformational transitions thus plays a crucial role in controlling binding mechanisms.     

Rhodopsin with 11-cis-locked chromophore is capable of forming an active state photoproduct

The visual pigment rhodopsin is characterized by an 11-cis retinal chromophore bound to Lys-296 via a protonated Schiff base. Following light absorption the C(11)=C(12) double bond isomerizes to trans configuration and triggers protein conformational alterations. These alterations lead to the formation of an active intermediate (Meta II), which binds and activates the visual G protein, transducin. We have examined by UV-visible and Fourier transform IR spectroscopy the photochemistry of a rhodopsin analogue with an 11-cis-locked chromophore, where cis to trans isomerization around the C(11)=C(12) double bond is prevented by a 6-member ring structure (Rh(6.10)). Despite this lock, the pigment was found capable of forming an active photoproduct with a characteristic protein conformation similar to that of native Meta II. This intermediate is further characterized by a protonated Schiff base and protonated Glu-113, as well as by its ability to bind a transducin-derived peptide previously shown to interact efficiently with native Meta II. The yield of this active photointermediate is pH-dependent and decreases with increasing pH. This study shows that with the C(11)=C(12) double bond being locked, isomerization around the C(9)=C(10) or the C(13)=C(14) double bonds may well lead to an activation of the receptor. Additionally, prolonged illumination at pH 7.5 produces a new photoproduct absorbing at 385 nm, which, however, does not exhibit the characteristic active protein conformation.