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.     

Relevance of rhodopsin studies for GPCR activation

Rhodopsin, the dim-light photoreceptor present in the rod cells of the retina, is both a retinal-binding protein and a G protein-coupled receptor (GPCR). Due to this conjunction, it benefits from an arsenal of spectroscopy techniques that can be used for its characterization, while being a model system for the important family of Class A (also referred to as “rhodopsin-like”) GPCRs. For instance, rhodopsin has been a crucial player in the field of GPCR structural biology. Until 2007, it was the only GPCR for which a high-resolution crystal structure was available, so all structure–activity analyses on GPCRs, from structure-based drug discovery to studies of structural changes upon activation, were based on rhodopsin. At present, about a third of currently available GPCR structures are still from rhodopsin. In this review, I show some examples of how these structures can still be used to gain insight into general aspects of GPCR activation. First, the analysis of the third intracellular loop in rhodopsin structures allows us to gain an understanding of the structural and dynamic properties of this region, which is absent (due to protein engineering or poor electron density) in most of the currently available GPCR structures. Second, a detailed analysis of the structure of the transmembrane domains in inactive, intermediate and active rhodopsin structures allows us to detect early conformational changes in the process of ligand-induced GPCR activation. Finally, the analysis of a conserved ligand-activated transmission switch in the transmembrane bundle of GPCRs in the context of the rhodopsin activation cycle, allows us to suggest that the structures of many of the currently available agonist-bound GPCRs may correspond to intermediate active states. While the focus in GPCR structural biology is inevitably moving away from rhodopsin, in other aspects rhodopsin is still at the forefront. For instance, the first studies of the structural basis of disease mutants in GPCRs, or the most detailed analysis of cellular GPCR signal transduction networks using a systems biology approach, have been carried out in rhodopsin. Finally, due again to its unique properties among GPCRs, rhodopsin will likely play an important role in the application of X-ray free electron laser crystallography to time-resolved structural biology in membrane proteins. Rhodopsin, thus, still remains relevant as a model system to study the molecular mechanisms of GPCR activation. This article is part of a Special Issue entitled: Retinal Proteins—You can teach an old dog new tricks.     

X-ray diffraction of heavy-atom labelled two-dimensional crystals of rhodopsin identifies the position of cysteine 140 in helix 3 and cysteine 316 in helix 8

We have used site-specific heavy-atom labelling and X-ray diffraction to localize single amino acid residues in the cytoplasmic domain of the integral membrane protein rhodopsin, the dim-light photoreceptor of retinal vertebrate rod cells. Two-dimensional orthorhombic crystals of the space group p22(1)2(1) (a=59.5(+/-1) A and b=82.7(+/-1.5) A) were produced from detergent-solubilized, partially delipidated rhodopsin. To obtain milligram amounts of two-dimensional crystals, which are required for X-ray diffraction, the yield of the crystalline material was significantly increased by reconstitution of rhodopsin in the presence of cholesterol (1:2 to 1:10 mol/mol) and by adding polar organic solvents to the dialysis buffer. The native cysteine residues C140 and C316 were then selectively labelled with mercury using the sulphydryl-specific reagent p-chloromercuribenzoate (1.6-2.1 mol Hg per mol rhodopsin). The labelling did not affect the unit cell dimensions. Optical absorption spectra of labelled and native two-dimensional rhodopsin crystals showed the characteristic 11-cis-retinal peak at 498 nm, which corresponds to the dark state of rhodopsin. The in-plane position of the mercury label was calculated at 9.5 A resolution from the intensity differences in the X-ray diffraction patterns of labelled and native crystals using Fourier difference methods and the phase information from electron crystallography. The label positions were in excellent agreement with the positions of C140 at the cytoplasmic end of helix 3 and of C316 in the cytoplasmic helix 8 recently obtained from three-dimensional rhodopsin crystals. Whereas these high-resolution diffraction studies were performed under cryogenic conditions (100 K), our results were obtained at room temperature with fully hydrated membranes and in the absence of loop-loop crystal contacts. To study the structural changes of the cytoplasmic loops involved in activation and signal transduction, our more physiological conditions offer important advantages. Furthermore, the localization of C316 is the first direct proof that the electron density on top of helix 1 observed by cryo-electron microscopy is a part of the C-terminal loop. Our approach is of particular interest for investigations of other membrane proteins, for which 3D crystals are not available. Structural constraints from heavy-atom labels at strategic sites enable the assignment of a position in the amino acid sequence to features visible in a low-resolution density map and the study of conformational changes associated with different functional states of the membrane protein.     

Structural studies of metarhodopsin II,the activated form of the G-protein coupled receptor,rhodopsin

The structural changes that accompany activation of a G-protein coupled receptor (GPCR) are not well understood. To better understand the activation of rhodopsin, the GPCR responsible for visual transduction, we report studies on the three-dimensional structure for the activated state of this receptor, metarhodopsin II. Differences between the three-dimensional structure of ground state rhodopsin and metarhodopsin II, particularly in the cytoplasmic face of the receptor, suggest how the receptor is activated to couple with transducin. In particular, activation opens a groove on the surface of the receptor that could bind the N-terminal helix of the G protein, transducin alpha.     

Retinal Flip in Rhodopsin Activation?

Rhodopsin is a well-characterized structural model of a G protein-coupled receptor. Photoisomerization of the covalently bound retinal triggers activation. Surprisingly, the x-ray crystal structure of the active Meta-II state has a 180° rotation about the long-axis of the retinal polyene chain. Unbiased microsecond-timescale all-atom molecular dynamics simulations show that the retinal cofactor can flip back to the orientation observed in the inactive state of rhodopsin under conditions favoring the Meta-I state. Our results provide, to our knowledge, the first evidence from molecular dynamics simulations showing how rotation of the retinal ligand within its binding pocket can occur in the activation mechanism of rhodopsin.     

A constitutively active Gα subunit provides insights into the mechanism of G protein activation

The activation of Gα subunits of heterotrimeric G proteins by G protein-coupled receptors (GPCRs) is a critical event underlying a variety of biological responses. Understanding how G proteins are activated will require structural and biochemical analyses of GPCRs complexed to their G protein partners, together with structure-function studies of Gα mutants that shed light on the different steps in the activation pathway. Previously, we reported that the substitution of a glycine for a proline at position 56 within the linker region connecting the helical and GTP-binding domains of a Gα chimera, designated αT*, yields a more readily exchangeable state for guanine nucleotides. Here we show that GDP-GTP exchange on αT*(G56P), in the presence of the light-activated GPCR, rhodopsin (R*), is less sensitive to the β1γ1 subunit complex than to wild-type αT*. We determined the X-ray crystal structure for the αT*(G56P) mutant and found that the G56P substitution leads to concerted changes that are transmitted to the conformationally sensitive switch regions, the α4-β6 loop, and the β6 strand. The α4-β6 loop has been proposed to be a GPCR contact site that signals to the TCAT motif and weakens the binding of the guanine ring of GDP, whereas the switch regions are the contact sites for the β1γ1 complex. Collectively, these biochemical and structural data lead us to suggest that αT*(G56P) may be adopting a conformation that is normally induced within Gα subunits by the combined actions of a GPCR and a Gβγ subunit complex during the G protein activation event.     

Studies on the structure of the G-protein-coupled receptor rhodopsin including the putative G-protein binding site in unactivated and activated forms

Activation of G-protein coupled receptors (GPCR) is not yet understood. A recent structure showed most of rhodopsin in the ground (not activated) state of the GPCR, but the cytoplasmic face, which couples to the G protein in signal transduction, was not well-defined. We have determined an experimental three-dimensional structure for rhodopsin in the unactivated state, which shows good agreement with the crystal structure in the transmembrane domain. This new structure defines the cytoplasmic face of rhodopsin. The G-protein binding site can be mapped. The same experimental approach yields a preliminary structure of the cytoplasmic face in the activated (metarhodopsin II) receptor. Differences between the two structures suggest how the receptor is activated to couple with transducin.     

Improvements in G protein-coupled receptor purification yield light stable rhodopsin crystals

G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling proteins and are the target of approximately half of all therapeutic agents. Agonist ligands bind their cognate GPCRs stabilizing the active conformation that is competent to bind G proteins, thus initiating a cascade of intracellular signaling events leading to modification of the cell activity. Despite their biomedical importance, the only known GPCR crystal structures are those of inactive rhodopsin forms. In order to understand how GPCRs are able to transduce extracellular signals across the plasma membrane, it is critical to determine the structure of these receptors in their ligand-bound, active state. Here, we report a novel combination of purification procedures that allowed the crystallization of rhodopsin in two new crystal forms and can be applicable to the purification and crystallization of other membrane proteins. Importantly, these new crystals are stable upon photoactivation and the preliminary X-ray diffraction analysis of both photoactivated and ground state rhodopsin crystals are also reported.     

The ring of the rhodopsin chromophore in a hydrophobic activation switch within the binding pocket

The current view that the beta-ionone ring of the rhodopsin chromophore vacates its binding pocket within the protein early in the photocascade has been adopted in efforts to provide structural models of photoreceptor activation. This event casts doubt on the ability of this covalently bonded ligand to participate directly in later stages involving activation of the photoreceptor and it is difficult to translate into predictions for the activation of related G protein-coupled receptors by diffusable ligands (e.g. neurotransmitters). The binding pocket fixes the formally equivalent pair of ring methyl groups (C16/C17) in different orientations that can be distinguished easily by (13)C NMR. Solid-state NMR observations on C16 and C17 are reported here that show instead that the ring is retained with strong selective interactions within the binding site into the activated state. We further show how increased steric interactions for this segment in the activated receptor can be explained by adjustment in the protein structure around the ring whilst it remains in its original location. This describes a plausible role for the ring in operating a hydrophobic switch from within the aromatic cluster of helix 6 of rhodopsin, which is coupled to electronic changes within the receptor through water-mediated, hydrogen-bonded networks between the conserved residues in G protein-coupled receptors.     

G-protein-coupled receptor dynamics: dimerization and activation models compared with experiment

Our previously derived models of the active state of the β2-adrenergic receptor are compared with recently published X-ray crystallographic structures of activated GPCRs (G-protein-coupled receptors). These molecular dynamics-based models using experimental data derived from biophysical experiments on activation were used to restrain the receptor to an active state that gave high enrichment for agonists in virtual screening. The β2-adrenergic receptor active model and X-ray structures are in good agreement over both the transmembrane region and the orthosteric binding site, although in some regions the active model is more similar to the active rhodopsin X-ray structures. The general features of the microswitches were well reproduced, but with minor differences, partly because of the unexpected X-ray results for the rotamer toggle switch. In addition, most of the interacting residues between the receptor and the G-protein were identified. This analysis of the modelling has also given important additional insight into GPCR dimerization: re-analysis of results on photoaffinity analogues of rhodopsin provided additional evidence that TM4 (transmembrane helix 4) resides at the dimer interface and that ligands such as bivalent ligands may pass between the mobile helices. A comparison, and discussion, is also carried out between the use of implicit and explicit solvent for active-state modelling.     

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.     

X-Ray diffraction analysis of three-dimensional crystals of bovine rhodopsin obtained from mixed micelles

Rhodopsin, a prototypic G protein-coupled receptor responsible for absorption of photons in retinal rod photoreceptor cells, was selectively extracted from bovine rod outer segment membranes, employing mixed micelles of nonyl beta-d-glucoside and heptanetriol. Highly purified rhodopsin was crystallized from solutions containing varying amounts of detergent and amphiphile. The crystals contained ground state rhodopsin molecules as judged by their red color and the linear dichroism originating from the 11-cis-retinal chromophore. However, when exposed to visible light, even at 4 degrees C, rhodopsin was bleached and the crystals decomposed. Reflections in the diffraction pattern were observed out to 3.5-A resolution at 100 K for the most ordered crystals. Diffraction data have been processed to 3.85-A resolution. The symmetry of the diffraction pattern and the systematic absences indicate that the crystals have tetragonal symmetry, space group P4(1)22 or P4(3)22, a = b = 96.51 A, c = 148.55 A. A value of 4.12 A(3)/Da for V(M) was obtained for one monomer in the asymmetric unit (eight molecules per unit cell). Our study is the first characterization of a three-dimensional crystal of a G protein-coupled receptor and may be valuable for future structural studies on related receptors of this important superfamily.     

G-protein coupled receptor structure

Because of their central role in regulation of cellular function, structure/function relationships for G-protein coupled receptors (GPCR) are of vital importance, yet only recently have sufficient data been obtained to begin mapping those relationships. GPCRs regulate a wide range of cellular processes, including the senses of taste, smell, and vision, and control a myriad of intracellular signaling systems in response to external stimuli. Many diseases are linked to GPCRs. A critical need exists for structural information to inform studies on mechanism of receptor action and regulation. X-ray crystal structures of only one GPCR, in an inactive state, have been obtained to date. However considerable structural information for a variety of GPCRs has been obtained using non-crystallographic approaches. This review begins with a review of the very earliest GPCR structural information, mostly derived from rhodopsin. Because of the difficulty in crystallizing GPCRs for X-ray crystallography, the extensive published work utilizing alternative approaches to GPCR structure is reviewed, including determination of three-dimensional structure from sparse constraints. The available X-ray crystallographic analyses on bovine rhodopsin are reviewed as the only available high-resolution structures for any GPCR. Structural information available on ligand binding to several receptors is included. The limited information on excited states of receptors is also reviewed. It is concluded that while considerable basic structural information has been obtained, more data are needed to describe the molecular mechanism of activation of a GPCR.     

Structural basis for ligand binding and specificity in adrenergic receptors: implications for GPCR-targeted drug discovery

Crystal structures of engineered human beta 2-adrenergic receptors (ARs) in complex with an inverse agonist ligand, carazolol, provide three-dimensional snapshots of the disposition of seven transmembrane helices and the ligand-binding site of an important G protein-coupled receptor (GPCR). As expected, beta 2-AR shares substantial structural similarities with rhodopsin, the dim-light photoreceptor of the rod cell. However, although carazolol and the 11- cis-retinylidene moiety of rhodopsin are situated in the same general binding pocket, the second extracellular (E2) loop structures are quite distinct. E2 in rhodopsin shows beta-sheet structure and forms part of the chromophore-binding site. In the beta 2-AR, E2 is alpha-helical and seems to be distinct from the receptor's active site, allowing a potential entry pathway for diffusible ligands. The structures, together with extensive structure-activity relationship (SAR) data from earlier studies, provide insight about possible structural determinants of ligand specificity and how the binding of agonist ligands might alter receptor conformation. We review key features of the new beta 2-AR structures in the context of recent complementary work on the conformational dynamics of GPCRs. We also report 600 ns molecular dynamics simulations that quantified beta 2-AR receptor mobility in a membrane bilayer environment and show how the binding of an agonist ligand, adrenaline (epinephrine), causes conformational changes to the ligand-binding pocket and neighboring helices.     

Crystals of native and modified bovine rhodopsins and their heavy atom derivatives

Rhodopsin, the pigment protein responsible for dim-light vision, is a G protein-coupled receptor that converts light absorption into the activation of a G protein, transducin, to initiate the visual response. We have crystallised detergent-solubilised bovine rhodopsin in the native form and after chemical modifications as needles 10-40 microm in cross-section. The crystals belong to the trigonal space group P3(1), with two molecules of rhodopsin per asymmetric unit, related by a non-crystallographic 2-fold axis parallel with the crystallographic screw axis along c (needle axis). The unit cell dimensions are a=103.8 A, c=76.6 A for native rhodopsin, but vary over a wide range after heavy atom derivatisation, with a between 101.5 A and 113.9 A, and c between 76.6 A and 79.2 A. Rhodopsin molecules are packed with the bundle of transmembrane helices tilted from the c-axis by about 100 degrees . The two molecules in the asymmetric unit form contacts along the entire length of their transmembrane helices 5 in an antiparallel orientation, and they are stacked along the needle axis according to the 3-fold screw symmetry. Hence hydrophobic contacts are prominent at protein interfaces both along and normal to the needle axis. The best crystals of native rhodopsin in this crystal form diffracted X-rays from a microfocused synchrotron source to 2.55 A maximum resolution. We describe steps taken to extend the diffraction limit from about 10 A to 2.6 A.     

Molecular simulations and solid-state NMR investigate dynamical structure in rhodopsin activation

Rhodopsin has served as the primary model for studying G protein-coupled receptors (GPCRs)-the largest group in the human genome, and consequently a primary target for pharmaceutical development. Understanding the functions and activation mechanisms of GPCRs has proven to be extraordinarily difficult, as they are part of a complex signaling cascade and reside within the cell membrane. Although X-ray crystallography has recently solved several GPCR structures that may resemble the activated conformation, the dynamics and mechanism of rhodopsin activation continue to remain elusive. Notably solid-state ((2))H NMR spectroscopy provides key information pertinent to how local dynamics of the retinal ligand change during rhodopsin activation. When combined with molecular mechanics simulations of proteolipid membranes, a new paradigm for the rhodopsin activation process emerges. Experiment and simulation both suggest that retinal isomerization initiates the rhodopsin photocascade to yield not a single activated structure, but rather an ensemble of activated conformational states. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Interaction of a G protein-coupled receptor with a G protein-derived peptide induces structural changes in both peptide and receptor: a Fourier-transform infrared study using isotopically labeled peptides

G protein-coupled receptor signaling involves productive interaction between agonist-activated receptor and G protein. We have used Fourier-transform infrared difference spectroscopy to examine the interaction between the active Meta II state of the visual pigment rhodopsin with a peptide analogue corresponding to the C terminus of the alpha-subunit of the G protein transducin. Formation of the receptor-peptide complex evokes a spectral signature consisting of conformationally sensitive amide I and amide II difference bands. In order to distinguish between amide backbone contributions of the peptide and of the receptor moiety to the vibrational spectra, we employed complete (13)C,(15)N-labeling of the peptide. This isotopic labeling downshifts selectively the bands of the peptide, which can thus be extracted. Our results show that formation of the complex between the activated Meta II receptor state and the peptide is accompanied by structural changes of the peptide, and of the receptor, indicating that the conformation of the Meta II.peptide complex is different from that of Meta II. This result implies that the activated receptor state has conformational flexibility. Binding of the peptide to the activated receptor state stabilizes a substate that deviates from that stabilized only by the agonist.     

First principles predictions of the structure and function of g-protein-coupled receptors: validation for bovine rhodopsin

G-protein-coupled receptors (GPCRs) are involved in cell communication processes and with mediating such senses as vision, smell, taste, and pain. They constitute a prominent superfamily of drug targets, but an atomic-level structure is available for only one GPCR, bovine rhodopsin, making it difficult to use structure-based methods to design receptor-specific drugs. We have developed the MembStruk first principles computational method for predicting the three-dimensional structure of GPCRs. In this article we validate the MembStruk procedure by comparing its predictions with the high-resolution crystal structure of bovine rhodopsin. The crystal structure of bovine rhodopsin has the second extracellular (EC-II) loop closed over the transmembrane regions by making a disulfide linkage between Cys-110 and Cys-187, but we speculate that opening this loop may play a role in the activation process of the receptor through the cysteine linkage with helix 3. Consequently we predicted two structures for bovine rhodopsin from the primary sequence (with no input from the crystal structure)-one with the EC-II loop closed as in the crystal structure, and the other with the EC-II loop open. The MembStruk-predicted structure of bovine rhodopsin with the closed EC-II loop deviates from the crystal by 2.84 A coordinate root mean-square (CRMS) in the transmembrane region main-chain atoms. The predicted three-dimensional structures for other GPCRs can be validated only by predicting binding sites and energies for various ligands. For such predictions we developed the HierDock first principles computational method. We validate HierDock by predicting the binding site of 11-cis-retinal in the crystal structure of bovine rhodopsin. Scanning the whole protein without using any prior knowledge of the binding site, we find that the best scoring conformation in rhodopsin is 1.1 A CRMS from the crystal structure for the ligand atoms. This predicted conformation has the carbonyl O only 2.82 A from the N of Lys-296. Making this Schiff base bond and minimizing leads to a final conformation only 0.62 A CRMS from the crystal structure. We also used HierDock to predict the binding site of 11-cis-retinal in the MembStruk-predicted structure of bovine rhodopsin (closed loop). Scanning the whole protein structure leads to a structure in which the carbonyl O is only 2.85 A from the N of Lys-296. Making this Schiff base bond and minimizing leads to a final conformation only 2.92 A CRMS from the crystal structure. The good agreement of the ab initio-predicted protein structures and ligand binding site with experiment validates the use of the MembStruk and HierDock first principles' methods. Since these methods are generic and applicable to any GPCR, they should be useful in predicting the structures of other GPCRs and the binding site of ligands to these proteins.     

Three-dimensional structure of an invertebrate rhodopsin and basis for ordered alignment in the photoreceptor membrane

Invertebrate rhodopsins activate a G-protein signalling pathway in microvillar photoreceptors. In contrast to the transducin-cyclic GMP phosphodiesterase pathway found in vertebrate rods and cones, visual transduction in cephalopod (squid, octopus, cuttlefish) invertebrates is signalled via Gq and phospholipase C. Squid rhodopsin contains the conserved residues of the G-protein coupled receptor (GPCR) family, but has only 35% identity with mammalian rhodopsins. Unlike vertebrate rhodopsins, cephalopod rhodopsin is arranged in an ordered lattice in the photoreceptor membranes. This organization confers sensitivity to the plane of polarized light and also provides the optimal orientation of the linear retinal chromophores in the cylindrical microvillar membranes for light capture. Two-dimensional crystals of squid rhodopsin show a rectilinear arrangement that is likely to be related to the alignment of rhodopsins in vivo.Here, we present a three-dimensional structure of squid rhodopsin determined by cryo-electron microscopy of two-dimensional crystals. Docking the atomic structure of bovine rhodopsin into the squid density map shows that the helix packing and extracellular plug structure are conserved. In addition, there are two novel structural features revealed by our map. The linear lattice contact appears to be made by the transverse C-terminal helix lying on the cytoplasmic surface of the membrane. Also at the cytoplasmic surface, additional density may correspond to a helix 5-6 loop insertion found in most GPCRs relative to vertebrate rhodopsins. The similarity supports the conservation in structure of rhodopsins (and other G-protein-coupled receptors) from phylogenetically distant organisms. The map provides the first indication of the structural basis for rhodopsin alignment in the microvillar membrane.     

Structural insights into agonist-induced activation of G-protein-coupled receptors

Recent years have seen tremendous breakthroughs in structure determination of G-protein-coupled receptors (GPCRs). In 2011, two agonist-bound active-state structures of rhodopsin have been published. Together with structures of several rhodopsin activation intermediates and a wealth of biochemical and spectroscopic information, they provide a unique structural framework on which to understand GPCR activation. Here we use this framework to compare the recent crystal structures of the agonist-bound active states of the β(2) adrenergic receptor (β(2)AR) and the A(2A) adenosine receptor (A(2A)AR). While activation of these three GPCRs results in rearrangements of TM5 and TM6, the extent of this conformational change varies considerably. Displacements of the cytoplasmic side of TM6 ranges between 3 and 8? depending on whether selective stabilizers of the active conformation are used (i.e. a G-protein peptide in the case of rhodopsin or a conformationally selective nanobody in the case of the β(2)AR) or not (A(2A)AR). The agonist-induced conformational changes in the ligand-binding pocket are largely receptor specific due to the different chemical nature of the agonists. However, several similarities can be observed, including a relocation of conserved residues W6.48 and F6.44 towards L5.51 and P5.50, and of I/L3.40 away from P5.50. This transmission switch links agonist binding to the movement of TM5 and TM6 through the rearrangement of the TM3-TM5-TM6 interface, and possibly constitutes a common theme of GPCR activation.