Comparative Sequence and Structural Analyses of G-Protein-Coupled Receptor Crystal Structures and Implications for Molecular Models

Background

Up until recently the only available experimental (high resolution) structure of a G-protein-coupled receptor (GPCR) was that of bovine rhodopsin. In the past few years the determination of GPCR structures has accelerated with three new receptors, as well as squid rhodopsin, being successfully crystallized. All share a common molecular architecture of seven transmembrane helices and can therefore serve as templates for building molecular models of homologous GPCRs. However, despite the common general architecture of these structures key differences do exist between them. The choice of which experimental GPCR structure(s) to use for building a comparative model of a particular GPCR is unclear and without detailed structural and sequence analyses, could be arbitrary. The aim of this study is therefore to perform a systematic and detailed analysis of sequence-structure relationships of known GPCR structures.

Methodology

We analyzed in detail conserved and unique sequence motifs and structural features in experimentally-determined GPCR structures. Deeper insight into specific and important structural features of GPCRs as well as valuable information for template selection has been gained. Using key features a workflow has been formulated for identifying the most appropriate template(s) for building homology models of GPCRs of unknown structure. This workflow was applied to a set of 14 human family A GPCRs suggesting for each the most appropriate template(s) for building a comparative molecular model.

Conclusions

The available crystal structures represent only a subset of all possible structural variation in family A GPCRs. Some GPCRs have structural features that are distributed over different crystal structures or which are not present in the templates suggesting that homology models should be built using multiple templates. This study provides a systematic analysis of GPCR crystal structures and a consistent method for identifying suitable templates for GPCR homology modelling that will help to produce more reliable three-dimensional models.     

Structure modeling of all identified G protein-coupled receptors in the human genome

G protein-coupled receptors (GPCRs), encoded by about 5% of human genes, comprise the largest family of integral membrane proteins and act as cell surface receptors responsible for the transduction of endogenous signal into a cellular response. Although tertiary structural information is crucial for function annotation and drug design, there are few experimentally determined GPCR structures. To address this issue, we employ the recently developed threading assembly refinement (TASSER) method to generate structure predictions for all 907 putative GPCRs in the human genome. Unlike traditional homology modeling approaches, TASSER modeling does not require solved homologous template structures; moreover, it often refines the structures closer to native. These features are essential for the comprehensive modeling of all human GPCRs when close homologous templates are absent. Based on a benchmarked confidence score, approximately 820 predicted models should have the correct folds. The majority of GPCR models share the characteristic seven-transmembrane helix topology, but 45 ORFs are predicted to have different structures. This is due to GPCR fragments that are predominantly from extracellular or intracellular domains as well as database annotation errors. Our preliminary validation includes the automated modeling of bovine rhodopsin, the only solved GPCR in the Protein Data Bank. With homologous templates excluded, the final model built by TASSER has a global C(alpha) root-mean-squared deviation from native of 4.6 angstroms, with a root-mean-squared deviation in the transmembrane helix region of 2.1 angstroms. Models of several representative GPCRs are compared with mutagenesis and affinity labeling data, and consistent agreement is demonstrated. Structure clustering of the predicted models shows that GPCRs with similar structures tend to belong to a similar functional class even when their sequences are diverse. These results demonstrate the usefulness and robustness of the in silico models for GPCR functional analysis. All predicted GPCR models are freely available for noncommercial users on our Web site (http://www.bioinformatics.buffalo.edu/GPCR).     

Exploring the structure of opioid receptors with homology modeling based on single and multiple templates and subsequent docking: A comparative study

Opioid receptors are the principal targets for opioids, which have been used as analgesics for centuries. Opioid receptors belong to the rhodopsin family of G-protein coupled receptors (GPCRs). In the absence of crystal structures of opioid receptors, 3D homology models have been reported with bovine rhodopsin as a template, though the sequence homology is low. Recently, it has been reported that use of multiple templates results in a better model for a target having low sequence identity with a single template. With the objective of carrying out a comparative study on the structural quality of the 3D models based on single and multiple templates, the homology models for opioid receptors (mu, delta and kappa) were generated using bovine rhodopsin as single template and the recently deposited crystal structures of squid rhodopsin, turkey β-1 and human β-2 adrenoreceptors along with bovine rhodopsin as multiple templates. In this paper we report the results of comparison between the refined 3D models based on multiple sequence alignment (MSA) and models built with bovine rhodopsin as template, using validation programs PROCHECK, PROSA, Verify 3D, Molprobity and docking studies. The results indicate that homology models of mu and kappa with multiple templates are better than those built with only bovine rhodopsin as template, whereas, in many aspects, the homology model of delta opioid receptor with single template is better with respect to the model based on multiple templates. Three nonselective ligands were docked to both the models of mu, delta and kappa opioid receptors using GOLD 3.1. The results of docking complied well with the pharamacophore, reported for nonspecific opioid ligands. The comparison of docking results for models with multiple templates and those with single template have been discussed in detail. Three selective ligands for each receptor were also docked. As the crystallographic structures are not yet known, this comparison will help in choosing better homology models of opioid receptors for studying ligand receptor interactions to design new potent opioid antagonists.     

Molecular dynamics simulations of bovine rhodopsin: influence of protonation states and different membrane-mimicking environments

G-protein coupled receptors (GPCRs) are a protein family of outstanding pharmaceutical interest. GPCR homology models, based on the crystal structure of bovine rhodopsin, have been shown to be valuable tools in the drug-design process. The initial model is often refined by molecular dynamics (MD) simulations, a procedure that has been recently discussed controversially. We therefore analyzed MD simulations of bovine rhodopsin in order to identify contacts that could serve as constraints in the simulation of homology models. Additionally, the effect of an N-terminal truncation, the nature of the membrane mimic, the influence of varying protonation states of buried residues and the importance of internal water molecules was analyzed. All simulations were carried out using the program-package GROMACS. While N-terminal truncation negatively influenced the overall protein stability, a stable simulation was possible in both solvent environments. As regards the protonation state of titratable sites, the experimental data could be reproduced by the program UHBD (University of Houston Brownian Dynamics), suggesting its application for studying homology models of GPCRs. A high flexibility was observed for internal water molecules at some sites. Finally, interhelical hydrogen-bonding interactions could be derived, which can now serve as constraints in the simulations of GPCR homology models.     

Protein‐based virtual screening of chemical databases. II. Are homology models of g‐protein coupled receptors suitable targets?

The aim of the current study is to investigate whether homology models of G-Protein-Coupled Receptors (GPCRs) that are based on bovine rhodopsin are reliable enough to be used for virtual screening of chemical databases. Starting from the recently described 2.8 A-resolution X-ray structure of bovine rhodopsin, homology models of an "antagonist-bound" form of three human GPCRs (dopamine D3 receptor, muscarinic M1 receptor, vasopressin V1a receptor) were constructed. The homology models were used to screen three-dimensional databases using three different docking programs (Dock, FlexX, Gold) in combination with seven scoring functions (ChemScore, Dock, FlexX, Fresno, Gold, Pmf, Score). Rhodopsin-based homology models turned out to be suitable, indeed, for virtual screening since known antagonists seeded in the test databases could be distinguished from randomly chosen molecules. However, such models are not accurate enough for retrieving known agonists. To generate receptor models better suited for agonist screening, we developed a new knowledge- and pharmacophore-based modeling procedure that might partly simulate the conformational changes occurring in the active site during receptor activation. Receptor coordinates generated by this new procedure are now suitable for agonist screening. We thus propose two alternative strategies for the virtual screening of GPCR ligands, relying on a different set of receptor coordinates (antagonist-bound and agonist-bound states).     

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.     

Crystal structure of rhodopsin: implications for vision and beyond

A heptahelical transmembrane bundle is a common structural feature of G-protein-coupled receptors (GPCRs) and bacterial retinal-binding proteins, two functionally distinct groups of membrane proteins. Rhodopsin, a photoreceptor protein involved in photopic (rod) vision, is a prototypical GPCR that contains 11-cis-retinal as its intrinsic chromophore ligand. Therefore, uniquely, rhodopsin is a GPCR and also a retinal-binding protein, but is not found in bacteria. Rhodopsin functions as a typical GPCR in processes that are triggered by light and photoisomerization of its ligand. Bacteriorhodopsin is a light-driven proton pump with an all-trans-retinal chromophore that photoisomerizes to 13-cis-retinal. The recent crystal structure determination of bovine rhodopsin revealed a structure that is not similar to previously established bacteriorhodopsin structures. Both groups of proteins have a heptahelical transmembrane bundle structure, but the helices are arranged differently. The activation of rhodopsin involves rapid cis-trans photoisomerization of the chromophore, followed by slower and incompletely defined structural rearrangements. For rhodopsin and related receptors, a common mechanism is predicted for the formation of an active state intermediate that is capable of interacting with G proteins.     

Role of rhodopsin N-terminus in structure and function of rhodopsin-bitter taste receptor chimeras

The bitter taste receptors (T2Rs) belong to the G protein-coupled receptor (GPCR) superfamily. In humans, bitter taste sensation is mediated by 25 T2Rs. Structure–function studies on T2Rs are impeded by the low-level expression of these receptors. Different lengths of rhodopsin N-terminal sequence inserted at the N-terminal region of T2Rs are commonly used to express these receptors in heterologous systems. While the additional sequences were reported, to enhance the expression of the T2Rs, the local structural perturbations caused by these sequences and its effect on receptor function or allosteric ligand binding were not characterized. In this study, we elucidated how different lengths of rhodopsin N-terminal sequence effect the structure and function of the bitter taste receptor, T2R4. Guided by molecular models of T2R4 built using a rhodopsin crystal structure as template, we constructed chimeric T2R4 receptors containing the rhodopsin N-terminal 33 and 38 amino acids. The chimeras were functionally characterized using calcium imaging, and receptor expression was determined by flow cytometry. Our results show that rhodopsin N-terminal 33 amino acids enhance expression of T2R4 by 2.5-fold and do not cause perturbations in the receptor structure.     

Engineering of an artificial light-modulated potassium channel

Ion Channel-Coupled Receptors (ICCRs) are artificial receptor-channel fusion proteins designed to couple ligand binding to channel gating. We previously validated the ICCR concept with various G protein-coupled receptors (GPCRs) fused with the inward rectifying potassium channel Kir6.2. Here we characterize a novel ICCR, consisting of the light activated GPCR, opsin/rhodopsin, fused with Kir6.2. To validate our two-electrode voltage clamp (TEVC) assay for activation of the GPCR, we first co-expressed the apoprotein opsin and the G protein-activated potassium channel Kir3.1(F137S) (Kir3.1*) in Xenopus oocytes. Opsin can be converted to rhodopsin by incubation with 11-cis retinal and activated by light-induced retinal cis→trans isomerization. Alternatively opsin can be activated by incubation of oocytes with all-trans-retinal. We found that illumination of 11-cis-retinal-incubated oocytes co-expressing opsin and Kir3.1* caused an immediate and long-lasting channel opening. In the absence of 11-cis retinal, all-trans-retinal also opened the channel persistently, although with slower kinetics. We then used the oocyte/TEVC system to test fusion proteins between opsin/rhodopsin and Kir6.2. We demonstrate that a construct with a C-terminally truncated rhodopsin responds to light stimulus independent of G protein. By extending the concept of ICCRs to the light-activatable GPCR rhodopsin we broaden the potential applications of this set of tools.     

Differences in binding sites of two melatonin receptors help to explain their selectivity to some melatonin analogs: a molecular modeling study

Numerous diseases have been linked to the malfunction of G-protein coupled receptors (GPCRs). Their adequate treatment requires rational design of new high-affinity and high-selectivity drugs targeting these receptors. In this work, we report three-dimensional models of the human MT(1) and MT(2) melatonin receptors, members of the GPCR family. The models are based on the X-ray structure of bovine rhodopsin. The computational approach employs an original procedure for optimization of receptor-ligand structures. It includes rotation of one of the transmembrane alpha-helices around its axis with simultaneous assessment of quality of the resulting complexes according to a number of criteria we have developed for this purpose. The optimal geometry of the receptor-ligand binding is selected based on the analysis of complementarity of hydrophobic/hydrophilic properties between the ligand and its protein environment in the binding site. The elaborated "optimized" models are employed to explore the details of protein-ligand interactions for melatonin and a number of its analogs with known affinity to MT(1) and MT(2) receptors. The models permit rationalization of experimental data, including those that were not used in model building. The perspectives opened by the constructed models and by the optimization procedure in the design of new drugs are discussed.     

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.     

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.     

Prediction of heterodimerization interfaces of G-protein coupled receptors with a new subtractive correlated mutation method

Recent studies employing differential epitope tagging, selective immunoprecipitation of receptor complexes and fluorescence or bioluminescence resonance energy transfer techniques provide direct evidence for heterodimerization between both closely and distantly related members of the G-protein coupled receptor (GPCR) family. Since heterodimerization appears to play a role in modulating agonist affinity, efficacy and/or trafficking properties, the molecular models of GPCRs required to understand receptor function must consider these oligomerization hypotheses. To advance knowledge in this field, we present here a computational approach based on correlated mutation analysis and the structural information contained in three-dimensional molecular models of the transmembrane regions of GPCRs built using the rhodopsin crystal structure as a template. The new subtractive correlated mutation method reveals likely heterodimerization interfaces amongst the different alternatives for the positioning of two tightly packed bundles of seven transmembrane domains next to each other in contact heterodimers of GPCRs. Predictions are applied to GPCRs in the class of opioid receptors. However, in the absence of a known structure of any GPCR dimer, the features of the method and predictions are also illustrated and analyzed for a dimeric complex of known structure.     

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.     

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.     

The structural evolution of a P2Y-like G-protein-coupled receptor

Based on the now available crystallographic data of the G-protein-coupled receptor (GPCR) prototype rhodopsin, many studies have been undertaken to build or verify models of other GPCRs. Here, we mined evolution as an additional source of structural information that may guide GPCR model generation as well as mutagenesis studies. The sequence information of 61 cloned orthologs of a P2Y-like receptor (GPR34) enabled us to identify motifs and residues that are important for maintaining the receptor function. The sequence data were compared with available sequences of 77 rhodopsin orthologs. Under a negative selection mode, only 17% of amino acid residues were preserved during 450 million years of GPR34 evolution. On the contrary, in rhodopsin evolution approximately 43% residues were absolutely conserved between fish and mammals. Despite major differences in their structural conservation, a comparison of structural data suggests that the global arrangement of the transmembrane core of GPR34 orthologs is similar to rhodopsin. The evolutionary approach was further applied to functionally analyze the relevance of common scaffold residues and motifs found in most of the rhodopsin-like GPCRs. Our analysis indicates that, in contrast to other GPCRs, maintaining the unique function of rhodopsin requires a more stringent network of relevant intramolecular constrains.     

Efficient coupling of transducin to monomeric rhodopsin in a phospholipid bilayer

G protein-coupled receptors (GPCRs) are seven transmembrane domain proteins that transduce extracellular signals across the plasma membrane and couple to the heterotrimeric family of G proteins. Like most intrinsic membrane proteins, GPCRs are capable of oligomerization, the function of which has only been established for a few different receptor systems. One challenge in understanding the function of oligomers relates to the inability to separate monomeric and oligomeric receptor complexes in membrane environments. Here we report the reconstitution of bovine rhodopsin, a GPCR expressed in the retina, into an apolipoprotein A-I phospholipid particle, derived from high density lipoprotein (HDL). We demonstrate that rhodopsin, when incorporated into these 10 nm reconstituted HDL (rHDL) particles, is monomeric and functional. Rhodopsin.rHDL maintains the appropriate spectral properties with respect to photoactivation and formation of the active form, metarhodopsin II. Additionally, the kinetics of metarhodopsin II decay is similar between rhodopsin in native membranes and rhodopsin in rHDL particles. Photoactivation of monomeric rhodopsin.rHDL also results in the rapid activation of transducin, at a rate that is comparable with that found in native rod outer segments and 20-fold faster than rhodopsin in detergent micelles. These data suggest that monomeric rhodopsin is the minimal functional unit in G protein activation and that oligomerization is not absolutely required for this process.     

Light causes phosphorylation of nonactivated visual pigments in intact mouse rod photoreceptor cells

Phosphorylation of G-protein-coupled receptors (GPCRs) is a required step in signal deactivation. Rhodopsin, a prototypical GPCR, exhibits high gain phosphorylation in vitro whereby a hundred-fold molar excess of phosphates are incorporated into the rhodopsin pool per molecule of activated rhodopsin. The extent by which high gain phosphorylation occurs in the intact mammalian photoreceptor cell, and the molecular mechanism underlying this reaction in vivo, is not known. Trans-phosphorylation is a mechanism proposed for high gain phosphorylation, whereby rhodopsin kinase, upon phosphorylating the activated receptor, continues to phosphorylate nearby nonactivated rhodopsin. We used two different transgenic mouse models to test whether trans-phosphorylation occurs in the intact photoreceptor cell. The first transgenic model expressed a murine cone pigment, S-opsin, together with the endogenous rhodopsin in the rod cell. We showed that selective stimulation of rhodopsin also led to phosphorylation of S-opsin. The second mouse model expressed the constitutively active human opsin mutant K296E. K296E, in the arrestin-/- background, also led to phosphorylation of endogenous mouse rhodopsin in the dark-adapted retina. Both mouse models provide strong support of trans-phosphorylation as an underlying mechanism of high gain phosphorylation, and provide evidence that a substantial fraction of nonactivated visual pigments becomes phosphorylated through this mechanism. Because activated, phosphorylated receptors exhibit decreased catalytic activity, our results suggest that dephosphorylation would be an important step in the full recovery of visual sensitivity during dark adaptation. These results may also have implications for other GPCR signaling pathways.     

Comparison of the transmembrane helices of bovine rhodopsin in the crystal structure and the C α template based on cryo-electron microscopy maps and sequence analysis of the G protein-coupled receptors

G protein-coupled receptors (GPCR) are activated by a diverse array of extracellular signals, ranging from light to polypeptide molecules. The receptors propagate these signals intracellularly using G protein secondary messenger pathways. A common feature in the architecture of these receptors is their seven transmembrane domains. The first crystal structure of a GPCR, bovine rhodopsin, has recently been solved at 2.8 Å. We compared the seven membrane-spanning helices (TMH) from the crystal structure of bovine rhodopsin with those from the low-resolution model of bovine rhodopsin based on the cryo-electron microscopy structure of frog rhodopsin developed by Dr Joyce Baldwin. The model developed by Baldwin used a consensus sequence approach to predict the rotational position of each helix with respect to the other six helices. Superposition of the entire helix bundle of the Baldwin model with the crystal structure gave a RMS difference (RMSD) of 3.2 Å for the 198 C f atoms which suggests a high level of similarity in the arrangement of the helices. Except for TMH IV (RMSD of 4.0 Å), the position of corresponding helices within the helix bundle overlapped well. The superposition of individual helices showed that the RMSD values over 3 Å in the global superposition were largely due to one or more of the following: (i) differences in the unraveling and kinks for these helices, (ii) translation of TMH perpendicular to the membrane and (iii) rotation of helices up to 31°, except for TMH IV in which an additional contribution to the RMSD came from the aforementioned observation. As other crystal structures of GPCRs become available, a comparison with the Baldwin consensus model may reveal larger differences than those observed here.     

Structural studies of the putative helix 8 in the human beta(2) adrenergic receptor: an NMR study

The recently reported crystal structure of bovine rhodopsin revealed a cytoplasmic helix (helix 8) in addition to the seven transmembrane helices. This domain is roughly perpendicular to the transmembrane bundle in the presence of an interface and may be a loop-like structure in the absence of an interface. Several studies carried out on this domain suggested that it might act as a conformational switch between the inactive and activated states of this G-protein coupled receptor (GPCR). These results raised the question whether helix 8 may be an important feature of other GPCRs as well. To explore this question, we determined the structure of a peptide representing the putative helix 8 domain in another receptor that belongs to the rhodopsin family of GPCRs, the human beta(2) adrenergic receptor (hbeta(2)AR), using two-dimensional (1)H nuclear magnetic resonance (NMR). The key results from this structural study are that the putative helix 8 domain is helical in detergent and in DMSO while in water this region is disordered; the conformation is therefore dependent upon the environment. Comparison of data from five GPCRs suggests that these observations may be generally important for GPCR structure and function.