The mammalian dim-light photoreceptor rhodopsin is a prototypic G protein coupled receptor (GPCR), interacting with the G protein, transducin, rhodopsin kinase, and arrestin. All of these proteins interact with rhodopsin at its cytoplasmic surface. Structural and modeling studies have provided in-depth descriptions of the respective interfaces. Overlap and thus competition for binding surfaces is a major regulatory mechanism for signal processing. Recently, it was found that the same surface is also targeted by small molecules. These ligands can directly interfere with the binding and activation of the proteins of the signal transduction cascade, but they can also allosterically modulate the retinal ligand binding pocket. Because the pocket that is targeted contains residues that are highly conserved across Class A GPCRs, these findings imply that it may be possible to target multiple GPCRs with the same ligand(s). This is desirable for example in complex diseases such as cancer where multiple GPCRs participate in the disease networks.
G protein coupled receptors; allostery; conformational changes; docking; protein-protein interactions
Recent solved structures of G protein-coupled receptors (GPCRs) provide insights into variation of the structure and molecular mechanisms of GPCR activation. In this review we provide evidence for the emerging paradigm of domain coupling facilitated by intrinsic disorder of the ligand-free state in GPCRs. The structure-function and dynamic studies suggest that ligand-bound GPCRs exhibit multiple active conformations in initiating cellular signals. Long-range intra-molecular and inter-molecular interactions at distant sites on the same receptor are crucial factors that modulate signaling function of GPCRs. Positive or negative coupling between the extracellular, the transmembrane and the intracellular domains facilitates cooperativity of activating “switches” as requirements for the functional plasticity of GPCRs. Awareness that allosteric ligands robustly affect domain coupling provides a novel mechanistic basis for rational drug development, small molecule antagonism and GPCR regulation by classical, as well as non-classical modes.
Building reliable structural models of G protein-coupled receptors (GPCRs) is a difficult task due to the paucity of suitable templates, low sequence identity, and the wide variety of ligand specificities within the superfamily. Template-based modeling is known to be the most successful method for protein structure prediction. However, refinement of homology models within 1–3 Å Cα RMSD of the native structure remains a major challenge. Here we address this problem by developing a novel protocol (foldGPCR) for modeling the transmembrane (TM) region of GPCRs in complex with a ligand, aimed to accurately model the structural divergence between the template and target in the TM helices. The protocol is based on predicted conserved inter-residue contacts between the template and target, and exploits an all-atom implicit membrane force field. The placement of the ligand in the binding pocket is guided by biochemical data. The foldGPCR protocol is implemented by a stepwise hierarchical approach, in which the TM helical bundle and the ligand are assembled by simulated annealing trials in the first step, and the receptor-ligand complex is refined with replica exchange sampling in the second step. The protocol is applied to model the human β2-adrenergic receptor (β2AR) bound to carazolol, using contacts derived from the template structure of bovine rhodopsin. Comparison to the X-ray crystal structure of the β2AR shows that our protocol is particularly successful in accurately capturing helix backbone irregularities and helix-helix packing interactions that distinguish rhodopsin from β2AR.
class A GPCR; structure prediction; simulated annealing; ligand binding; implicit solvent; membrane protein
The adenosine class of G protein-coupled receptors mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A2A adenosine receptor in complex with a high affinity subtype-selective antagonist, ZM241385, to 2.6 Å resolution. Four disulfide bridges in the extracellular domain combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures defines a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extended conformation perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core in ligand recognition by this class of GPCRs, and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors.
G-protein coupled receptors (GPCRs) are involved in many different physiological processes and their function can be modulated by small molecules which bind in the transmembrane (TM) domain. Because of their structural and sequence conservation, the TM domains are often used in bioinformatics approaches to first create a multiple sequence alignment (MSA) and subsequently identify ligand binding positions. So far methods have been developed to predict the common ligand binding residue positions for class A GPCRs.
Here we present 1) ss-TEA, a method to identify specific ligand binding residue positions for any receptor, predicated on high quality sequence information. 2) The largest MSA of class A non olfactory GPCRs in the public domain consisting of 13324 sequences covering most of the species homologues of the human set of GPCRs. A set of ligand binding residue positions extracted from literature of 10 different receptors shows that our method has the best ligand binding residue prediction for 9 of these 10 receptors compared to another state-of-the-art method.
The combination of the large multi species alignment and the newly introduced residue selection method ss-TEA can be used to rapidly identify subfamily specific ligand binding residues. This approach can aid the design of site directed mutagenesis experiments, explain receptor function and improve modelling. The method is also available online via GPCRDB at http://www.gpcr.org/7tm/.
Multiple sequence alignments become biologically meaningful only if conserved and functionally important residues and
secondary structural elements preserved can be identified at equivalent positions. This is particularly important for transmembrane
proteins like G-protein coupled receptors (GPCRs) with seven transmembrane helices. TM-MOTIF is a software package and an
effective alignment viewer to identify and display conserved motifs and amino acid substitutions (AAS) at each position of the
aligned set of homologous sequences of GPCRs. The key feature of the package is to display the predicted membrane topology for
seven transmembrane helices in seven colours (VIBGYOR colouring scheme) and to map the identified motifs on its respective
helices /loop regions. It is an interactive package which provides options to the user to submit query or pre-aligned set of GPCR
sequences to align with a reference sequence, like rhodopsin, whose structure has been solved experimentally. It also provides the
possibility to identify the nearest homologue from the available inbuilt GPCR or Olfactory Receptor cluster dataset whose
association is already known for its receptor type.
The database is available for free at email@example.com
Transmembrane helices; Membrane topology; Amino acid conservation and substitutions; GPCR cluster association
G protein coupled receptors (GPCRs) are seven transmembrane proteins that mediate the majority of cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs1,2,3,4,5 reveal structural conservation extending from the orthosteric ligand binding site in the transmembrane core to the cytoplasmic G protein coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse, and therefore represents an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the β2 adrenergic receptor: a salt bridge linking extracellular loops (ECLs) 2 and 3. Small molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G protein activation (agonist, neutral antagonist, and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide new insight into the dynamic behavior of GPCRs not addressable by static, inactive-state crystal structures.
The majority of transmembrane signal transduction in response to diverse external stimuli is mediated by G-protein coupled receptors (GPCRs) and are the principal signal transducers. GPCRs are characterized by seven membrane-spanning domains with an extracellular N-terminus and a cytoplasmic C-terminus which functions along with GTP-binding protein in a highly coordinated fashion. Role of heterotrimeric G-proteins in abiotic stresses has been reported, but the response of GPCR is not yet well characterized. In the present study we report the isolation of one putative GPCR (966 bp) from Indica rice (Oryza sativa cv. Indica group Swarna) and described its transcriptional regulation under abiotic stresses. Amino acid sequence analyses shows the presence of typical heptahelical transmembrane spanning domains with extracellular N-terminus involved in ligand binding and cytoplasm facing C-terminus that binds with heterotrimeric G-protein. Sequence analysis also confirmed the presence of all signature motifs required for functional GPCR. Domain and site prediction shows the presence of myristoylation sites for membrane association and protein kinase C sites for its desensitization. The transcript levels of rice GPCR was induced following NaCl and ABA treatments. However, in drought condition the expression profile of GPCR upregulated during early exposure which subsequently decreased. On the other hand it seems no significant effect due to cold and heat stress. These findings provide a direct evidence for transcriptional regulation of rice GPCR under abiotic stress conditions. These findings also suggest that GPCR can be exploited for promoting stress tolerance in plants.
abiotic stress; G-protein coupled receptor; myristoylation; protein kinase C; real-time PCR; rice; signal transduction
Heterotrimeric G proteins (Gα, Gβ/Gγ subunits) constitute one of the most important components of cell signaling cascade. G Protein Coupled Receptors (GPCRs) perceive many extracellular signals and transduce them to heterotrimeric G proteins, which further transduce these signals intracellular to appropriate downstream effectors and thereby play an important role in various signaling pathways. GPCRs exist as a superfamily of integral membrane protein receptors that contain seven transmembrane α-helical regions, which bind to a wide range of ligands. Upon activation by a ligand, the GPCR undergoes a conformational change and then activate the G proteins by promoting the exchange of GDP/GTP associated with the Gα subunit. This leads to the dissociation of Gβ/Gγ dimer from Gα. Both these moieties then become free to act upon their downstream effectors and thereby initiate unique intracellular signaling responses. After the signal propagation, the GTP of Gα-GTP is hydrolyzed to GDP and Gα becomes inactive (Gα-GDP), which leads to its re-association with the Gβ/Gγ dimer to form the inactive heterotrimeric complex. The GPCR can also transduce the signal through G protein independent pathway. GPCRs also regulate cell cycle progression. Till to date thousands of GPCRs are known from animal kingdom with little homology among them, but only single GPCR has been identified in plant system. The Arabidopsis GPCR was reported to be cell cycle regulated and also involved in ABA and in stress signaling. Here I have described a general mechanism of signal transduction through GPCR/G proteins, structure of GPCRs, family of GPCRs and plant GPCR and its role.
heterotrimeric G proteins; GPCRs; seven-transmembrane receptors; signal transduction; stress signaling
G-protein coupled receptors (GPCRs) are a class of drug targets of primary importance. However, receptor assays are based on measurement of either ligand displacement or downstream functional responses, rather than direct observation of ligand binding. Issues of allosteric modulation, probe dependence, and functional selectivity create challenges in selecting suitable assays formats. Therefore, a method that directly measures GPCR–ligand interactions, independent of binding site, probe, and signaling pathway would be a useful primary and orthogonal screening method. We have developed a GPCR biosensor assay protocol that offers the opportunity for high-throughput label-free screening that directly measures GPCR–ligand interactions. The biosensor-based direct screening method identifies the interaction of both orthosteric and allosteric ligands with solubilized, native GPCRs, in a label-free and cell-free environment, thus overcoming the limitations of indirect and displacement assay methods. We exemplify the method by the discovery of novel ligands for the chemokine receptor, CCR5, that are ligand efficient fragments.
Surface plasmon resonance; G-protein coupled receptors; CCR5; allosteric; fragments
G protein-coupled receptors (GPCRs) are a superfamily of seven transmembrane-spanning proteins involved in a wide array of physiological functions and are the most common targets of pharmaceuticals. This study aims to identify a cohort or clique of positions that share high mutual information. Using a multiple sequence alignment of the transmembrane (TM) domains, we calculated the mutual information between all inter-TM pairs of aligned positions and ranked the pairs by mutual information. A mutual information graph was constructed with vertices that corresponded to TM positions and edges between vertices were drawn if the mutual information exceeded a threshold of statistical significance. Positions with high degree (i.e. had significant mutual information with a large number of other positions) were found to line a well defined inter-TM ligand binding cavity for class A as well as class C GPCRs. Although the natural ligands of class C receptors bind to their extracellular N-terminal domains, the possibility of modulating their activity through ligands that bind to their helical bundle has been reported. Such positions were not found for class B GPCRs, in agreement with the observation that there are not known ligands that bind within their TM helical bundle. All identified key positions formed a clique within the MI graph of interest. For a subset of class A receptors we also considered the alignment of a portion of the second extracellular loop, and found that the two positions adjacent to the conserved Cys that bridges the loop with the TM3 qualified as key positions. Our algorithm may be useful for localizing topologically conserved regions in other protein families.
This review of the current literature on mutations in G protein-coupled receptors (GPCRs) of the rhodopsin-related family intends to draw inferences from amino acid sequences for single receptors and multiple sequence alignments with regard to the molecular architecture of this class of receptors. For this purpose a comprehensive list of mutations within the transmembrane helical regions (TMs; over 390 mutations from 38 different receptor subtypes) and their effects on function was compiled, and an alignment of known GPCR sequences (over 150 separate sequences) was made. Regions most prominently involved in ligand binding are located in TMs 3, 5, 6, and 7. Position 3.32 in TM3 is occupied by a D in all biogenic amine receptors (sequence conservation) but may be occupied by uncharged residues in other receptors while its role in ligand binding is analogous (function conservation). TMs 5, 6, and 7 display considerable sequence conservation throughout the majority of GPCRs investigated, but not necessarily at those positions involved in ligand binding. However, considerable function conservation is observed for positions 5.42 (frequently hydrophilic), 5.46 (small amino acids required for agonist binding to “small ligand” receptors), 6.52 and 7.39 (high variability), and 7.43 (frequently aromatic). A general conclusion of this review is that there is overwhelming conservation of structure-function correlates among GPCRs. Thus, it is now possible to cross-correlate the results of mutagenesis studies between GPCRs of different subfamilies, and to use those results to predict the function of specific residues in new GPCR sequences.
G protein-coupled receptors; GPCRs; transmembrane domains; mutagenesis; sequence alignment; identifiers; molecular architecture; molecular structure
Gremlin-1, a bone morphogenetic protein (BMP) antagonist, is overexpressed in various cancerous tissues but its role in carcinogenesis has not been established. Here, we report that gremlin-1 binds various cancer cell lines and this interaction is inhibited by our newly developed gremlin-1 antibody, GRE1. Gremlin-1 binding to cancer cells was unaffected by the presence of BMP-2, BMP-4, and BMP-7. In addition, the binding was independent of vascular endothelial growth factor receptor-2 (VEGFR2) expression on the cell surface. Addition of gremlin-1 to A549 cells induced a fibroblast-like morphology and decreased E-cadherin expression. In a scratch wound healing assay, A549 cells incubated with gremlin-1 or transfected with gremlin-1 showed increased migration, which was inhibited in the presence of the GRE1 antibody. Gremlin-1 transfected A549 cells also exhibited increased invasiveness as well as an increased growth rate. These effects were also inhibited by the addition of the GRE1 antibody. In conclusion, this study demonstrates that gremlin-1 directly interacts with cancer cells in a BMP- and VEGFR2-independent manner and can induce cell migration, invasion, and proliferation.
The superfamily of the seven transmembrane G-protein-coupled receptors (7TM/GPCRs) is the largest family of membrane-associated receptors. GPCRs are involved in the pathophysiology of numerous human diseases, and they constitute an estimated 30–40% of all drug targets. During the last two decades, GPCR oligomerization has been extensively studied using methods like bioluminescence resonance energy transfer (BRET) and today, receptor–receptor interactions within the GPCR superfamily is a well-established phenomenon. Evidence of the impact of GPCR oligomerization on, e.g., ligand binding, receptor expression, and signal transduction indicates the physiological and pharmacological importance of these receptor interactions. In contrast to the larger and more thoroughly studied GPCR subfamilies A and C, the B1 subfamily is small and comprises only 15 members, including, e.g., the secretin receptor, the glucagon receptor, and the receptors for parathyroid hormone (PTHR1 and PTHR2). The dysregulation of several family B1 receptors is involved in diseases, such as diabetes, chronic inflammation, and osteoporosis which underlines the pathophysiological importance of this GPCR subfamily. In spite of this, investigation of family B1 receptor oligomerization and especially its pharmacological importance is still at an early stage. Even though GPCR oligomerization is a well-established phenomenon, there is a need for more investigations providing a direct link between these interactions and receptor functionality in family B1 GPCRs. One example of the functional effects of GPCR oligomerization is the facilitation of allosterism including cooperativity in ligand binding to GPCRs. Here, we review the currently available data on family B1 GPCR homo- and heteromerization, mainly based on BRET investigations. Furthermore, we cover the functional influence of oligomerization on ligand binding as well as the link between oligomerization and binding cooperativity.
GPCRs; family B1; oligomerization; BRET; binding cooperativity
Numerous genes encode G protein-coupled receptors (GPCRs)-a main molecular target for drug therapy. Estimates indicate that the human genome contains approximately 600 GPCR genes. This article addresses therapeutic implications of sequence variations in GPCR genes. A number of inactivating and activating receptor mutations have been shown to cause a variety of (mostly rare) genetic disorders. However, pharmacogenetic and pharmacogenomic studies on GPCRs are scarce, and therapeutic relevance of variant receptor alleles often remains unclear. Confounding factors in assessing the therapeutic relevance of variant GPCR alleles include 1) interaction of a single drug with multiple closely related receptors, 2) poorly defined binding pockets that can accommodate drug ligands in different orientations or at alternative receptor domains, 3) possibility of multiple receptor conformations with distinct functions, and 4) multiple signaling pathways engaged by a single receptor. For example, antischizophrenic drugs bind to numerous receptors, several of which might be relevant to therapeutic outcome. Without knowing accurately what role a given receptor subtype plays in clinical outcome and how a sequence variation affects drug-induced signal transduction, we cannot predict the therapeutic relevance of a receptor variant. Genome-wide association studies with single nucleotide polymorphisms could identify critical target receptors for disease susceptibility and drug efficacy or toxicity.
G Protein-Coupled; Receptors; Drug Therapy; Pharmacogenomics; Pharmacogenetics
Controlling enzyme activity by ligand binding to a regulatory domain of choice may have many applications e.g. as biosensors and as tools in regulating cellular functions. However, until now only a small number of ligand-binding domains have been successfully linked to enzyme activity. G protein-coupled receptors (GPCR) are capable of recognizing an extraordinary structural variety of extracellular signals including inorganic and organic molecules. Ligand binding to GPCR results in conformational changes involving the transmembrane helices. Here, we assessed whether ligand-induced conformational changes within the GPCR helix bundle can be utilized to control the activity of an integrated enzyme.
As a proof of principle, we inserted the luciferase amino acid sequence into the third intracellular loop of the M3 muscarinic acetylcholine receptor. This fusion protein retained both receptor and enzyme function. Receptor blockers slightly but significantly reduced enzyme activity. By successive deletion mutagenesis the enzyme activity was optimally coupled to ligand-induced conformational helix movements.
Our results demonstrate that in engineered GPCR-enzyme chimeras, intracellular enzyme activity can be directly controlled by a GPCR serving as the extracellular ligand-binding domain.
Sphingosine 1-phosphate (S1P), a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type, is an endogenous ligand of five G protein-coupled receptors (GPCRs) in the endothelial differentiation gene family. The lack of GPCR crystal structures sets serious limitations to rational drug design and in silico searches for subtype-selective ligands. Here we report on the experimental validation of a computational model of the ligand binding pocket of the S1P1 GPCR surrounding the aliphatic portion of S1P. The extensive mutagenesis-based validation confirmed 18 residues lining the hydrophobic ligand binding pocket, which, combined with the previously validated three head group-interacting residues, now complete the mapping of the S1P ligand recognition site. We identified six mutants (L3.43G/L3.44G, L3.43E/L3.44E, L5.52A, F5.48G, V6.40L, and F6.44G) that maintained wild type [32P]S1P binding with abolished ligand-dependent activation by S1P. These data suggest a role for these amino acids in the conformational transition of S1P1 to its activated state. Three aromatic mutations (F5.48Y, F6.44G, and W6.48A) result in differential activation, by S1P or SEW2871, indicating that structural differences between the two agonists can partially compensate for differences in the amino acid side chain. The now validated ligand binding pocket provided us with a pharmacophore model, which was used for in silico screening of the NCI, National Institutes of Health, Developmental Therapeutics chemical library, leading to the identification of two novel nonlipid agonists of S1P1.
G- Protein coupled receptors (GPCRs) comprise the largest group of eukaryotic cell surface receptors with great pharmacological interest. A broad range of native ligands interact and activate GPCRs, leading to signal transduction within cells. Most of these responses are mediated through the interaction of GPCRs with heterotrimeric GTP-binding proteins (G-proteins). Due to the information explosion in biological sequence databases, the development of software algorithms that could predict properties of GPCRs is important. Experimental data reported in the literature suggest that heterotrimeric G-proteins interact with parts of the activated receptor at the transmembrane helix-intracellular loop interface. Utilizing this information and membrane topology information, we have developed an intensive exploratory approach to generate a refined library of statistical models (Hidden Markov Models) that predict the coupling preference of GPCRs to heterotrimeric G-proteins. The method predicts the coupling preferences of GPCRs to Gs, Gi/o and Gq/11, but not G12/13 subfamilies.
Using a dataset of 282 GPCR sequences of known coupling preference to G-proteins and adopting a five-fold cross-validation procedure, the method yielded an 89.7% correct classification rate. In a validation set comprised of all receptor sequences that are species homologues to GPCRs with known coupling preferences, excluding the sequences used to train the models, our method yields a correct classification rate of 91.0%. Furthermore, promiscuous coupling properties were correctly predicted for 6 of the 24 GPCRs that are known to interact with more than one subfamily of G-proteins.
Our method demonstrates high correct classification rate. Unlike previously published methods performing the same task, it does not require any transmembrane topology prediction in a preceding step. A web-server for the prediction of GPCRs coupling specificity to G-proteins available for non-commercial users is located at .
Purpose of review
Class A G protein-coupled receptors, including the chemokine receptors, CCR5 and CXCR4, share a seven transmembrane-spanning α-helix architecture that accommodates signal propagation from across biological membranes. CXCR4 and CCR5 are utilized as co-receptors during HIV viral entry and therefore crystal structures of GPCRs aid in the understanding of their function in viral entry.
Recent progress in structure determination of class A GPCRs, which include vertebrate and invertebrate rhodopsin as well as adrenergic and adenosine receptors, provide molecular templates for how this diverse group of transmembrane receptors functions. Each of these GPCRs differs in how specific ligands bind to the transmembrane core, underscoring that additional structures of GPCRs from other sub-families are needed to facilitate rational drug design. More recent studies also indicate a need to consider the more complex character of GPCRs, such as their oligomerization and dynamics.
Recently the atomic structures of invertebrate rhodopsin, β1- and β2-adrenergic receptors and the A2A-adenosine receptor have been solved via X-ray crystallography. The impact that these structures have on the biochemistry of viral entry and signal transduction is discussed. Because the chemokine receptors have proven refractory to structural studies thus far, further structural study of the chemokine receptors will be essential to understanding ligand binding, activation and function as co-receptors during viral entry.
rhodopsin; G protein-coupled receptor; membrane protein crystallography; GPCR; CCR5; CXCR4
G Protein-Coupled Receptors (GPCRs) comprise a large family of seven-helix transmembrane proteins which regulate cellular signaling by sensing light, ligands, and binding proteins. The GPCR activation process, however, is not a simple on-off switch; current models suggest a complex conformational landscape in which the active, signaling state includes multiple conformations with similar downstream activity. The present study probes the conformational dynamics of single β2-Adrenergic Receptors (β2ARs) in the solution phase by Anti-Brownian ELectrokinetic (ABEL) trapping. The ABEL trap uses fast electrokinetic feedback in a microfluidic configuration to allow direct observation of a single fluorescently-labeled β2AR for hundreds of milliseconds to seconds. By choosing a reporter dye and labeling site sensitive to ligand binding, we observe a diversity of discrete fluorescence intensity and lifetime levels in single β2ARs, indicating a varying radiative lifetime and a range of discrete conformational states with dwell times of hundreds of milliseconds. We find that binding of agonist increases the dwell times of these states, and furthermore, we observe millisecond fluctuations within states. The intensity autocorrelations of these faster fluctuations are well-described by stretched exponential functions with stretching exponent β ~ 0.5, suggesting protein dynamics over a range of timescales.
The lack of a crystallographically derived structure for all but three G (TP [guanosine triphosphate]-binding) protein-coupled receptor (GPCRs) proteins necessitates the use of computationally derived methods to determine their structures. Computational methodologies allow a mechanistic glimpse into GPCR–ligand interactions at a molecular level to better understand the initial steps leading to a protein's biologic functions, ie, protecting the ligands that activate, deactivate, or inhibit the protein, stabilizing protein structure in the membrane's lipid bilayer, and ensuring that the hydrophilic environment within the GPCR-binding pocket is maintained. Described here is a formalism that quantifies the amphiphilic nature of a helix, by determining the effective hydrophobicity (or hydrophilicity) at specific positions around it. This formalism will enable computational protein modelers to position helices so that the functional aspects of GPCRs are adequately represented in the model. Hydro-Eff®, an online tool, allows users to calculate effective helical hydrophobicities.
GPCR; effective hydrophobicity; amphiphilicity
G-protein coupled receptors (GPCRs) are targets of nearly one third of the drugs at the current pharmaceutical market. Despite their importance in many cellular processes the crystal structures are available for less than 20 unique GPCRs of the Rhodopsin-like class. Fortunately, even though involved in different signaling cascades, this large group of membrane proteins has preserved a uniform structure comprising seven transmembrane helices that allows quite reliable comparative modeling. Nevertheless, low sequence similarity between the GPCR family members is still a serious obstacle not only in template selection but also in providing theoretical models of acceptable quality. An additional level of difficulty is the prediction of kinks and bulges in transmembrane helices. Usage of multiple templates and generation of alignments based on sequence profiles may increase the rate of success in difficult cases of comparative modeling in which the sequence similarity between GPCRs is exceptionally low. Here, we present GPCRM, a novel method for fast and accurate generation of GPCR models using averaging of multiple template structures and profile-profile comparison. In particular, GPCRM is the first GPCR structure predictor incorporating two distinct loop modeling techniques: Modeller and Rosetta together with the filtering of models based on the Z-coordinate. We tested our approach on all unique GPCR structures determined to date and report its performance in comparison with other computational methods targeting the Rhodopsin-like class. We also provide a database of precomputed GPCR models of the human receptors from that class.
GPCRM server and database: http://gpcrm.biomodellab.eu
Recently available G-protein coupled receptor (GPCR) structures and biophysical studies suggest that the difference between the effects of various agonists and antagonists cannot be explained by single structures alone, but rather that the conformational ensembles of the proteins need to be considered. Here we use an elastic network model-guided molecular dynamics simulation protocol to generate an ensemble of conformers of a prototypical GPCR, β2-adrenergic receptor (β2AR). The resulting conformers are clustered into groups based on the conformations of the ligand binding site, and distinct conformers from each group are assessed for their binding to known agonists of β2AR. We show that the select ligands bind preferentially to different predicted conformers of β2AR, and identify a role of β2AR extracellular region as an allosteric binding site for larger drugs such as salmeterol. Thus, drugs and ligands can be used as “computational probes” to systematically identify protein conformers with likely biological significance.
G protein-coupled receptors play a major role in transmembrane signalling in higher organisms and many are important drug targets. We report the 2.7 Å resolution crystal structure of a β1-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey receptor had been selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane α-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilised by two disulphide bonds and a sodium ion. Cyanopindolol binding to the β1-adrenergic receptor and carazolol binding to the β2-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the β2-adrenergic receptor, directly interacts via a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.
The trace amine-associated receptor 1 (TAAR1) is an aminergic G protein-coupled receptor (GPCR) potently activated by 3-iodothyronamine (1), an endogenous derivative of thyroid hormone. Structure activity relationship studies on 1 and related agonists showed that the rat and mouse species of TAAR1 accommodated structural modifications and functional groups on the ethylamine portion and the biaryl ether moiety of the molecule. However, the two receptors clearly exhibited distinct, species-specific ligand preferences despite being remarkably similar with 93% sequence similarity. In this study, we generated single and double mutants of rat and mouse TAAR1 to probe the molecular recognition of agonists and the underlying basis for the ligand selectivity of rat and mouse TAAR1. Key, non-conserved specificity determinant residues in transmembranes helices 4 and 7 within the ligand binding site appear to be the primary source of a number of the observed ligand preferences. Residue 7.39 in transmembrane 7 dictated the preference for a β-phenyl ring while residue 4.56 in transmembrane 4 was partially responsible for the lower potency of 1 and tyramine for the mouse receptor. Additionally, 1 and tyramine were found to have the same binding mode in rat TAAR1 despite structure activity relationship data suggesting the possibility of each molecule having different binding orientations. These findings provide valuable insights into the critical binding site residues involved in the ligand-receptor interaction that can influence compound selectivity and functional activity of aminergic GPCRs.