Prediction of the odorant binding site of olfactory receptor proteins by human-mouse comparisons

Olfactory receptors (ORs) are a large family of proteins involved in the recognition and discrimination of numerous odorants. These receptors belong to the G-protein coupled receptor (GPCR) hyperfamily, for which little structural data are available. In this study we predict the binding site residues of OR proteins by analyzing a set of 1441 OR protein sequences from mouse and human. The central insight utilized is that functional contact residues would be conserved among pairs of orthologous receptors, but considerably less conserved among paralogous pairs. Using judiciously selected subsets of 218 ortholog pairs and 518 paralog pairs, we have identified 22 sequence positions that are both highly conserved among the putative orthologs and variable among paralogs. These residues are disposed on transmembrane helices 2 to 7, and on the second extracellular loop of the receptor. Strikingly, although the prediction makes no assumption about the location of the binding site, these amino acid positions are clustered around a pocket in a structural homology model of ORs, mostly facing the inner lumen. We propose that the identified positions constitute the odorant binding site. This conclusion is supported by the observation that all but one of the predicted binding site residues correspond to ligand-contact positions in other rhodopsin-like GPCRs.     

Classification of G-protein coupled receptors by alignment-independent extraction of principal chemical properties of primary amino acid sequences

We have developed an alignment-independent method for classification of G-protein coupled receptors (GPCRs) according to the principal chemical properties of their amino acid sequences. The method relies on a multivariate approach where the primary amino acid sequences are translated into vectors based on the principal physicochemical properties of the amino acids and transformation of the data into a uniform matrix by applying a modified autocross-covariance transform. The application of principal component analysis to a data set of 929 class A GPCRs showed a clear separation of the major classes of GPCRs. The application of partial least squares projection to latent structures created a highly valid model (cross-validated correlation coefficient, Q(2) = 0.895) that gave unambiguous classification of the GPCRs in the training set according to their ligand binding class. The model was further validated by external prediction of 535 novel GPCRs not included in the training set. Of the latter, only 14 sequences, confined in rapidly expanding GPCR classes, were mispredicted. Moreover, 90 orphan GPCRs out of 165 were tentatively identified to GPCR ligand binding class. The alignment-independent method could be used to assess the importance of the principal chemical properties of every single amino acid in the protein sequences for their contributions in explaining GPCR family membership. It was then revealed that all amino acids in the unaligned sequences contributed to the classifications, albeit to varying extent; the most important amino acids being those that could also be determined to be conserved by using traditional alignment-based methods.     

Insights from the analysis of conserved motifs and permitted amino acid exchanges in the human, the fly and the worm GPCR clusters

G-protein coupled receptors (GPCRs) belong to biologically important and functionally diverse and largest super family of membrane proteins. GPCRs retain a characteristic membrane topology of seven alpha helices with three intracellular, three extracellular loops and flanking N' and C' terminal residues. Subtle differences do exist in the helix boundaries (TM-domain), loop lengths, sequence features such as conserved motifs, and substituting amino acid patterns and their physiochemical properties amongst these sequences (clusters) at intra-genomic and inter-genomic level (please re-phrase into 2 statements for clarity). In the current study, we employ prediction of helix boundaries and scores derived from amino acid substitution exchange matrices to identify the conserved amino acid residues (motifs) as consensus in aligned set of homologous GPCR sequences. Co-clustered GPCRs from human and other genomes, organized as 32 clusters, were employed to study the amino acid conservation patterns and species-specific or cluster-specific motifs. Critical analysis on sequence composition and properties provide clues to connect functional relevance within and across genome for vast practical applications such as design of mutations and understanding of disease-causing genetic abnormalities.     

The impact of cryo-EM on determining allosteric modulator-bound structures of G protein-coupled receptors

G-protein coupled receptors (GPCRs) are important therapeutic targets for the treatment of human disease. Although GPCRs are highly successful drug targets, there are many challenges associated with the discovery and translation of small molecule ligands that target the endogenous ligand-binding site for GPCRs. Allosteric modulators are a class of ligands that target alternative binding sites known as allosteric sites and offer fresh opportunities for the development of new therapeutics. However, only a few allosteric modulators have been approved as drugs. Advances in GPCR structural biology enabled by the cryogenic electron microscopy (cryo-EM) revolution have provided new insights into the molecular mechanism and binding location of small molecule allosteric modulators. This review highlights the latest findings from allosteric modulator-bound structures of Class A, B, and C GPCRs with a focus on small molecule ligands. Emerging methods that will facilitate cryo-EM structures of more difficult ligand-bound GPCR complexes are also discussed. The results of these studies are anticipated to aid future structure-based drug discovery efforts across many different GPCRs.     

Prediction of GPCRs with pseudo amino acid composition: employing composite features and grey incidence degree based classification

G-protein coupled receptor (GPCR) is a protein family that is found only in the Eukaryotes. They are used for the interfacing of cell to the outside world and are involved in many physiological processes. Their role in drug development is evident. Hence, the prediction of GPCRs is very much demanding. Because of the unavailability of 3D structures of most of the GPCRs; the statistical and machine learning based prediction of GPCRs is much demanding. The GPCRs are classified into family, sub family and sub-sub family levels in the proposed approach. We have extracted features using the hybrid combination of Pseudo amino acid, Fast Fourier Transform and Split amino acid techniques. The overall feature vector is then reduced using Principle component analysis. Mostly, GPCRs are composed of two or more sub units. The arrangement and number of sub units forming a GPCR are referred to as quaternary structure. The functions of GPCRs are closely related to their quaternary structure. The classification in the present research is performed using grey incidence degree (GID) measure, which can efficiently analyze the numerical relation between various components of GPCRs. The GID measure based classification has shown remarkable improvement in predicting GPCRs.     

Allosteric activation of metabotropic glutamate receptor 5

Abstract

Metabotropic glutamate receptor 5 (mGluR5) is a class C G protein-coupled receptor (GPCR) with both an extracellular ligand binding site and an allosteric intrahelical chamber located similarly to the orthosteric ligand binding site of Class A GPCRs. Ligands binding to this ancestral site of mGluR5 can act as positive (PAM), negative (NAM) or silent (SAM) allosteric modulators, and their medicinal chemistry optimization is notoriously difficult, as subtle structural changes may cause significant variation in activity and switch in the functional response. Here we present all atom molecular dynamics simulations of NAM, SAM and PAM complexes formed by closely related ligands and analyse the structural differences of the complexes. Several residues involved in the activation are identified and the formation of a continuous water channel in the active complex but not in the inactive ones is recognized. Our results suggest that the mechanism of mGluR5 activation is similar to that of class A GPCRs.

Communicated by Ramaswamy H. Sarma     

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.     

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.     

Proteomic applications of automated GPCR classification

The G-protein coupled receptor (GPCR) superfamily fulfils various metabolic functions and interacts with a diverse range of ligands. There is a lack of sequence similarity between the six classes that comprise the GPCR superfamily. Moreover, most novel GPCRs found have low sequence similarity to other family members which makes it difficult to infer properties from related receptors. Many different approaches have been taken towards developing efficient and accurate methods for GPCR classification, ranging from motif-based systems to machine learning as well as a variety of alignment-free techniques based on the physiochemical properties of their amino acid sequences. This review describes the inherent difficulties in developing a GPCR classification algorithm and includes techniques previously employed in this area.     

G蛋白偶联受体及其类型的预测

G蛋白偶联受体是非常重要的信号分子受体,其功能失调会导致许多疾病的产生。在前期工作的基础上,作者将序列特征分析与支持向量机技术结合起来,通过分析序列的特征差异,对G蛋白偶联受体分子及其类型进行识别。首次提取了G蛋白偶联受体对应的mRNA序列的绝对密码子使用频率作为特征,这主要因为它既包含了基因密码子使用偏性的信息,也包含了基因所编码蛋白的氨基酸组成信息。结果显示：在G蛋白偶联受体序列及其类型预测的问题中,设计支持向量机分类器时,最好选择使用包含基因序列绝对密码子使用频率和蛋白序列双联氨基酸使用频率两部分信息的组合特征作为特征,同时采用径向基核作为核函数。     

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.     

Molecular basis for activation of G protein-coupled receptor kinases

G protein‐coupled receptor (GPCR) kinases (GRKs) selectively recognize and are allosterically regulated by activated GPCRs, but the molecular basis for this interaction is not understood. Herein, we report crystal structures of GRK6 in which regions known to be critical for receptor phosphorylation have coalesced to stabilize the kinase domain in a closed state and to form a likely receptor docking site. The crux of this docking site is an extended N‐terminal helix that bridges the large and small lobes of the kinase domain and lies adjacent to a basic surface of the protein proposed to bind anionic phospholipids. Mutation of exposed, hydrophobic residues in the N‐terminal helix selectively inhibits receptor, but not peptide phosphorylation, suggesting that these residues interact directly with GPCRs. Our structural and biochemical results thus provide an explanation for how receptor recognition, phospholipid binding, and kinase activation are intimately coupled in GRKs.     

The importance of valine 114 in ligand binding in β2‐adrenergic receptor

G‐protein coupled receptors (GPCRs) are transmembrane signaling molecules, with a majority of them performing important physiological roles. β₂‐Adrenergic receptor (β₂‐AR) is a well‐studied GPCRs that mediates natural responses to the hormones adrenaline and noradrenaline. Analysis of the ligand‐binding region of β₂‐AR using the recently solved high‐resolution crystal structures revealed a number of highly conserved amino acids that might be involved in ligand binding. However, detailed structure‐function studies on some of these residues have not been performed, and their role in ligand binding remains to be elucidated. In this study, we have investigated the structural and functional role of a highly conserved residue valine 114, in hamster β₂‐AR by site‐directed mutagenesis. We replaced V114 in hamster β₂‐AR with a number of amino acid residues carrying different functional groups. In addition to the complementary substitutions V114I and V114L, the V114C and V114E mutants also showed significant ligand binding and agonist dependent G‐protein activation. However, the V114G, V114T, V114S, and V114W mutants failed to bind ligand in a specific manner. Molecular modeling studies were conducted to interpret these results in structural terms. We propose that the replacement of V114 influences not only the interaction of the ethanolamine side‐chains but also the aryl‐ring of the ligands tested. Results from this study show that the size and orientation of the hydrophobic residue at position V114 in β₂‐AR affect binding of both agonists and antagonists, but it does not influence the receptor expression or folding.     

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.     

Dimerization in aminergic G-protein-coupled receptors: application of a hidden-site class model of evolution

G-Protein-coupled receptors (GPCRs) are an important superfamily of transmembrane proteins involved in cellular communication. Recently, it has been shown that dimerization is a widely occurring phenomenon in the GPCR superfamily, with likely important physiological roles. Here we use a novel hidden-site class model of evolution as a sequence analysis tool to predict possible dimerization interfaces in GPCRs. This model aims to simulate the evolution of proteins at the amino acid level, allowing the analysis of their sequences in an explicitly evolutionary context. Applying this model to aminergic GPCR sequences, we first validate the general reasoning behind the model. We then use the model to perform a family specific analysis of GPCRs. Accounting for the family structure of these proteins, this approach detects different evolutionarily conserved and accessible patches on transmembrane (TM) helices 4-6 in different families. On the basis of these findings, we propose an experimentally testable dimerization mechanism, involving interactions among different combinations of these helices in different families of aminergic GPCRs.     

The anatomy of mammalian sweet taste receptors

All sweet‐tasting compounds are detected by a single G‐protein coupled receptor (GPCR), the heterodimer T1R2‐T1R3, for which no experimental structure is available. The sweet taste receptor is a class C GPCR, and the recently published crystallographic structures of metabotropic glutamate receptor (mGluR) 1 and 5 provide a significant step forward for understanding structure‐function relationships within this family. In this article, we recapitulate more than 600 single point site‐directed mutations and available structural data to obtain a critical alignment of the sweet taste receptor sequences with respect to other class C GPCRs. Using this alignment, a homology 3D‐model of the human sweet taste receptor is built and analyzed to dissect out the role of key residues involved in ligand binding and those responsible for receptor activation. Proteins 2017; 85:332–341. © 2016 Wiley Periodicals, Inc.     

Genetic analysis of the first and third extracellular loops of the C5a receptor reveals an essential WXFG motif in the first loop

The extracellular loops of G protein-coupled receptors (GPCRs) frequently contain binding sites for peptide ligands. However, the mechanism of receptor activation following ligand binding and the influence of the extracellular loops in other aspects of receptor function are poorly understood. Here we report a structure-function analysis of the first and third extracellular loops of the human C5a receptor, a GPCR that binds a 74-amino acid peptide ligand. Amino acid substitutions were randomly incorporated into each loop, and functional receptors were identified in yeast. The first extracellular loop contains a large number of positions that cannot tolerate amino acid substitutions, especially residues within the WXFG motif found in many rhodopsin-like GPCRs, yet disruption of these residues does not alter C5a binding affinity. These results demonstrate an unanticipated role for the first extracellular loop, and the WXFG motif in particular, in ligand-mediated activation of the C5a receptor. This motif likely serves a similar role in other GPCRs. The third extracellular loop, in contrast, contains far fewer preserved residues and appears to play a less essential role in receptor activation.     

Ligand specificity of odorant receptors

Odorant receptors belong to class A of the G protein-coupled receptors (GPCRs) and detect a large number of structurally diverse odorant molecules. A recent structural bioinformatic analysis suggests that structural features are conserved across class A of GPCRs in spite of their low sequence identity. Based on this work, we have aligned the sequences of 29 ORs for which ligand binding data are available. Recent site-directed mutagenesis experiments on one such receptor (MOR174-9) provide information that helped to identify nine amino-acid residues involved in ligand binding. Our modeling provides a rationale for amino acids in equivalent positions in most of the odorant receptors considered and helps to identify other amino acids that could be important for ligand binding. Our findings are consistent with most of the previous models and allow predictions for site-directed mutagenesis experiments, which could also validate our model.     

Determination of network of residues that regulate allostery in protein families using sequence analysis

Allosteric interactions between residues that are spatially apart and well separated in sequence are important in the function of multimeric proteins as well as single-domain proteins. This observation suggests that, among the residues that are involved in long-range communications, mutation at one site should affect interactions at a distant site. By adopting a sequence-based approach, we present an automated approach that uses a generalization of the familiar sequence entropy in conjunction with a coupled two-way clustering algorithm, to predict the network of interactions that trigger allosteric interactions in proteins. We use the method to identify the subset of dynamically important residues in three families, namely, the small PDZ family, G protein-coupled receptors (GPCR), and the Lectins, which are cell-adhesion receptors that mediate the tethering and rolling of leukocytes on inflamed endothelium. For the PDZ and GPCR families, our procedure predicts, in agreement with previous studies, a network containing a small number of residues that are involved in their function. Application to the Lectin family reveals a network of residues interspersed throughout the C-terminal end of the structure that are responsible for binding to ligands. Based on our results and previous studies, we propose that functional robustness requires that only a small subset of distantly connected residues be involved in transmitting allosteric signals in proteins.     

Are specific nonannular cholesterol binding sites present in G-protein coupled receptors?

The G-protein coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across membranes, and represent major drug targets in all clinical areas. Membrane cholesterol has been reported to have a modulatory role in the function of a number of GPCRs. Interestingly, recently reported crystal structures of GPCRs have shown structural evidence of cholesterol binding sites. Two possible mechanisms have been previously suggested by which membrane cholesterol could influence the structure and function of GPCRs (i) through a direct/specific interaction with GPCRs, which could induce a conformational change in the receptor, or (ii) through an indirect way by altering the membrane physical properties in which the receptor is embedded or due to a combination of both. We discuss here a novel mechanism by which membrane cholesterol could affect structure and function of GPCRs and propose that cholesterol binding sites in GPCRs could represent ‘nonannular’ binding sites. Interestingly, previous work from our laboratory has demonstrated that membrane cholesterol is required for the function of the serotonin_1A receptor, which could be due to specific interaction of the receptor with cholesterol. Based on these results, we envisage that there could be specific/nonannular cholesterol binding site(s) in the serotonin_1A receptor. We have analyzed putative cholesterol binding sites from protein databases in the serotonin_1A receptor, a representative GPCR, for which we have previously demonstrated specific requirement of membrane cholesterol for receptor function. Our analysis shows that cholesterol binding sites are inherent characteristic features of serotonin_1A receptors and are conserved over evolution. Progress in deciphering molecular details of the nature of GPCR-cholesterol interaction in the membrane would lead to better insight into our overall understanding of GPCR function in health and disease, thereby enhancing our ability to design better therapeutic strategies to combat diseases related to malfunctioning of GPCRs.