Critical analysis of the successes and failures of homology models of G protein‐coupled receptors

We present a critical assessment of the performance of our homology model refinement method for G protein‐coupled receptors (GPCRs), called LITICon that led to top ranking structures in a recent structure prediction assessment GPCRDOCK2010. GPCRs form the largest class of drug targets for which only a few crystal structures are currently available. Therefore, accurate homology models are essential for drug design in these receptors. We submitted five models each for human chemokine CXCR4 (bound to small molecule IT1t and peptide CVX15) and dopamine D3DR (bound to small molecule eticlopride) before the crystal structures were published. Our models in both CXCR4/IT1t and D3/eticlopride assessments were ranked first and second, respectively, by ligand RMSD to the crystal structures. For both receptors, we developed two types of protein models: homology models based on known GPCR crystal structures, and ab initio models based on the prediction method MembStruk. The homology‐based models compared better to the crystal structures than the ab initio models. However, a robust refinement procedure for obtaining high accuracy structures is needed. We demonstrate that optimization of the helical tilt, rotation, and translation is vital for GPCR homology model refinement. As a proof of concept, our in‐house refinement program LITiCon captured the distinct orientation of TM2 in CXCR4, which differs from that of adrenoreceptors. These findings would be critical for refining GPCR homology models in future. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Assessment of the transmembrane domain structures in GPCR Dock 2013 models

The community-wide blind prediction of G-protein coupled receptor (GPCR) structures and ligand docking has been conducted three times and the quality of the models was primarily assessed by the accuracy of ligand binding modes. The seven transmembrane (TM) helices of the receptors were taken as a whole; thus the model quality within the 7TM domains has not been evaluated. Here we evaluate the 7TM domain structures in the models submitted for the last round of prediction – GPCR Dock 2013. Applying the 7?×?7 RMSD matrix analysis described in our prior work, we show that the models vary widely in prediction accuracy of the 7TM structures, exhibiting diverse structural differences from the targets. For the prediction of the 5-hydroxytryptamine receptors, the top 7TM models are rather close to the targets, which however are not ranked top by ligand-docking. On the other hand, notable deviations of the TMs are found in in the previously identified top docking models that closely resemble other receptors. We further reveal reasons of success and failure in ligand docking for the models. This current assessment not only complements the previous assessment, but also provides important insights into the current status of GPCR modeling and ligand docking.     

Determination of key receptor–ligand interactions of dopaminergic arylpiperazines and the dopamine D2 receptor homology model

Interest in structure-based G-protein-coupled receptor (GPCR) ligand discovery is huge, given that almost 30 % of all approved drugs belong to this category of active compounds. The GPCR family includes the dopamine receptor subtype D2 (D2DR), but unfortunately—as is true of most GPCRs—no experimental structures are available for these receptors. In this publication, we present the molecular model of D2DR based on the previously published crystal structure of the dopamine D3 receptor (D3DR). A molecular modeling study using homology modeling and docking simulation provided a rational explanation for the behavior of the arylpiperazine ligand. The observed binding modes and receptor–ligand interactions provided us with fresh clues about how to optimize selectivity for D2DR receptors.

Intracellular cross-talk between the GPCR CXCR1 and CXCR2: role of carboxyl terminus phosphorylation sites

In the present study, we used the human chemokine receptors CXCR1 and CXCR2 as a model system for the study of intracellular cross-talk between two closely related G protein-coupled receptors (GPCR). In cells expressing either CXCR1 or CXCR2, exposure to the CXCL8 ligand resulted in prominent reduction in cell surface expression of the receptors. We have shown previously that the reduction in cell surface expression of CXCR1 and CXCR2, to be termed herein "down-regulation", is significantly lower in cells expressing both receptors together. Now we show that reduced receptor down-regulation was specific to the CXCR1+CXCR2 pair. Also, CXCR2 carboxyl terminus phosphorylation sites were required for inducing inhibition of CXCR1 down-regulation, and vice versa. Accordingly, phosphorylation of CXCR2 carboxyl terminus domain was intact when expressed together with CXCR1. Moreover, specific carboxyl terminus phosphorylation sites on each of the wild type receptors protected them from more severe inhibition of down-regulation, induced by joint expression with the other receptor. When concomitantly expressed, CXCR1 and CXCR2 were impaired in recycling to the plasma membrane, despite their undergoing intact dephosphorylation. Overall, we show that cross-talk between two GPCR is manifested by impairment of their intracellular trafficking, primarily of ligand-induced down-regulation, via carboxyl terminus phosphorylation sites.     

Elastic network normal mode dynamics reveal the GPCR activation mechanism

G‐protein‐coupled receptors (GPCR) are a family of membrane‐embedded metabotropic receptors which translate extracellular ligand binding into an intracellular response. Here, we calculate the motion of several GPCR family members such as the M2 and M3 muscarinic acetylcholine receptors, the A_2A adenosine receptor, the β₂‐adrenergic receptor, and the CXCR4 chemokine receptor using elastic network normal modes. The normal modes reveal a dilation and a contraction of the GPCR vestibule associated with ligand passage, and activation, respectively. Contraction of the vestibule on the extracellular side is correlated with cavity formation of the G‐protein binding pocket on the intracellular side, which initiates intracellular signaling. Interestingly, the normal modes of rhodopsin do not correlate well with the motion of other GPCR family members. Electrostatic potential calculation of the GPCRs reveal a negatively charged field around the ligand binding site acting as a siphon to draw‐in positively charged ligands on the membrane surface. Altogether, these results expose the GPCR activation mechanism and show how conformational changes on the cell surface side of the receptor are allosterically translated into structural changes on the inside. Proteins 2014; 82:579–586. © 2013 Wiley Periodicals, Inc.     

Adaptor Protein-2 Interaction with Arrestin Regulates GPCR Recycling and Apoptosis

G protein-coupled receptors (GPCRs) are integral to cellular function in nearly all physiologic and many pathologic processes. GPCR signaling represents an intricate balance between receptor activation, inactivation (desensitization, internalization and degradation) and resensitization (recycling and de novo synthesis). Complex formation between phosphorylated GPCRs, arrestins and an ever-increasing number of effector molecules is known to regulate cellular function. Previous studies have demonstrated that, although N -formyl peptide receptor (FPR) internalization occurs in the absence of arrestins, FPR recycling is arrestin-dependent. Furthermore, FPR stimulation in the absence of arrestins leads to receptor accumulation in perinuclear endosomes and apoptosis. In this study, we show that the interaction of GPCR-bound arrestin with adaptor protein-2 (AP-2) is a critical anti-apoptotic event. In addition, AP-2 associates with the receptor-arrestin complex in perinuclear endosomes and is required for proper post-endocytic GPCR trafficking. Finally, we observed that depletion of endogenous AP-2 results in the initiation of apoptosis upon stimulation of multiple GPCRs, including P2Y purinergic receptors and CXCR2, but not CXCR4. We propose a model in which the abnormal accumulation of internalized GPCR-arrestin complexes in recycling endosomes, resulting from defective arrestin-AP-2 interactions, leads to the specific initiation of aberrant signaling pathways and apoptosis.     

Conformational Antibody Binding to a Native,Cell-Free Expressed GPCR in Block Copolymer Membranes

G-protein coupled receptors (GPCRs) play a key role in physiological processes and are attractive drug targets. Their biophysical characterization is, however, highly challenging because of their innate instability outside a stabilizing membrane and the difficulty of finding a suitable expression system. We here show the cell-free expression of a GPCR, CXCR4, and its direct embedding in diblock copolymer membranes. The polymer-stabilized CXCR4 is readily immobilized onto biosensor chips for label-free binding analysis. Kinetic characterization using a conformationally sensitive antibody shows the receptor to exist in the correctly folded conformation, showing binding behaviour that is commensurate with heterologously expressed CXCR4.     

Can molecular dynamics simulations improve the structural accuracy and virtual screening performance of GPCR models?

The determination of G protein-coupled receptor (GPCR) structures at atomic resolution has improved understanding of cellular signaling and will accelerate the development of new drug candidates. However, experimental structures still remain unavailable for a majority of the GPCR family. GPCR structures and their interactions with ligands can also be modelled computationally, but such predictions have limited accuracy. In this work, we explored if molecular dynamics (MD) simulations could be used to refine the accuracy of in silico models of receptor-ligand complexes that were submitted to a community-wide assessment of GPCR structure prediction (GPCR Dock). Two simulation protocols were used to refine 30 models of the D₃ dopamine receptor (D₃R) in complex with an antagonist. Close to 60 μs of simulation time was generated and the resulting MD refined models were compared to a D₃R crystal structure. In the MD simulations, the receptor models generally drifted further away from the crystal structure conformation. However, MD refinement was able to improve the accuracy of the ligand binding mode. The best refinement protocol improved agreement with the experimentally observed ligand binding mode for a majority of the models. Receptor structures with improved virtual screening performance, which was assessed by molecular docking of ligands and decoys, could also be identified among the MD refined models. Application of weak restraints to the transmembrane helixes in the MD simulations further improved predictions of the ligand binding mode and second extracellular loop. These results provide guidelines for application of MD refinement in prediction of GPCR-ligand complexes and directions for further method development.     

Towards Improved Quality of GPCR Models by Usage of Multiple Templates and Profile-Profile Comparison

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.

Availability

GPCRM server and database: http://gpcrm.biomodellab.eu     

Mapping the ligand-binding site on a G protein-coupled receptor (GPCR) using genetically encoded photocrosslinkers

We developed a general cell-based photocrosslinking approach to investigate the binding interfaces necessary for the formation of G protein-coupled receptor (GPCR) signaling complexes. The two photoactivatable unnatural amino acids p-benzoyl-L-phenylalanine and p-azido-L-phenylalanine were incorporated by amber codon suppression technology into CXC chemokine receptor 4 (CXCR4). We then probed the ligand-binding site for the HIV-1 coreceptor blocker, T140, using a fluorescein-labeled T140 analogue. Among eight amino acid positions tested, we found a unique UV-light-dependent crosslink specifically between residue 189 and T140. These results are evaluated with molecular modeling using the crystal structure of CXCR4 bound to CVX15.     

GPR88 Reveals a Discrete Function of Primary Cilia as Selective Insulators of GPCR Cross-Talk

A number of G protein-coupled receptors (GPCRs) localize to primary cilia but the functional significance of cilia to GPCR signaling remains incompletely understood. We investigated this question by focusing on the D1 dopamine receptor (D1R) and beta-2 adrenergic receptor (B2AR), closely related catecholamine receptors that signal by stimulating production of the diffusible second messenger cyclic AMP (cAMP) but differ in localization relative to cilia. D1Rs robustly concentrate on cilia of IMCD3 cells, as shown previously in other ciliated cell types, but disrupting cilia did not affect D1R surface expression or ability to mediate a concentration-dependent cAMP response. By developing a FRET-based biosensor suitable for resolving intra- from extra- ciliary cAMP changes, we found that the D1R-mediated cAMP response is not restricted to cilia and extends into the extra-ciliary cytoplasm. Conversely the B2AR, which we show here is effectively excluded from cilia, also generated a cAMP response in both ciliary and extra-ciliary compartments. We identified a distinct signaling effect of primary cilia through investigating GPR88, an orphan GPCR that is co-expressed with the D1R in brain, and which we show here is targeted to cilia similarly to the D1R. In ciliated cells, mutational activation of GPR88 strongly reduced the D1R-mediated cAMP response but did not affect the B2AR-mediated response. In marked contrast, in non-ciliated cells, GPR88 was distributed throughout the plasma membrane and inhibited the B2AR response. These results identify a discrete ‘insulating’ function of primary cilia in conferring selectivity on integrated catecholamine signaling through lateral segregation of receptors, and suggest a cellular activity of GPR88 that might underlie its effects on dopamine-dependent behaviors.     

Evolution of the class C GPCR Venus flytrap modules involved positive selected functional divergence

Background  

Class C G protein-coupled receptors (GPCRs) represent a distinct group of the GPCR family, which structurally possess a characteristically distinct extracellular domain inclusive of the Venus flytrap module (VFTM). The VFTMs of the class C GPCRs is responsible for ligand recognition and binding, and share sequence similarity with bacterial periplasmic amino acid binding proteins (PBPs). An extensive phylogenetic investigation of the VFTMs was conducted by analyzing for functional divergence and testing for positive selection for five typical groups of the class C GPCRs. The altered selective constraints were determined to identify the sites that had undergone functional divergence via positive selection. In order to structurally demonstrate the pattern changes during the evolutionary process, three-dimensional (3D) structures of the GPCR VFTMs were modelled and reconstructed from ancestral VFTMs.     

Structural biology of G protein‐coupled receptor signaling complexes

G protein‐coupled receptors (GPCRs) constitute the largest family of cell surface receptors that mediate numerous cell signaling pathways, and are targets of more than one‐third of clinical drugs. Thanks to the advancement of novel structural biology technologies, high‐resolution structures of GPCRs in complex with their signaling transducers, including G‐protein and arrestin, have been determined. These 3D complex structures have significantly improved our understanding of the molecular mechanism of GPCR signaling and provided a structural basis for signaling‐biased drug discovery targeting GPCRs. Here we summarize structural studies of GPCR signaling complexes with G protein and arrestin using rhodopsin as a model system, and highlight the key features of GPCR conformational states in biased signaling including the sequence motifs of receptor TM6 that determine selective coupling of G proteins, and the phosphorylation codes of GPCRs for arrestin recruitment. We envision the future of GPCR structural biology not only to solve more high‐resolution complex structures but also to show stepwise GPCR signaling complex assembly and disassembly and dynamic process of GPCR signal transduction.     

Multiplex Detection of Homo- and Heterodimerization of G Protein-Coupled Receptors by Proximity Biotinylation

Dimerization of G protein-coupled receptors (GPCRs) represents a potential mechanism by which GPCR functions are regulated. Several resonance energy transfer (RET)-based methods have revealed GPCR homo- and heterodimerization. However, interpretation of an increase in FRET efficiency could be attributed to either dimerization/oligomerization events or conformational changes within an already dimerized/oligomerized receptor complex. Furthermore, RET-based methods can only measure pairwise dimerization, and cannot easily achieve multiplex detection. In this study, we applied proximity-based biotinylation for detecting receptor dimerization by utilizing a specific enzyme-substrate pair that are fused to GPCRs. The biotin ligase BirA is fused to CXCR4 and site-specifically biotinylates an acceptor peptide (AP) in the presence of biotin. As a test case for our newly developed assay, we have characterized the homo-dimerization of chemokine receptor CXCR4 and heterodimerization of CXCR4 with CCR2 or CCR5. The degree of biotinylation varies with the amount of GPCR-AP as well as biotinylation time. Using enzyme/substrate receptor pairs and measuring receptor biotinylation, we demonstrate that CXCR4 can homo-dimerize and hetero-dimerize with CCR2 and CCR5. The effect of CXCL12, agonist for CXCR4, was found to decrease surface biotinylation of CXCR4-AP. This effect is due to a combination of CXCR4 endocytosis and stabilization of CXCR4 homodimers. Finally, when CXCR4-AP, CCR2-AP, and CCR5-AP were expressed together, we observed CXCR4-CXCR4 homodimers and CXCR4-CCR2 and CXCR4-CCR5 heterodimers. The newly developed assay opens new opportunity for multiplex detection for GPCR homo- and heterodimerization within the same cellular context.     

Direct assessment of CXCR4 mutant conformations reveals complex link between receptor structure and G(alpha)(i) activation

Ligand binding to G protein-coupled receptors (GPCRs) is thought to induce changes in receptor conformation that translate into activation of downstream effectors. The link between receptor conformation and activity is still insufficiently understood, as current models of GPCR activation fail to take an increasing amount of experimental data into account. To elucidate structure-function relationships in GPCR activation, we used bioluminescence resonance energy transfer to directly assess the conformation of mutants of the chemokine receptor CXCR4. We analyzed substitutions in the arginine cage DRY motif and in the conserved asparagine N(3.35)119, which are pivotal molecular switches for receptor conformation and activation. G(alpha)(i) activation of the mutants was either similar to wild-type CXCR4 (D133N, Y135A, and N119D) or resulted in loss of activity (R134A and N119K). Mutant N119S was constitutively active but further activated by agonist. Bioluminescence resonance energy transfer analysis suggested no simple correlation between conformational changes in response to ligand binding and activation of G(alpha)(i) by the mutants. Different conformations of active receptors were detected (for wild-type CXCR4, D133N, and N119S), suggesting that different receptor conformations are able to trigger G(alpha)(i) activity. Several conformations were also found for inactive mutants. These data provide biophysical evidence for different receptor conformations being active with respect to a single readout. They support models of GPCR structure-activity relationships that take this conformational flexibility of active receptors into account.     

CODA-RET reveals functional selectivity as a result of GPCR heteromerization

Here we present a new method that combines protein complementation with resonance energy transfer to study conformational changes in response to activation of a defined G protein-coupled receptor heteromer, and we apply the approach to the putative dopamine D1-D2 receptor heteromer. Remarkably, the potency of the D2 dopamine receptor (D2R) agonist R-(-)-10,11-dihydroxy-N-n-propylnoraporphine (NPA) to change the Gα(i) conformation via the D2R protomer in the D1-D2 heteromer was enhanced ten-fold relative to its potency in the D2R homomer. In contrast, the potencies of the D2R agonists dopamine and quinpirole were the same in the homomer and heteromer. Thus, we have uncovered a molecular mechanism for functional selectivity in which a drug acts differently at a G protein-coupled receptor (GPCR) protomer depending on the identity of the second protomer participating in the formation of the signaling unit--opening the door to enhancing pharmacological specificity by targeting differences between homomeric and heteromeric signaling.     

Insights into unbound–bound states of GPR142 receptor in a membrane-aqueous system using molecular dynamics simulations

G protein coupled receptors (GPCRs) are source machinery in signal transduction pathways and being one of the major therapeutic targets play a significant in drug discovery. GPR142, an orphan GPCR, has been implicated in the regulation of insulin, thereby having a crucial role in Type II diabetes management. Deciphering of the structures of orphan, GPCRs (O-GPCRs) offer better prospects for advancements in research in ion translocation and transduction of extracellular signals. As the crystallographic structure of GPR142 is not available in PDB, therefore, threading and ab initio-based approaches were used for 3D modeling of GPR142. Molecular dynamic simulations (900 ns) were performed on the 3D model of GPR142 and complexes of GPR142 with top five hits, obtained through virtual screening, embedded in lipid bilayer with aqueous system using OPLS force field. Compound 1, 3, and 4 may act as scaffolds for designing potential lead agonists for GPR142. The finding of GPR142 MD simulation study provides more comprehensive representation of the functional properties. The concern for Type II diabetes is increasing worldwide and successful treatment of this disease demands novel drugs with better efficacy.     

S-nitrosylation-regulated GPCR signaling

G protein-coupled receptors (GPCRs) are the most numerous and diverse type of cell surface receptors, accounting for about 1% of the entire human genome and relaying signals from a variety of extracellular stimuli that range from lipid and peptide growth factors to ions and sensory inputs. Activated GPCRs regulate a multitude of target cell functions, including intermediary metabolism, growth and differentiation, and migration and invasion. The GPCRs contain a characteristic 7-transmembrane domain topology and their activation promotes complex formation with a variety of intracellular partner proteins, which form basis for initiation of distinct signaling networks as well as dictate fate of the receptor itself. Both termination of active GPCR signaling and removal from the plasma membrane are controlled by protein post-translational modifications of the receptor itself and its interacting partners. Phosphorylation, acylation and ubiquitination are the most studied post-translational modifications involved in GPCR signal transduction, subcellular trafficking and overall expression. Emerging evidence demonstrates that protein S-nitrosylation, the covalent attachment of a nitric oxide moiety to specified cysteine thiol groups, of GPCRs and/or their associated effectors also participates in the fine-tuning of receptor signaling and expression. This newly appreciated mode of GPCR system modification adds another set of controls to more precisely regulate the many cellular functions elicited by this large group of receptors. This article is part of a Special Issue entitled: Regulation of cellular processes by S-nitrosylation.     

The ubiquitin ligase deltex-3l regulates endosomal sorting of the G protein–coupled receptor CXCR4

G protein–coupled receptor (GPCR) sorting into the degradative pathway is important for limiting the duration and magnitude of signaling. Agonist activation of the GPCR CXCR4 induces its rapid ubiquitination and sorting to lysosomes via the endosomal sorting complex required for transport (ESCRT) pathway. We recently reported that ESCRT-0 ubiquitination is linked to the efficiency with which CXCR4 is sorted for lysosomal degradation; however mechanistic insight is lacking. Here we define a novel role for the really interesting new gene–domain E3 ubiquitin ligase deltex-3-like (DTX3L) in regulating CXCR4 sorting from endosomes to lysosomes. We show that DTX3L localizes to early endosomes upon CXCR4 activation and interacts directly with and inhibits the activity of the E3 ubiquitin ligase atrophin-1 interacting protein 4. This serves to limit the extent to which ESCRT-0 is ubiquitinated and is able to sort CXCR4 for lysosomal degradation. Therefore we define a novel role for DTX3L in GPCR endosomal sorting and reveal an unprecedented link between two distinct E3 ubiquitin ligases to control the activity of the ESCRT machinery.