Structural determinants of the supramolecular organization of G protein-coupled receptors in bilayers

The G protein-coupled receptor (GPCR) rhodopsin self-assembles into supramolecular structures in native bilayers, but the structural determinants of receptor oligomerization are not known. We carried out multiple self-assembly coarse-grained molecular dynamics (CGMD) simulations of model membranes containing up to 64 molecules of the visual receptor rhodopsin over time scales reaching 100 μs. The simulations show strong preferential interaction modes between receptors. Two primary modes of receptor-receptor interactions are consistent with umbrella sampling/potential of mean force (PMF) calculations as a function of the distance between a pair of receptors. The preferential interfaces, involving helices (H) 1/8, 4/5 and 5, present no energy barrier to forming a very stable receptor dimer. Most notably, the PMFs show that the preferred rhodopsin dimer exists in a tail-to-tail conformation, with the interface comprising transmembrane H1/H2 and amphipathic H8 at the extracellular and cytoplasmic surfaces, respectively. This dimer orientation is in line with earlier electron microscopy, X-ray, and cross-linking experiments of rhodopsin and other GPCRs. Less stable interfaces, involving H4 and H6, have a free energy barrier for desolvation (delipidation) of the interfaces and appear to be designed to stabilize "lubricated" (i.e., lipid-coated) dimers. The overall CGMD strategy used here is general and can be applied to study the homo- and heterodimerization of GPCRs and other transmembrane proteins. Systematic extension of the work will deepen our understanding of the forces involved in the membrane organization of integral membrane proteins.     

Structural Insights into Ligand—Receptor Interactions Involved in Biased Agonism of G-Protein Coupled Receptors

G protein-coupled receptors (GPCRs) are versatile signaling proteins that mediate complex cellular responses to hormones and neurotransmitters. Ligand directed signaling is observed when agonists, upon binding to the same receptor, trigger significantly different configuration of intracellular events. The current work reviews the structurally defined ligand – receptor interactions that can be related to specific molecular mechanisms of ligand directed signaling across different receptors belonging to class A of GPCRs. Recent advances in GPCR structural biology allow for mapping receptors’ binding sites with residues particularly important in recognition of ligands’ structural features that are responsible for biased signaling. Various studies show particular role of specific residues lining the extended ligand binding domains, biased agonists may alternatively affect their interhelical interactions and flexibility what can be translated into intracellular loop rearrangements. Studies on opioid and angiotensin receptors indicate importance of residues located deeper within the binding cavity and direct interactions with receptor residues linking the ortosteric ligand binding site with the intracellular transducer binding domain. Collection of results across different receptors may suggest elements of common molecular mechanisms which are responsible for passing alternative signals from biased agonists.     

Dynamic models of G-protein coupled receptor dimers: indications of asymmetry in the rhodopsin dimer from molecular dynamics simulations in a POPC bilayer

Based on the growing evidence that G-protein coupled receptors (GPCRs) form homo- and hetero-oligomers, models of GPCR signaling are now considering macromolecular assemblies rather than monomers, with the homo-dimer regarded as the minimal oligomeric arrangement required for functional coupling to the G-protein. The dynamic mechanisms of such signaling assemblies are unknown. To gain some insight into properties of GPCR dimers that may be relevant to functional mechanisms, we study their current structural prototype, rhodopsin. We have carried out nanosecond time-scale molecular dynamics (MD) simulations of a rhodopsin dimer and compared the results to the monomer simulated in the same type of bilayer membrane model composed of an equilibrated unit cell of hydrated palmitoyl-oleoyl-phosphatidyl choline (POPC). The dynamic representation of the homo-dimer reveals the location of structural changes in several regions of the monomeric subunits. These changes appear to be more pronounced at the dimerization interface that had been shown to be involved in the activation process [Proc Natl Acad Sci USA 102:17495, 2005]. The results are consistent with a model of GPCR activation that involves allosteric modulation through a single GPCR subunit per dimer.     

Membrane interactive alpha-helices in GPCRs as a novel drug target

G-Protein Coupled Receptors (GPCRs) are one of the most important targets for pharmaceutical drug design. Over the past 30 years, mounting evidence has suggested the existence of homo and hetero dimers or higher-order complexes (oligomers) that are involved in signal transduction and some diseases. The number of reports describing GPCR oligomerization has increased, and in 2003, the organization of mouse rhodopsin into two-dimensional arrays of dimers was determined by an atomic force microscopic analysis. The analysis of the mouse rhodopsin complex has enabled us to discuss the oligomerization based on structural data. Although many unsolved problems still remains, the idea that GPCRs directly interact to form oligomers has been gradually accepted. One of the recent findings in the GPCR investigations is the clarification of the mechanisms of GPCR oligomerization at a molecular level. Most of these studies have suggested the importance of transmembrane alpha-helices for GPCR oligomerization. In this review, we will first summarize the importance of GPCR oligomerization and the functions of GPCRs. Then, we will explain the involvement of transmembrane alpha-helices in the oligomerization and a drug design strategy that targets these regions for GPCR oligomerization. Considering the current drug design methods, which are based on the modification of the protein-protein interactions of soluble regions of proteins, a "peptide mimic approach" that targets the transmembrane alpha-helices constituting the interfaces would be promising in drug discovery for GPCR oligomerization. For that purpose, we must know the positions of the interfaces. However, problems specific to membrane proteins have made it difficult to identify the positions of the interfaces experimentally. Therefore, information about the interfaces predicted by bioinformatics approaches is valuable. At the end of this review, several bioinformatics approaches toward interface prediction for oligomerization are introduced. The benefits and the pitfalls of these approaches are also discussed.     

Snooker: a structure-based pharmacophore generation tool applied to class A GPCRs

G-protein coupled receptors (GPCRs) are important drug targets for various diseases and of major interest to pharmaceutical companies. The function of individual members of this protein family can be modulated by the binding of small molecules at the extracellular side of the structurally conserved transmembrane (TM) domain. Here, we present Snooker, a structure-based approach to generate pharmacophore hypotheses for compounds binding to this extracellular side of the TM domain. Snooker does not require knowledge of ligands, is therefore suitable for apo-proteins, and can be applied to all receptors of the GPCR protein family. The method comprises the construction of a homology model of the TM domains and prioritization of residues on the probability of being ligand binding. Subsequently, protein properties are converted to ligand space, and pharmacophore features are generated at positions where protein ligand interactions are likely. Using this semiautomated knowledge-driven bioinformatics approach we have created pharmacophore hypotheses for 15 different GPCRs from several different subfamilies. For the beta-2-adrenergic receptor we show that ligand poses predicted by Snooker pharmacophore hypotheses reproduce literature supported binding modes for ～75% of compounds fulfilling pharmacophore constraints. All 15 pharmacophore hypotheses represent interactions with essential residues for ligand binding as observed in mutagenesis experiments and compound selections based on these hypotheses are shown to be target specific. For 8 out of 15 targets enrichment factors above 10-fold are observed in the top 0.5% ranked compounds in a virtual screen. Additionally, prospectively predicted ligand binding poses in the human dopamine D3 receptor based on Snooker pharmacophores were ranked among the best models in the community wide GPCR dock 2010.     

Bivalent ligands of CXCR4 with rigid linkers for elucidation of the dimerization state in cells

To date, challenges in the design of bivalent ligands for G protein-coupled receptors (GPCRs) have revealed difficulties stemming from lack of knowledge of the state of oligomerization of the GPCR. The synthetic bivalent ligands with rigid linkers that are presented here can predict the dimer form of CXCR4 and be applied to molecular probes in cancerous cells. This "molecular ruler" approach would be useful in elucidating the details of CXCR4 oligomer formation.     

Over-expression, solubilization, and purification of G protein-coupled receptors for structural biology

With the advent of the recent determination of high-resolution crystal structures of bovine rhodopsin and human beta2 adrenergic receptor (beta2AR), there are still many structure-function relationships to be learned from other G protein-coupled receptors (GPCRs). Many of the pharmaceutically interesting GPCRs cannot be modeled because of their amino acid sequence divergence from bovine rhodopsin and beta2AR. Structure determination of GPCRs can provide new avenues for engineering drugs with greater potency and higher specificity. Several obstacles need to be overcome before membrane protein structural biology becomes routine: over-expression, solubilization, and purification of milligram quantities of active and stable GPCRs. Coordinated iterative efforts are required to generate any significant GPCR over-expression. To formulate guidelines for GPCR purification efforts, we review published conditions for solubilization and purification using detergents and additives. A discussion of sample preparation of GPCRs in detergent phase, bicelles, nanodiscs, or low-density lipoproteins is presented in the context of potential structural biology applications. In addition, a review of the solubilization and purification of successfully crystallized bovine rhodopsin and beta2AR highlights tools that can be used for other GPCRs.     

New strategies in drug discovery for GPCRs: high throughput detection of cellular ERK phosphorylation

G-protein coupled receptors (GPCRs) are a large family of receptors for a wide range of stimulants, including hormones, neurotransmitters, and taste and olfactory chemicals. Due to their broad involvement in cellular responses, GPCRs affect many important body functions both in health and disease. Compared to other receptor families, the GPCRs have been a rich source of extracellularly-acting pharmaceuticals, due largely to the fact that many GPCR ligands are small molecules when compared with ligands for other receptors, such as the tyrosine kinase receptor family. This has allowed the development of small molecule modulators of receptor function that act on specific GPCRs, such as those involved in cardiovascular regulation. However, at several levels, current screening technologies of drug development for GPCRs are lacking. Firstly, responses from many GPCRs, such as the Gi-coupled GPCRs, are not easily measured in large screening programs by current techniques. Secondly, there are few options for detecting agonists of orphan GPCRs. Thirdly, it is now clear that the signaling from GPCRs is more complex than once thought, and the measurement of Ca(2+) and cAMP can account for only a fraction of the biological information emanating from an activated GPCR. Studies of the discrete and sometimes separable activation of the Ras/Raf/Mek/ERK cascade by many GPCRs is likely to offer development of new agonists and antagonists, contribute to new pharmacologies from receptors, and raise the potential for novel drug candidates in this important area of biology. Downstream activation of the ERK pathway, with or without transactivation of growth factor receptors, has not been measurable by high throughput methodologies. This article presents recent advances and associated applications for screening of GPCRs and other receptor species through the rapid measurement of protein phosphorylation events, such as ERK phosphorylation, as new readouts for drug discovery.     

Solid-state NMR evidence for a protonation switch in the binding pocket of the H1 receptor upon binding of the agonist histamine

G protein coupled receptors (GPCRs) represent a major superfamily of transmembrane receptor proteins that are crucial in cellular signaling and are major pharmacological targets. While the activity of GPCRs can be modulated by agonist binding, the mechanisms that link agonist binding to G protein coupling are poorly understood. Here we present a method to accurately examine the activity of ligands in their bound state, even at low affinity, by solid-state NMR dipolar correlation spectroscopy and confront this method with the human H1 receptor. The analysis reveals two different charge states of the bound agonist, dicationic with a charged imidazole ring and monocationic with a neutral imidazole ring, with the same overall conformation. The combination of charge difference and pronounced heterogeneity agrees with converging evidence that the active and inactive states of the GPCR represent a dynamic equilibrium of substates and that proton transfer between agonist and protein side chains can shift this equilibrium by stabilizing the active receptor population relative to the inactive one. In fact, the data suggest a global functional analogy between H1 receptor activation and the meta I/meta II charge/discharge equilibrium in rhodopsin (GPCR). This corroborates current ideas on unifying principles in GPCR structure and function.     

Membrane protein microarrays

This paper describes the fabrication of microarrays consisting of G protein-coupled receptors (GPCRs) on surfaces coated with gamma-aminopropylsilane (GAPS). Microspots of model membranes on GAPS-coated surfaces were observed to have several desired properties-high mechanical stability, long range lateral fluidity, and a thickness corresponding to a lipid bilayer in the bulk of the microspot. GPCR arrays were obtained by printing membrane preparations containing GPCRs using a quill-pin printer. To demonstrate specific binding of ligands, arrays presenting neurotensin (NTR1), adrenergic (beta1), and dopamine (D1) receptors were treated with fluorescently labeled neurotensin (BT-NT). Fluorescence images revealed binding only to microspots corresponding to the neurotensin receptor; this specificity was further demonstrated by the inhibition of binding in the presence of excess unlabeled neurotensin. The ability of GPCR arrays to enable selectivity studies between the different subtypes of a receptor was examined by printing arrays consisting of three subtypes of the adrenergic receptor: beta1, beta2, and alpha2A. When treated with fluorescently labeled CGP 12177, a cognate antagonist analogue specific to beta-adrenergic receptors, binding was only observed to microspots of the beta1 and beta2 receptors. Furthermore, binding of labeled CGP 12177 was inhibited when the arrays were incubated with solutions also containing ICI 118551, and in a manner consistent with the higher affinity of ICI 118551 for the beta2 receptor relative to that for the beta1 receptor. The ability to estimate binding affinities of compounds using GPCR arrays was examined using a competitive binding assay with BT-NT and unlabeled neurotensin on NTR1 arrays. The estimated IC(50) value (2 nM) for neurotensin is in agreement with the literature; this agreement suggests that the receptor -G protein complex is preserved in the microspot. This first ever demonstration of direct pin-printing of membrane proteins and ligand-binding assays thereof fills a significant void in protein microchip technology--the lack of practical microarray-based methods for membrane proteins.     

High-throughput modeling of human G-protein coupled receptors: amino acid sequence alignment, three-dimensional model building, and receptor library screening

The current study describes the development of a computer package (GPCRmod) aimed at the high-throughput modeling of the therapeutically important family of human G-protein coupled receptors (GPCRs). GPCRmod first proposes a reliable alignment of the seven transmembrane domains (7 TMs) of most druggable human GPCRs based on pattern/motif recognition for each of the 7 TMs that are considered independently. It then converts the alignment into knowledge-based three-dimensional (3-D) models starting from a set of 3-D backbone templates and two separate rotamer libraries for side chain positioning. The 7 TMs of 277 human GPCRs have been accurately aligned, unambiguously clustered in three different classes (rhodopsin-like, secretin-like, metabotropic glutamate-like), and converted into high-quality 3-D models at a remarkable throughput (ca. 3s/model). A 3-D GPCR target library of 277 receptors has consequently been setup. Its utility for "in silico" inverse screening purpose has been demonstrated by recovering among top scorers the receptor of a selective GPCR antagonist as well as the receptors of a promiscuous antagonist. The current GPCR target library thus constitutes a 3-D database of choice to address as soon as possible the "virtual selectivity" profile of any GPCR antagonist or inverse agonist in an early hit optimization process.     

G protein betagamma subunits as targets for small molecule therapeutic development

G proteins mediate the action of G protein coupled receptors (GPCRs), a major target of current pharmaceuticals and a major target of interest in future drug development. Most pharmaceutical interest has been in the development of selective GPCR agonists and antagonists that activate or inhibit specific GPCRs. Some recent thinking has focused on the idea that some pathologies are the result of the actions of an array of GPCRs suggesting that targeting single receptors may have limited efficacy. Thus, targeting pathways common to multiple GPCRs that control critical pathways involved in disease has potential therapeutic relevance. G protein betagamma subunits released from some GPCRs upon receptor activation regulate a variety of downstream pathways to control various aspects of mammalian physiology. There is evidence from cell- based and animal models that excess Gbetagamma signaling can be detrimental and blocking Gbetagamma signaling has salutary effects in a number of pathological models. Gbetagamma regulates downstream pathways through modulation of enzymes that produce cellular second messengers or through regulation of ion channels by direct protein-protein interactions. Thus, blocking Gbetagamma functions requires development of small molecule agents that disrupt Gbetagamma protein interactions with downstream partners. Here we discuss evidence that small molecule targeting Gbetagamma could be of therapeutic value. The concept of disruption of protein-protein interactions by targeting a "hot spot" on Gbetagamma is delineated and the biochemical and virtual screening strategies for identification of small molecules that selectively target Gbetagamma functions are outlined. Evaluation of the effectiveness of virtual screening indicates that computational screening enhanced identification of true Gbetagamma binding molecules. However, further refinement of the approach could significantly improve the yield of Gbetagamma binding molecules from this screen that could result in multiple candidate leads for future drug development.     

Direct interaction between an allosteric agonist pepducin and the chemokine receptor CXCR4

Cell surface heptahelical G protein-coupled receptors (GPCRs) mediate critical cellular signaling pathways and are important pharmaceutical drug targets. (1) In addition to traditional small-molecule approaches, lipopeptide-based GPCR-derived pepducins have emerged as a new class of pharmaceutical agents. (2, 3) To better understand how pepducins interact with targeted receptors, we developed a cell-based photo-cross-linking approach to study the interaction between the pepducin agonist ATI-2341 and its target receptor, chemokine C-X-C-type receptor 4 (CXCR4). A pepducin analogue, ATI-2766, formed a specific UV-light-dependent cross-link to CXCR4 and to mutants with truncations of the N-terminus, the known chemokine docking site. These results demonstrate that CXCR4 is the direct binding target of ATI-2341 and suggest a new mechanism for allosteric modulation of GPCR activity. Adaptation and application of our findings should prove useful in further understanding pepducin modulation of GPCRs as well as enable new experimental approaches to better understand GPCR signal transduction.     

Classifying G-protein coupled receptors with bagging classification tree

G-protein coupled receptors (GPCRs) play a key role in different biological processes, such as regulation of growth, death and metabolism of cells. They are major therapeutic targets of numerous prescribed drugs. However, the ligand specificity of many receptors is unknown and there is little structural information available. Bioinformatics may offer one approach to bridge the gap between sequence data and functional knowledge of a receptor. In this paper, we use a bagging classification tree algorithm to predict the type of the receptor based on its amino acid composition. The prediction is performed for GPCR at the sub-family and sub-sub-family level. In a cross-validation test, we achieved an overall predictive accuracy of 91.1% for GPCR sub-family classification, and 82.4% for sub-sub-family classification. These results demonstrate the applicability of this relative simple method and its potential for improving prediction accuracy.     

Molecular Alliance—From Orthosteric and Allosteric Ligands to Dualsteric/Bitopic Agonists at G Protein Coupled Receptors

Cell‐membrane‐spanning G protein coupled receptors (GPCRs) belong to the most important therapeutic target structures. Endogenous transmitters bind from the outer side of the membrane to the “orthosteric” binding site either deep in the binding pocket or at the extracellular N‐terminal end of the receptor protein. Exogenous modulators that utilize a different, “allosteric”, binding site unveil a pathway to receptor subtype‐selectivity. However, receptor activation through the orthosteric area is often more powerful. Recently there has been evidence that orthosteric/allosteric, in other words “dualsteric”, hybrid compounds unite subtype selectivity and receptor activation. These “bitopic” modulators channelreceptor activation and subsequent intracellular signaling into a subset of possible routes. This concept offers access to GPCR modulators with an unprecedented receptor‐subtype and signaling selectivity profile and, as a consequence, to drugs with fewer side effects.     

Activation of G-protein-coupled receptors in cell-derived plasma membranes supported on porous beads

G-protein-coupled receptors (GPCRs) are ubiquitous mediators of signal transduction across cell membranes and constitute a very important class of therapeutic targets. In order to study the complex biochemical signaling network coupling to the intracellular side of GPCRs, it is necessary to engineer and control the downstream signaling components, which is difficult to realize in living cells. We have developed a bioanalytical platform enabling the study of GPCRs in their native membrane transferred inside-out from live cells to lectin-coated beads, with both membrane sides of the receptor being accessible for molecular interactions. Using heterologously expressed adenosine A(2A) receptor carrying a yellow fluorescent protein, we showed that the tethered membranes comprised fully functional receptors in terms of ligand and G protein binding. The interactions between the different signaling partners during the formation and subsequent dissociation of the ternary signaling complex on single beads could be observed in real time using multicolor fluorescence microscopy. This approach of tethering inside-out native membranes accessible from both sides is straightforward and readily applied to other transmembrane proteins. It represents a generic platform suitable for ensemble as well as single-molecule measurements to investigate signaling processes at plasma membranes.     

Homo‐molecular Fluorescence Complementation for Direct Visualization of Receptor Oligomerization in Living Cells

Cell surface receptor oligomerization is an attractive target process for drug screening. However, simple but reliable (and thus high‐throughput) visualization methods for receptor oligomerization are still lacking. Herein, we report on a new single‐construct homo‐molecular fluorescence complementation (Homo‐FC) probe, which shows strong fluorescence signals by oligomerization of fused receptors in living cells with unexpectedly low background signals. Importantly, this high signal‐to‐noise ratio was not affected by expression level variations of fused receptors. The Homo‐FC probe was developed by optimized flopped fusion of split fragments of superfolder green fluorescence protein and subsequent surface charge engineering. Homo‐FC reliably visualized the oligomerization of diverse natural receptors such as GPCR, EGFR, and even cytosolic DAI.     

G-Protein-Coupled Receptor–Membrane Interactions Depend on the Receptor Activation State

G-protein-coupled receptors (GPCRs) are the largest family of human membrane proteins and serve as primary targets of approximately one-third of currently marketed drugs. In particular, adenosine A₁ receptor (A₁AR) is an important therapeutic target for treating cardiac ischemia–reperfusion injuries, neuropathic pain, and renal diseases. As a prototypical GPCR, the A₁AR is located within a phospholipid membrane bilayer and transmits cellular signals by changing between different conformational states. It is important to elucidate the lipid–protein interactions in order to understand the functional mechanism of GPCRs. Here, all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method were performed on both the inactive (antagonist bound) and active (agonist and G-protein bound) A₁AR, which was embedded in a 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) lipid bilayer. In the GaMD simulations, the membrane lipids played a key role in stabilizing different conformational states of the A₁AR. Our simulations further identified important regions of the receptor that interacted distinctly with the lipids in highly correlated manner. Activation of the A₁AR led to differential dynamics in the upper and lower leaflets of the lipid bilayer. In summary, GaMD enhanced simulations have revealed strongly coupled dynamics of the GPCR and lipids that depend on the receptor activation state. © 2019 Wiley Periodicals, Inc.     

GPCR–drug interactions prediction using random forest with drug-association-matrix-based post-processing procedure

G-protein-coupled receptors (GPCRs) are important targets of modern medicinal drugs. The accurate identification of interactions between GPCRs and drugs is of significant importance for both protein function annotations and drug discovery. In this paper, a new sequence-based predictor called TargetGDrug is designed and implemented for predicting GPCR–drug interactions. In TargetGDrug, the evolutionary feature of GPCR sequence and the wavelet-based molecular fingerprint feature of drug are integrated to form the combined feature of a GPCR–drug pair; then, the combined feature is fed to a trained random forest (RF) classifier to perform initial prediction; finally, a novel drug-association-matrix-based post-processing procedure is applied to reduce potential false positive or false negative of the initial prediction. Experimental results on benchmark datasets demonstrate the efficacy of the proposed method, and an improvement of 15% in the Matthews correlation coefficient (MCC) was observed over independent validation tests when compared with the most recently released sequence-based GPCR–drug interactions predictor. The implemented webserver, together with the datasets used in this study, is freely available for academic use at http://csbio.njust.edu.cn/bioinf/TargetGDrug.