A crystal clear solution for determining G-protein-coupled receptor structures

G-protein-coupled receptors (GPCRs) are medically important membrane proteins that are targeted by over 30% of small molecule drugs. At the time of writing, 15 unique GPCR structures have been determined, with 77 structures deposited in the PDB database, which offers new opportunities for drug development and for understanding the molecular mechanisms of GPCR activation. Many different factors have contributed to this success, but if there is one single factor that can be singled out as the foundation for producing well-diffracting GPCR crystals, it is the stabilisation of the detergent-solubilised receptor-ligand complex. This review will focus predominantly on one of the successful strategies for the stabilisation of GPCRs, namely the thermostabilisation of GPCRs using systematic mutagenesis coupled with thermostability assays. Structures of thermostabilised GPCRs bound to a wide variety of ligands have been determined, which has led to an understanding of ligand specificity; why some ligands act as agonists as opposed to partial or inverse agonists; and the structural basis for receptor activation.     

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

Background

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

Methodology

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

Conclusions

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

New insights into GPCR coupling and dimerisation from cryo-EM structures

Over the past three years (2020–2022) more structures of GPCRs have been determined than in the previous twenty years (2000–2019), primarily of GPCR complexes that are large enough for structure determination by single-particle cryo-EM. This review will present some structural highlights that have advanced our molecular understanding of promiscuous G protein coupling, how a G protein receptor kinase and β-arrestins couple to GPCRs, and GPCR dimerisation. We will also discuss advances in the use of gene fusions, nanobodies, and F_ab fragments to facilitate the structure determination of GPCRs in the inactive state that, on their own, are too small for structure determination by single-particle cryo-EM.     

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.     

Relevance of rhodopsin studies for GPCR activation

Rhodopsin, the dim-light photoreceptor present in the rod cells of the retina, is both a retinal-binding protein and a G protein-coupled receptor (GPCR). Due to this conjunction, it benefits from an arsenal of spectroscopy techniques that can be used for its characterization, while being a model system for the important family of Class A (also referred to as “rhodopsin-like”) GPCRs. For instance, rhodopsin has been a crucial player in the field of GPCR structural biology. Until 2007, it was the only GPCR for which a high-resolution crystal structure was available, so all structure–activity analyses on GPCRs, from structure-based drug discovery to studies of structural changes upon activation, were based on rhodopsin. At present, about a third of currently available GPCR structures are still from rhodopsin. In this review, I show some examples of how these structures can still be used to gain insight into general aspects of GPCR activation. First, the analysis of the third intracellular loop in rhodopsin structures allows us to gain an understanding of the structural and dynamic properties of this region, which is absent (due to protein engineering or poor electron density) in most of the currently available GPCR structures. Second, a detailed analysis of the structure of the transmembrane domains in inactive, intermediate and active rhodopsin structures allows us to detect early conformational changes in the process of ligand-induced GPCR activation. Finally, the analysis of a conserved ligand-activated transmission switch in the transmembrane bundle of GPCRs in the context of the rhodopsin activation cycle, allows us to suggest that the structures of many of the currently available agonist-bound GPCRs may correspond to intermediate active states. While the focus in GPCR structural biology is inevitably moving away from rhodopsin, in other aspects rhodopsin is still at the forefront. For instance, the first studies of the structural basis of disease mutants in GPCRs, or the most detailed analysis of cellular GPCR signal transduction networks using a systems biology approach, have been carried out in rhodopsin. Finally, due again to its unique properties among GPCRs, rhodopsin will likely play an important role in the application of X-ray free electron laser crystallography to time-resolved structural biology in membrane proteins. Rhodopsin, thus, still remains relevant as a model system to study the molecular mechanisms of GPCR activation. This article is part of a Special Issue entitled: Retinal Proteins—You can teach an old dog new tricks.     

Nanobody stabilization of G protein-coupled receptor conformational states

Remarkable progress has been made in the field of G protein-coupled receptor (GPCR) structural biology during the past four years. Several obstacles to generating diffraction quality crystals of GPCRs have been overcome by combining innovative methods ranging from protein engineering to lipid-based screens and microdiffraction technology. The initial GPCR structures represent energetically stable inactive-state conformations. However, GPCRs signal through different G protein isoforms or G protein-independent effectors upon ligand binding suggesting the existence of multiple ligand-specific active states. These active-state conformations are unstable in the absence of specific cytosolic signaling partners representing new challenges for structural biology. Camelid single chain antibody fragments (nanobodies) show promise for stabilizing active GPCR conformations and as chaperones for crystallogenesis.     

Emerging structural biology of lipid G protein‐coupled receptors

The first crystal structure of a G protein‐coupled receptor (GPCR) was that of the bovine rhodopsin, solved in 2000, and is a light receptor within retina rode cells that enables vision by transducing a conformational signal from the light‐induced isomerization of retinal covalently bound to the receptor. More than 7 years after this initial discovery and following more than 20 years of technological developments in GPCR expression, stabilization, and crystallography, the high‐resolution structure of the adrenaline binding β₂‐adrenergic receptor, a ligand diffusible receptor, was discovered. Since then, high‐resolution structures of more than 53 unique GPCRs have been determined leading to a significant improvement in our understanding of the basic mechanisms of ligand‐binding and ligand‐mediated receptor activation that revolutionized the field of structural molecular pharmacology of GPCRs. Recently, several structures of eight unique lipid‐binding receptors, one of the most difficult GPCR families to study, have been reported. This review presents the outstanding structural and pharmacological features that have emerged from these new lipid receptor structures. The impact of these findings goes beyond mechanistic insights, providing evidence of the fundamental role of GPCRs in the physiological integration of the lipid signaling system, and highlighting the importance of sustained research into the structural biology of GPCRs for the development of new therapeutics targeting lipid receptors.     

G蛋白偶联受体的结构生物学研究

G蛋白偶联受体(G protein-coupled receptor,GPCR)在细胞信号转导过程中发挥关键的生理学功能,是极其重要的药物靶标,其三维结构信息对功能研究以及新药研发具有十分重要的意义。近年来,新技术的发展和应用使GPCR的结构生物学研究发生了跨越式的发展,本文简要回顾这些新的技术和方法以及已解析的GPCR三维结构,并以CCR5和P2Y12R两种受体的结构为例来具体阐明现阶段GPCR结构生物学研究的内容和意义。     

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.     

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.     

Increasingly accurate dynamic molecular models of G-protein coupled receptor oligomers: Panacea or Pandora's box for novel drug discovery?

For years, conventional drug design at G-protein coupled receptors (GPCRs) has mainly focused on the inhibition of a single receptor at a usually well-defined ligand-binding site. The recent discovery of more and more physiologically relevant GPCR dimers/oligomers suggests that selectively targeting these complexes or designing small molecules that inhibit receptor–receptor interactions might provide new opportunities for novel drug discovery. To uncover the fundamental mechanisms and dynamics governing GPCR dimerization/oligomerization, it is crucial to understand the dynamic process of receptor–receptor association, and to identify regions that are suitable for selective drug binding. This minireview highlights current progress in the development of increasingly accurate dynamic molecular models of GPCR oligomers based on structural, biochemical, and biophysical information that has recently appeared in the literature. In view of this new information, there has never been a more exciting time for computational research into GPCRs than at present. Information-driven modern molecular models of GPCR complexes are expected to efficiently guide the rational design of GPCR oligomer-specific drugs, possibly allowing researchers to reach for the high-hanging fruits in GPCR drug discovery, i.e. more potent and selective drugs for efficient therapeutic interventions.     

Structural Features of β2 Adrenergic Receptor: Crystal Structures and Beyond

The beta2-adrenergic receptor (β2AR) family, which is the largest family of cell surface receptors in humans. Extra attention has been focused on the human GPCRs because they have been studied as important protein targets for pharmaceutical drug development. In fact, approximately 40% of marketed drugs directly work on GPCRs. GPCRs respond to various extracellular stimuli, such as sensory signals, neurotransmitters, chemokines, and hormones, to induce structural changes at the cytoplasmic surface, activating downstream signaling pathways, primarily through interactions with heterotrimeric G proteins or through G-protein independent pathways, such as arrestin. Most GPCRs, except for rhodhopsin, which contains covalently linked 11 cis-retinal, bind to diffusible ligands, having various conformational states between inactive and active structures. The first human GPCR structure was determined using an inverse agonist bound β2AR in 2007 and since then, more than 20 distinct GPCR structures have been solved. However, most GPCR structures were solved as inactive forms, and an agonist bound fully active structure is still hard to obtain. In a structural point of view, β2AR is relatively well studied since its fully active structure as a complex with G protein as well as several inactive structures are available. The structural comparison of inactive and active states gives an important clue in understanding the activation mechanism of β2AR. In this review, structural features of inactive and active states of β2AR, the interaction of β2AR with heterotrimeric G protein, and the comparison with β1AR will be discussed.     

G‐protein‐coupled receptor structures were not built in a day

Among the most exciting recent developments in structural biology is the structure determination of G‐protein‐coupled receptors (GPCRs), which comprise the largest class of membrane proteins in mammalian cells and have enormous importance for disease and drug development. The GPCR structures are perhaps the most visible examples of a nascent revolution in membrane protein structure determination. Like other major milestones in science, however, such as the sequencing of the human genome, these achievements were built on a hidden foundation of technological developments. Here, we describe some of the methods that are fueling the membrane protein structure revolution and have enabled the determination of the current GPCR structures, along with new techniques that may lead to future structures.     

Allostery in GPCRs: 'MWC' revisited

G protein-coupled receptors (GPCRs) constitute the largest family of receptors in the genome and are the targets for at least 30% of current medicines. In recent years, there has been a dramatic increase in the discovery of allosteric modulators of GPCR activity and a growing appreciation of the diverse modes by which GPCRs can be regulated by both orthosteric and allosteric ligands. Interestingly, some of the contemporary views of GPCR function reflect characteristics that are shared by prototypical allosteric proteins, as encompassed in the classic Monod-Wyman-Changeux (MWC) model initially proposed for enzymes and subsequently extended to other protein families. In this review, we revisit the MWC model in the context of emerging structural, functional and operational data on GPCR allostery.     

Monomeric G-protein-coupled receptor as a functional unit

Rhodopsin, the first purified G-protein-coupled receptor (GPCR), was characterized as a functional monomer 30 year ago, but dimerization of GPCRs recently became the new paradigm of signal transduction. It has even been claimed, on the basis of recent biophysical and biochemical studies, that this new concept could be extended to higher-order oligomerization. Here this view is challenged. The new studies of rhodopsin and other simple (class 1a) GPCRs solubilized in detergent are re-assessed and are compared to the earlier classical studies of rhodopsin and other membrane proteins solubilized in detergent. The new studies are found to strengthen rather than invalidate the conclusions of the early ones and to support a monomeric model for rhodopsin and other class 1a GPCRs. A molecular model is proposed for the functional coupling of a rhodopsin monomeric unit with a G-protein heterotrimer. This model should be valid even for GPCRs that exist as structural dimers.     

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

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

Improving the apo-state detergent stability of NTS1 with CHESS for pharmacological and structural studies

The largest single class of drug targets is the G protein-coupled receptor (GPCR) family. Modern high-throughput methods for drug discovery require working with pure protein, but this has been a challenge for GPCRs, and thus the success of screening campaigns targeting soluble, catalytic protein domains has not yet been realized for GPCRs. Therefore, most GPCR drug screening has been cell-based, whereas the strategy of choice for drug discovery against soluble proteins is HTS using purified proteins coupled to structure-based drug design. While recent developments are increasing the chances of obtaining GPCR crystal structures, the feasibility of screening directly against purified GPCRs in the unbound state (apo-state) remains low. GPCRs exhibit low stability in detergent micelles, especially in the apo-state, over the time periods required for performing large screens. Recent methods for generating detergent-stable GPCRs, however, offer the potential for researchers to manipulate GPCRs almost like soluble enzymes, opening up new avenues for drug discovery. Here we apply cellular high-throughput encapsulation, solubilization and screening (CHESS) to the neurotensin receptor 1 (NTS₁) to generate a variant that is stable in the apo-state when solubilized in detergents. This high stability facilitated the crystal structure determination of this receptor and also allowed us to probe the pharmacology of detergent-solubilized, apo-state NTS₁ using robotic ligand binding assays. NTS₁ is a target for the development of novel antipsychotics, and thus CHESS-stabilized receptors represent exciting tools for drug discovery.     

Sequence,Structure and Ligand Binding Evolution of Rhodopsin-Like G Protein-Coupled Receptors: A Crystal Structure-Based Phylogenetic Analysis

G protein-coupled receptors (GPCRs) form the largest family of membrane receptors in the human genome. Advances in membrane protein crystallization so far resulted in the determination of 24 receptors available as high-resolution atomic structures. We performed the first phylogenetic analysis of GPCRs based on the available set of GPCR structures. We present a new phylogenetic tree of known human rhodopsin-like GPCR sequences based on this structure set. We can distinguish the three separate classes of small-ligand binding GPCRs, peptide binding GPCRs, and olfactory receptors. Analyzing different structural subdomains, we found that small molecule binding receptors most likely have evolved from peptide receptor precursors, with a rhodopsin/S1PR1 ancestor, most likely an ancestral opsin, forming the link between both classes. A light-activated receptor therefore seems to be the origin of the small molecule hormone receptors of the central nervous system. We find hints for a common evolutionary path of both ligand binding site and central sodium/water binding site. Surprisingly, opioid receptors exhibit both a binding cavity and a central sodium/water binding site similar to the one of biogenic amine receptors instead of peptide receptors, making them seemingly prone to bind small molecule ligands, e.g. opiates. Our results give new insights into the relationship and the pharmacological properties of rhodopsin-like GPCRs.     

The impact of G-protein-coupled receptor hetero-oligomerization on function and pharmacology

Although highly controversial just a few years ago, the idea that G-protein-coupled receptors (GPCRs) may undergo homo-oligomerization or hetero-oligomerization has recently gained considerable attention. The recognition that GPCRs may exhibit either dimeric or oligomeric structures is based on a number of different biochemical and biophysical approaches. Although much effort has been spent to demonstrate the mechanism(s) by which GPCRs interact with each other, the physiological relevance of this phenomenon remains elusive. An additional source of uncertainty stems from the realization that homo-oligomerization and hetero-oligomerization of GPCRs may affect receptor binding and activity in different ways, depending on the type of interacting receptors. In this brief review, the functional and pharmacological effects of the hetero-oligomerization of GPCR on binding and cell signaling are critically analyzed.     

New tools for G-protein coupled receptor (GPCR) drug discovery: combination of baculoviral expression system and solid state NMR

Biotechnology using molecular biology, biochemistry, biophysics, and computational approaches provides an alternative approach for classical pharmacological screening to look at ligand-receptor interactions and receptor specificity, which should support the design of selective drugs based on detailed structural principles. This review addresses specific approaches to study function, structure and relevance of a major pharmaceutical target, namely the G-Protein Coupled Receptors (GPCRs). The main aim of this review has been to exploit and combine GPCR over-expression in a baculoviral expression system with solid-state MAS NMR (ssNMR) approaches for the elucidation of electronic structures of the coordinating ligands/drugs and their modes of interactions with the GPCRs. This review summarizes the approaches, possible future experiments and developments using the above combination of tools for GPCR drug discovery.