G Protein-coupled Receptors Structure Research Articles

Structural Insights and Characterization of a Novel Transmembrane Binding Site for Potential Allosteric Modulation of the Mu‐opioid Receptor

Objective Opioids such as morphine, codeine, and oxycodone represent the gold standard for the treatment of pain. These drugs elicit their analgesic action by activating the mu-opioid receptor (mu-OR), a class A G protein-coupled receptor (GPCR). These agonists suffer from serious side effects, including respiratory depression, constipation, tolerance, and drug abuse. Recent advances in the structural biology of GPCRs revealed novel transmembrane lipid-facing binding pockets for allosteric modulators of the beta-2-adrenergic and cannabinoid (CB1) receptors. Allosteric ligands offer distinct advantages such as greater receptor subtype selectivity and the potential to “finetune” endogenous agonism while preserving the temporal and spatial characteristics of the signaling. Most importantly, allosteric modulators can offer unique safety by providing a “ceiling level” for their effects. Our central hypothesis is that a druggable transmembrane allosteric site exists for mu-OR, and high-affinity small-molecule ligands that stabilize the receptor in an active conformation can be identified. In this study, we identified such a novel transmembrane allosteric binding site on mu-OR and several high-affinity positive allosteric modulators through a comprehensive multidisciplinary (computational and experimental) approach. Our results provide the structural and mechanistic basis for the allosteric modulation of the receptor through this site and offer insights into the key binding site residues and membrane interactions that are critical for ligand affinity and receptor stabilization in the active state. Methods We employed a comprehensive computational approach that includes a sequence- and structure-based alignment of several experimental structures of GPCRs bound to their allosteric modulators to identify and characterize lipid-facing transmembrane binding sites. Structure-based pharmacophore models were developed, refined, and used for virtual screening of commercially available compound libraries to identify potential ligands using MOE software. Molecular docking, molecular dynamics simulations, and funnel metadynamics were used to assess the relative binding energies, using GROMACS and PLUMED software. Top ligands were evaluated by multiple in vitro assays for the positive allosteric modulation of mu-OR. Results A lipid-facing transmembrane binding site distinct from the orthosteric site was identified, and several high-affinity small-molecule allosteric modulators with drug-like properties were obtained by a combined computational and experimental approach. A variety of activation signature distances monitored were able to distinguish an active receptor from an inactive. The molecules will be further validated by in vivo methods for potential analgesic actions. Conclusion Our results illustrate a potential allosteric binding site on the transmembrane helices of the µ-opioid receptor that requires the active participation of membrane lipids for stable binding of ligands. The obtained ligands may translate into a safe and potential therapeutic alternative for opioids in the treatment of pain.

Development of an Antigen-Antibody Co-Display System for Detecting Interaction of G-Protein-Coupled Receptors and Single-Chain Variable Fragments.

G-protein-coupled receptors (GPCRs), especially chemokine receptors, are ideal targets for monoclonal antibody drugs. Considering the special multi-pass transmembrane structure of GPCR, it is often a laborious job to obtain antibody information about off-targets and epitopes on antigens. To accelerate the process, a rapid and simple method needs to be developed. The split-ubiquitin-based yeast two hybrid system (YTH) was used as a blue script for a new method. By fusing with transmembrane peptides, scFv antibodies were designed to be anchored on the cytomembrane, where the GPCR was co-displayed as well. The coupled split-ubiquitin system transformed the scFv-GPCR interaction signal into the expression of reporter genes. By optimizing the topological structure of scFv fusion protein and key elements, including signal peptides, transmembrane peptides, and flexible linkers, a system named Antigen-Antibody Co-Display (AACD) was established, which rapidly detected the interactions between antibodies and their target GPCRs, CXCR4 and CXCR5, while also determining the off-target antibodies and antibody-associated epitopes. The AACD system can rapidly determine the association between GPCRs and their candidate antibodies and shorten the research period for off-target detection and epitope identification. This system should improve the process of GPCR antibody development and provide a new strategy for GPCRs antibody screening.

BIO-GATS: A Tool for Automated GPCR Template Selection Through a Biophysical Approach for Homology Modeling.

G protein-coupled receptors (GPCRs) are the largest family of membrane proteins with more than 800 members. GPCRs are involved in numerous physiological functions within the human body and are the target of more than 30% of the United States Food and Drug Administration (FDA) approved drugs. At present, over 400 experimental GPCR structures are available in the Protein Data Bank (PDB) representing 76 unique receptors. The absence of an experimental structure for the majority of GPCRs demand homology models for structure-based drug discovery workflows. The generation of good homology models requires appropriate templates. The commonly used methods for template selection are based on sequence identity. However, there exists low sequence identity among the GPCRs. Sequences with similar patterns of hydrophobic residues are often structural homologs, even with low sequence identity. Extending this, we propose a biophysical approach for template selection based principally on hydrophobicity correspondence between the target and the template. Our approach takes into consideration other relevant parameters, including resolution, similarity within the orthosteric binding pocket of GPCRs, and structure completeness, for template selection. The proposed method was implemented in the form of a free tool called Bio-GATS, to provide the user with easy selection of the appropriate template for a query GPCR sequence. Bio-GATS was successfully validated with recent published benchmarking datasets. An application to an olfactory receptor to select an appropriate template has also been provided as a case study.

Computational evaluation of interactions between olfactory receptor OR2W1 and its ligands.

Mammalian olfactory receptors are a family of G protein‒coupled receptors (GPCRs) that occupy a large part of the genome. In human genes, olfactory receptors account for more than 40% of all GPCRs. Several types of GPCR structures have been identified, but there is no single olfactory receptor whose structure has been determined experimentally to date. The aim of this study was to model the interactions between an olfactory receptor and its ligands at the molecular level to provide hints on the binding modes between the OR2W1 olfactory receptor and its agonists and inverse agonists. The results demonstrated the modes of ligand binding in a three-dimensional model of OR2W1 and showed a statistically significant difference in binding affinity to the olfactory receptor between agonists and inverse agonists.

The Open Question of How GPCRs Interact with GPCR Kinases (GRKs).

G protein-coupled receptors (GPCRs), which regulate a vast number of eukaryotic processes, are desensitized by various mechanisms but, most importantly, by the GPCR kinases (GRKs). Ever since GRKs were first identified, investigators have sought to determine which structural features of GRKs are used to select for the agonist-bound states of GPCRs and how this binding event in turn enhances GRK catalytic activity. Despite a wealth of molecular information from high-resolution crystal structures of GRKs, the mechanisms driving activation have remained elusive, in part because the GRK N-terminus and active site tether region, previously proposed to serve as a receptor docking site and to be key to kinase domain closure, are often disordered or adopt inconsistent conformations. However, two recent studies have implicated other regions of GRKs as being involved in direct interactions with active GPCRs. Atomic resolution structures of GPCR–GRK complexes would help refine these models but are, so far, lacking. Here, we assess three distinct models for how GRKs recognize activated GPCRs, discuss limitations in the approaches used to generate them, and then experimentally test a hypothetical GPCR interaction site in GRK2 suggested by the two newest models.

Engineering of Challenging G Protein-Coupled Receptors for Structure Determination and Biophysical Studies.

Membrane proteins such as G protein-coupled receptors (GPCRs) exert fundamental biological functions and are involved in a multitude of physiological responses, making these receptors ideal drug targets. Drug discovery programs targeting GPCRs have been greatly facilitated by the emergence of high-resolution structures and the resulting opportunities to identify new chemical entities through structure-based drug design. To enable the determination of high-resolution structures of GPCRs, most receptors have to be engineered to overcome intrinsic hurdles such as their poor stability and low expression levels. In recent years, multiple engineering approaches have been developed to specifically address the technical difficulties of working with GPCRs, which are now beginning to make more challenging receptors accessible to detailed studies. Importantly, successfully engineered GPCRs are not only valuable in X-ray crystallography, but further enable biophysical studies with nuclear magnetic resonance spectroscopy, surface plasmon resonance, native mass spectrometry, and fluorescence anisotropy measurements, all of which are important for the detailed mechanistic understanding, which is the prerequisite for successful drug design. Here, we summarize engineering strategies based on directed evolution to reduce workload and enable biophysical experiments of particularly challenging GPCRs.

Allosteric coupling and biased agonism in G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are essential cell membrane signaling molecules and represent the most important class of drug targets. Some signaling pathways downstream of a GPCR may be responsible for drug adverse effects, while others mediate therapeutic efficacy. Biased ligands preferentially activate only a subset of all GPCR signaling pathways. They hold great potential to become next-generation GPCR drugs with less side effects due to their potential to exclusively activate desired signaling pathways. However, the molecular basis of biased agonism is poorly understood. GPCR activation occurs through allosteric coupling, the propagation of conformational changes from the extracellular ligand-binding pocket to the intracellular G protein-binding interface. Comparison of GPCR structures in complex with G proteins or β-arrestin reveals that intracellular transducer coupling results in closure of the ligand-binding pocket trapping the agonist inside its binding site. Allosteric coupling appears to be transducer-specific offering the possibility of harnessing this mechanism for the design of biased ligands. Here, we review the biochemical, pharmacological, structural, and biophysical evidence for allosteric coupling and delineate that biased agonism should be a consequence of preferential allosteric coupling from the ligand-binding pocket to one transducer-binding site. As transducer binding leads to large structural rearrangements in the extracellular ligand-binding pocket, we survey biased ligands with an extended binding mode that interact with extracellular receptor domains. We propose that biased ligands use ligand-specific triggers inside the binding pocket that are relayed through preferential allosteric coupling to a specific transducer, eventually leading to biased signaling.

Predictable cholesterol binding sites in GPCRs lack consensus motifs

A rich diversity of transmembrane G protein-coupled receptors (GPCRs) are used by eukaryotes to sense physical and chemical signals. In humans alone, 800 GPCRs comprise the largest and most therapeutically targeted receptor class. Recent advances in GPCR structural biology have produced hundreds of GPCR structures solved by X-ray diffraction and increasingly, cryo-electron microscopy (cryo-EM). Many of these structures are stabilized by site-specific cholesterol binding, but it is unclear whether these interactions are a product of recurring cholesterol-binding motifs and if observed patterns of cholesterol binding differ by experimental technique. Here, we comprehensively analyze the location and composition of cholesterol binding sites in the current set of 473 human GPCR structural chains. Our findings establish that cholesterol binds similarly in cryo-EM and X-ray structures and show that 92% of cholesterol molecules on GPCR surfaces reside in predictable locations that lack discernable cholesterol-binding motifs.

Homology Modeling of Class A G-Protein-Coupled Receptors in the Age of the Structure Boom.

With 700 members, G protein-coupled receptors (GPCRs) of the rhodopsin family (class A) form the largest membrane receptor family in humans and are the target of about 30% of presently available pharmaceutical drugs. The recent boom in GPCR structures led to the structural resolution of 57 unique receptors in different states (39 receptors in inactive state only, 2 receptors in active state only and 16 receptors in different activation states). In spite of these tremendous advances, most computational studies on GPCRs, including molecular dynamics simulations, virtual screening and drug design, rely on GPCR models obtained by homology modeling. In this protocol, we detail the different steps of homology modeling with the MODELLER software, from template selection to model evaluation. The present structure boom provides closely related templates for most receptors. If, in these templates, some of the loops are not resolved, in most cases, the numerous available structures enable to find loop templates with similar length for equivalent loops. However, simultaneously, the large number of putative templates leads to model ambiguities that may require additional information based on multiple sequence alignments or molecular dynamics simulations to be resolved. Using the modeling of the human bradykinin receptor B1 as a case study, we show how several templates are managed by MODELLER, and how the choice of template(s) and of template fragments can improve the quality of the models. We also give examples of how additional information and tools help the user to resolve ambiguities in GPCR modeling.

New small molecule fluorescent probes for G protein-coupled receptors: valuable tools for drug discovery.

G protein-coupled receptors (GPCRs) are essential signaling proteins and tractable therapeutic targets. To develop new drug candidates, GPCR drug discovery programs require versatile, sensitive pharmacological tools for ligand binding and compound screening. With the availability of new imaging modalities and proximity-based ligand binding technologies, fluorescent ligands offer many advantages and are increasingly being used, yet labeling small molecules remains considerably more challenging relative to peptides. Focusing on recent fluorescent small molecule studies for family A GPCRs, this review addresses some of the key challenges, synthesis approaches and structure-activity relationship considerations, and discussesadvantages of using high-resolution GPCR structures to inform conjugation strategies. While no single approach guarantees successful labeling without loss of affinity or selectivity, the choice of fluorophore, linker type and site of attachment have proved to be critical factors that can significantly affect their utility in drug discovery programs, and as discussed, can sometimes lead to very unexpected results.

GPCRdb in 2021: integrating GPCR sequence, structure and function.

G protein-coupled receptors (GPCRs) form both the largest family of membrane proteins and drug targets, mediating the action of one-third of medicines. The GPCR database, GPCRdb serves >4 000 researchers every month and offers reference data, analysis of own or literature data, experiment design and dissemination of published datasets. Here, we describe new and updated GPCRdb resources with a particular focus on integration of sequence, structure and function. GPCRdb contains all human non-olfactory GPCRs (and >27 000 orthologs), G-proteins and arrestins. It includes over 2 000 drug and in-trial agents and nearly 200 000 ligands with activity and availability data. GPCRdb annotates all published GPCR structures (updated monthly), which are also offered in a refined version (with re-modeled missing/distorted regions and reverted mutations) and provides structure models of all human non-olfactory receptors in inactive, intermediate and active states. Mutagenesis data in the GPCRdb spans natural genetic variants, GPCR-G protein interfaces, ligand sites and thermostabilising mutations. A new sequence signature tool for identification of functional residue determinants has been added and two data driven tools to design ligand site mutations and constructs for structure determination have been updated extending their coverage of receptors and modifications. The GPCRdb is available at https://gpcrdb.org.

Post-Translational Modifications of G Protein-Coupled Receptors Control Cellular Signaling Dynamics in Space and Time.

G protein-coupled receptors (GPCRs) are a large family comprising >800 signaling receptors that regulate numerous cellular and physiologic responses. GPCRs have been implicated in numerous diseases and represent the largest class of drug targets. Although advances in GPCR structure and pharmacology have improved drug discovery, the regulation of GPCR function by diverse post-translational modifications (PTMs) has received minimal attention. Over 200 PTMs are known to exist in mammalian cells, yet only a few have been reported for GPCRs. Early studies revealed phosphorylation as a major regulator of GPCR signaling, whereas later reports implicated a function for ubiquitination, glycosylation, and palmitoylation in GPCR biology. Although our knowledge of GPCR phosphorylation is extensive, our knowledge of the modifying enzymes, regulation, and function of other GPCR PTMs is limited. In this review we provide a comprehensive overview of GPCR post-translational modifications with a greater focus on new discoveries. We discuss the subcellular location and regulatory mechanisms that control post-translational modifications of GPCRs. The functional implications of newly discovered GPCR PTMs on receptor folding, biosynthesis, endocytic trafficking, dimerization, compartmentalized signaling, and biased signaling are also provided. Methods to detect and study GPCR PTMs as well as PTM crosstalk are further highlighted. Finally, we conclude with a discussion of the implications of GPCR PTMs in human disease and their importance for drug discovery. SIGNIFICANCE STATEMENT: Post-translational modification of G protein-coupled receptors (GPCRs) controls all aspects of receptor function; however, the detection and study of diverse types of GPCR modifications are limited. A thorough understanding of the role and mechanisms by which diverse post-translational modifications regulate GPCR signaling and trafficking is essential for understanding dysregulated mechanisms in disease and for improving and refining drug development for GPCRs.

Structure, dynamics and lipid interactions of serotonin receptors: excitements and challenges.

Serotonin (5-hydroxytryptamine, 5-HT) is an intrinsically fluorescent neurotransmitter found in organisms spanning a wide evolutionary range. Serotonin exerts its diverse actions by binding to distinct cell membrane receptors which are classified into many groups. Serotonin receptors are involved in regulating a diverse array of physiological signaling pathways and belong to the family of either G protein-coupled receptors (GPCRs) or ligand-gated ion channels. Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive and behavioral functions such as sleep, mood, pain, anxiety, depression, aggression, and learning. Serotonin receptors act as drug targets for a number of diseases, particularly neuropsychiatric disorders. The signaling mechanism and efficiency of serotonin receptors depend on their amazing ability to rapidly access multiple conformational states. This conformational plasticity, necessary for the wide variety of functions displayed by serotonin receptors, is regulated by binding to various ligands. In this review, we provide a succinct overview of recent developments in generating and analyzing high-resolution structures of serotonin receptors obtained using crystallography and cryo-electron microscopy. Capturing structures of distinct conformational states is crucial for understanding the mechanism of action of these receptors, which could provide important insight for rational drug design targeting serotonin receptors. We further provide emerging information and insight from studies on interactions of membrane lipids (such as cholesterol) with serotonin receptors. We envision that a judicious combination of analysis of high-resolution structures and receptor-lipid interaction would allow a comprehensive understanding of GPCR structure, function and dynamics, thereby leading to efficient drug discovery.

Activation Microswitches in Adenosine Receptor A2A Function as Rheostats in the Cell Membrane.

Although multiple components of the cell membrane modulate the stability and activation of G protein-coupled receptors (GPCRs), insights into the dynamics of GPCR structures come from biophysical studies conducted in detergents. This is because of the challenges of studying activation in a multicomponent lipid bilayer. To understand the role of cellular membrane lipids and cations in GPCR activation, we performed multiscale molecular dynamics simulations (56 μs) on three different conformational states of adenosine receptor A2AR, in both the cell membrane-like lipid bilayer and in detergent micelles. Molecular dynamics (MD) simulations show that the phosphatidylinositol bisphosphate (PIP2) interacts with the basic residues in the intracellular regions of A2AR, thereby reducing the flexibility of the receptor in the inactive state and limiting the transition to the active-intermediate state. In the G protein-coupled fully active state, PIP2 stabilizes the GPCR:G protein complex. Such stiffening effects are absent in non-ionic detergent micelles, and therefore, more transitions have been observed in detergents. The inter-residue distances that change significantly upon GPCR activation are known as activation microswitches. The activation microswitches show different levels of activation in the cell membrane, in the pure POPC bilayer, and in detergents. Thus, the temporal heat map of different activation microswitches calculated from the MD simulations suggests a rheostat model of GPCR activation microswitches rather than the binary switch model. These simulation results connect the chemistry of cell membrane lipids to receptor activity, which is useful for the design of detergents mimicking the cell membrane.

Illuminating the Path to Target GPCR Structures and Functions.

G-Protein-coupled receptors (GPCRs) are ubiquitous within eukaryotes, responsible for a wide array of physiological and pathological processes. Indeed, the fact that they are the most drugged target in the human genome is indicative of their importance. Despite the clear interest in GPCRs, most information regarding their activity has been so far obtained by analyzing the response from a "bulk medium". As such, this Perspective summarizes some of the common methods for this indirect observation. Nonetheless, by inspecting approaches applying super-resolution imaging, we argue that imaging is perfectly situated to obtain more detailed structural and spatial information, assisting in the development of new GPCR-targeted drugs and clinical strategies. The benefits of direct optical visualization of GPCRs are analyzed in the context of potential future directions in the field.

Efficiency of Homology Modeling Assisted Molecular Docking in G-protein Coupled Receptors.

Molecular docking is in regular practice to assess ligand affinity on a target protein crystal structure. In the absence of protein crystal structure, the homology modeling or comparative modeling is the best alternative to elucidate the relationship details between a ligand and protein at the molecular level. The development of accurate homology modeling (HM) and its integration with molecular docking (MD) is essential for successful, rational drug discovery. The G-protein coupled receptors (GPCRs) are attractive therapeutic targets due to their immense role in human pharmacology. The GPCRs are membrane-bound proteins with the complex constitution, and the understanding of their activation and inactivation mechanisms is quite challenging. Over the past decade, there has been a rapid expansion in the number of solved G-protein-coupled receptor (GPCR) crystal structures; however, the majority of the GPCR structures remain unsolved. In this context, HM guided MD has been widely used for structure-based drug design (SBDD) of GPCRs. The focus of this review is on the recent (i) developments on HM supported GPCR drug discovery in the absence of GPCR crystal structures and (ii) application of HM in understanding the ligand interactions at the binding site, virtual screening, determining receptor subtype selectivity and receptor behaviour in comparison with GPCR crystal structures. The HM in GPCRs has been extremely challenging due to the scarcity in template structures. In such a scenario, it is difficult to get accurate HM that can facilitate understanding of the ligand-receptor interactions. This problem has been alleviated to some extent by developing refined HM based on incorporating active /inactive ligand information and inducing protein flexibility. In some cases, HM proteins were found to outscore crystal structures. The developments in HM have been highly operative to gain insights about the ligand interaction at the binding site and receptor functioning at the molecular level. Thus, HM guided molecular docking may be useful for rational drug discovery for the GPCRs mediated diseases.

Benchmarking the performance of MM/PBSA in virtual screening enrichment using the GPCR-Bench dataset.

Recent breakthroughs in G protein-coupled receptor (GPCR) crystallography and the subsequent increase in number of solved GPCR structures has allowed for the unprecedented opportunity to utilize their experimental structures for structure-based drug discovery applications. As virtual screening represents one of the primary computational methods used for the discovery of novel leads, the GPCR-Bench dataset was created to facilitate comparison among various virtual screening protocols. In this study, we have benchmarked the performance of Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) in improving virtual screening enrichment in comparison to docking with Glide, using the entire GPCR-Bench dataset of 24 GPCR targets and 254,646 actives and decoys. Reranking the top 10% of the docked dataset using MM/PBSA resulted in improvements for six targets at EF1% and nine targets at EF5%, with the gains in enrichment being more pronounced at the EF1% level. We additionally assessed the utility of rescoring the top ten poses from docking and the ability of short MD simulations to refine the binding poses prior to MM/PBSA calculations. There was no clear trend of the benefit observed in both cases, suggesting that utilizing a single energy minimized structure for MM/PBSA calculations may be the most computationally efficient approach in virtual screening. Overall, the performance of MM/PBSA rescoring in improving virtual screening enrichment obtained from docking of the GPCR-Bench dataset was found to be relatively modest and target-specific, highlighting the need for validation of MM/PBSA-based protocols prior to prospective use.

How Do Molecular Dynamics Data Complement Static Structural Data of GPCRs.

G protein-coupled receptors (GPCRs) are implicated in nearly every physiological process in the human body and therefore represent an important drug targeting class. Advances in X-ray crystallography and cryo-electron microscopy (cryo-EM) have provided multiple static structures of GPCRs in complex with various signaling partners. However, GPCR functionality is largely determined by their flexibility and ability to transition between distinct structural conformations. Due to this dynamic nature, a static snapshot does not fully explain the complexity of GPCR signal transduction. Molecular dynamics (MD) simulations offer the opportunity to simulate the structural motions of biological processes at atomic resolution. Thus, this technique can incorporate the missing information on protein flexibility into experimentally solved structures. Here, we review the contribution of MD simulations to complement static structural data and to improve our understanding of GPCR physiology and pharmacology, as well as the challenges that still need to be overcome to reach the full potential of this technique.

Benchmarking GPCR homology model templateselection in combination with de novo loop generation.

G protein-coupled receptors (GPCR) comprise the largest family of membrane proteins and are of considerable interest as targets for drug development. However, many GPCR structures remain unsolved. To address the structural ambiguity of these receptors, computational tools such as homology modeling and loop modeling are often employed to generate predictive receptor structures. Here we combined both methods to benchmark a protocol incorporating homology modeling based on a locally selected template and extracellular loop modeling that additionally evaluates the presence of template ligands during these modeling steps. Ligands were also docked using three docking methods and two pose selection methods to elucidate an optimal ligand pose selection method. Results suggest that local template-based homology models followed by loop modeling produce more accurate and predictive receptor models than models produced without loop modeling, with decreases in average receptor and ligand RMSD of 0.54Å and 2.91Å, respectively. Ligand docking results showcased the ability of MOE induced fit docking to produce ligand poses with atom root-mean-square deviation (RMSD) values at least 0.20Å lower (on average) than the other two methods benchmarked in this study. In addition, pose selection methods (software-based scoring, ligand complementation) selected lower RMSD poses with MOE induced fit docking than either of the other methods (averaging at least 1.57Å lower), indicating that MOE induced fit docking is most suited for docking into GPCR homology models in our hands. In addition, target receptor models produced with a template ligand present throughout the modeling process most often produced target ligand poses with RMSD values ≤ 4.5Å and Tanimoto coefficients > 0.6 after selection based on ligand complementation than target receptor models produced in the absence of template ligands. Overall, the findings produced by this study support the use of local template homology modeling in combination with de novo ECL2 modeling in the presence of a ligand from the template crystal structure to generate GPCR models intended to study ligand binding interactions.

The discovery of a new antibody for BRIL-fused GPCR structure determination

G-protein-coupled receptors (GPCRs)—the largest family of cell-surface membrane proteins—mediate the intracellular signal transduction of many external ligands. Thus, GPCRs have become important drug targets. X-ray crystal structures of GPCRs are very useful for structure-based drug design (SBDD). Herein, we produced a new antibody (SRP2070) targeting the thermostabilised apocytochrome b562 from Escherichia coli M7W/H102I/R106L (BRIL). We found that a fragment of this antibody (SRP2070Fab) facilitated the crystallisation of the BRIL-tagged, ligand bound GPCRs, 5HT1B and AT2R. Furthermore, the electron densities of the ligands were resolved, suggesting that SPR2070Fab is versatile and adaptable for GPCR SBDD. We anticipate that this new tool will significantly accelerate structure determination of other GPCRs and the design of small molecular drugs targeting them.