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Adenosine receptors (ARs) comprise the P1 class of purinergic receptors and belong to the largest family of integral membrane proteins in the human genome, the G protein-coupled receptors (GPCRs). ARs are classified into four subtypes, A1, A2A, A2B, and A3, which are all activated by extracellular adenosine, and play central roles in a broad range of physiological processes, including sleep regulation, angiogenesis and modulation of the immune system. ARs are potential therapeutic targets in a variety of pathophysiological conditions, including sleep disorders, cancer, and dementia, which has made them important targets for structural biology. Over a decade of research and innovation has culminated with the publication of more than 30 crystal structures of the human adenosine A2A receptor (A2AR), making it one of the best structurally characterized GPCRs at the atomic level. In this review we analyze the structural data reported for A2AR that described for the first time the binding of mode of antagonists, including newly developed drug candidates, synthetic and endogenous agonists, sodium ions and an engineered G protein. These structures have revealed the key conformational changes induced upon agonist and G protein binding that are central to signal transduction by A2AR, and have highlighted both similarities and differences in the activation mechanism of this receptor compared to other class A GPCRs. Finally, comparison of A2AR with the recently solved structures of A1R has provided the first structural insight into the molecular determinants of ligand binding specificity in different AR subtypes.

Adenosine A 2A receptor structure Adenosine receptors are G protein-coupled receptors that are found in the heart and the brain, and adenosine is the endogenous ligand for this class of transmembrane receptor. Lebon et al . present two X-ray crystal structures of a thermostabilized human adenosine A 2A receptor bound to its endogenous agonist adenosine and the synthetic agonist NECA. Comparison of the agonist-bound structures of A 2A receptor with the agonist-bound structures of β-adrenoceptors suggests that the contraction of the ligand binding pocket caused by the inward motion of several helices may be a common feature in the activation of all G protein-coupled receptors. Adenosine receptors and β-adrenoceptors are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins on binding the agonists adenosine 1 or noradrenaline 2 , respectively. GPCRs have similar structures consisting of seven transmembrane helices that contain well-conserved sequence motifs, indicating that they are probably activated by a common mechanism 3 , 4 . Recent structures of β-adrenoceptors highlight residues in transmembrane region 5 that initially bind specifically to agonists rather than to antagonists, indicating that these residues have an important role in agonist-induced activation of receptors 5 , 6 , 7 . Here we present two crystal structures of the thermostabilized human adenosine A 2A receptor (A 2A R-GL31) bound to its endogenous agonist adenosine and the synthetic agonist NECA. The structures represent an intermediate conformation between the inactive and active states, because they share all the features of GPCRs that are thought to be in a fully activated state, except that the cytoplasmic end of transmembrane helix 6 partially occludes the G-protein-binding site. The adenine substituent of the agonists binds in a similar fashion to the chemically related region of the inverse agonist ZM241385 (ref. 8 ). Both agonists contain a ribose group, not found in ZM241385, which extends deep into the ligand-binding pocket where it makes polar interactions with conserved residues in H7 (Ser 277 7.42 and His 278 7.43 ; superscripts refer to Ballesteros–Weinstein numbering 9 ) and non-polar interactions with residues in H3. In contrast, the inverse agonist ZM241385 does not interact with any of these residues and comparison with the agonist-bound structures indicates that ZM241385 sterically prevents the conformational change in H5 and therefore it acts as an inverse agonist. Comparison of the agonist-bound structures of A 2A R with the agonist-bound structures of β-adrenoceptors indicates that the contraction of the ligand-binding pocket caused by the inward motion of helices 3, 5 and 7 may be a common feature in the activation of all GPCRs.

A highly conserved rearrangement of residue contacts functions as a common step in the activation pathways of diverse G-protein-coupled receptors. Structural convergence in GPCRs A comprehensive structural analysis of 27 class A G-protein-coupled receptors (GPCRs) reveals that, despite the extensive diversity in the activation pathways between receptors, the pathways converge near the G-protein-coupling region. The convergence is mediated by a highly conserved structural rearrangement of residue contacts between transmembrane helices. These findings may explain how the activation steps initiated by diverse ligands enable GPCRs to bind a common repertoire of G proteins, and will have implications for the modelling and engineering of GPCRs for structure-based drug discovery. Class A G-protein-coupled receptors (GPCRs) are a large family of membrane proteins that mediate a wide variety of physiological functions, including vision, neurotransmission and immune responses 1 , 2 , 3 , 4 . They are the targets of nearly one-third of all prescribed medicinal drugs 5 such as beta blockers and antipsychotics. GPCR activation is facilitated by extracellular ligands and leads to the recruitment of intracellular G proteins 3 , 6 . Structural rearrangements of residue contacts in the transmembrane domain serve as ‘activation pathways’ that connect the ligand-binding pocket to the G-protein-coupling region within the receptor. In order to investigate the similarities in activation pathways across class A GPCRs, we analysed 27 GPCRs from diverse subgroups for which structures of active, inactive or both states were available. Here we show that, despite the diversity in activation pathways between receptors, the pathways converge near the G-protein-coupling region. This convergence is mediated by a highly conserved structural rearrangement of residue contacts between transmembrane helices 3, 6 and 7 that releases G-protein-contacting residues. The convergence of activation pathways may explain how the activation steps initiated by diverse ligands enable GPCRs to bind a common repertoire of G proteins.

Heterodimerization of metabotropic glutamate receptors (mGlus) generates functional units that modulate the synapse activity, and displays strong therapeutic potential for treating brain disorders and psychiatry diseases. Here, Wang et al. solved the cryo-EM structures of mGlu2–mGlu3, and mGlu2–mGlu4 heterodimers in various conformational states, revealing the role of each subunit in the asymmetric signaling of mGlu heterodimers and the molecular basis of their allosteric modulation, and giving a rationale to understand which subunit activates the G protein.

The metabotropic glutamate receptors (mGlus) are class C G protein-coupled receptors (GPCR) that form obligate dimers activated by the major excitatory neurotransmitter L-glutamate. The architecture of mGlu receptor comprises an extracellular Venus-Fly Trap domain (VFT) connected to the transmembrane domain (7TM) through a Cysteine-Rich Domain (CRD). The binding of L-glutamate in the VFTs and subsequent conformational change results in the signal being transmitted to the 7TM inducing G protein binding and activation. The mGlu receptors signal transduction can be allosterically potentiated by positive allosteric modulators (PAMs) binding to the 7TMs, which are of therapeutic interest in various neurological disorders. Here, we report the cryoEM structures of metabotropic glutamate receptor 5 (mGlu 5 ) purified with three chemically and pharmacologically distinct PAMs. We find that the PAMs modulate the receptor equilibrium through their different binding modes, revealing how their interactions in the 7TMs impact the mGlu 5 receptor conformational landscape and function. In addition, we identified a PAM-free but agonist-bound intermediate state that also reveals interactions mediated by intracellular loop 2. The activation of mGlu 5 receptor is a multi-step process in which the binding of the PAMs in the 7TM modulates the equilibrium towards the active state. Allosteric control of metabotropic glutamate receptor is of therapeutic value in the treatment of neurological disorders. Here, using cryoEM, the authors describe the diversity of positive allosteric modulation on the metabotropic glutamate receptor, mGlu 5 .

Magnani et al . describe a protocol to generate thermostable membrane proteins for structural analysis. This approach combines mutagenesis with a rapid, radioligand-based thermostability assay to screen and identify mutants with optimal stability. The thermostability of an integral membrane protein (MP) in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals that are suitable for structure determination. However, many mammalian MPs are too unstable for crystallization. We developed a thermostabilization strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes ∼6–12 months to thermostabilize a G-protein-coupled receptor (GPCR) containing 300 amino acid (aa) residues. The resulting thermostabilized MPs are more easily crystallized and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs because it is possible to determine multiple structures of the thermostabilized receptors bound to low-affinity ligands. Protocols and advice are given on how to develop thermostability assays for MPs and how to combine mutations to make an optimally stable mutant suitable for structural studies. The steps in the procedure include the generation of ∼300 site-directed mutants by Ala/Leu scanning mutagenesis, the expression of each mutant in mammalian cells by transient transfection and the identification of thermostable mutants using a thermostability assay that is based on binding of an 125 I-labeled radioligand to the unpurified, detergent-solubilized MP. Individual thermostabilizing point mutations are then combined to make an optimally stable MP that is suitable for structural biology and other biophysical studies.

The activation rate of downstream G proteins imprints receptor signaling G protein–coupled receptors (GPCRs) are eukaryotic plasma membrane receptors that are organized into four classes in humans: A, B, C, and Frizzled. They internalize extracellular stimuli by activating a common pool of intracellular signaling partners such as the heterotrimeric G proteins (composed of Gα, β, and γ subunits) that subsequently induce an appropriate cellular response. Recent advances in cryo–electron microscopy (cryo-EM) enables challenging structures of GPCR signaling complexes to be solved, providing unprecedented insights about the molecular basis of their signal transduction ( 1 ). Qiao et al. ( 2 ) reported two cryo-EM structures of the class B human glucagon receptor (GCGR) G s and G i complexes, which helped clarify GCGR G protein selectivity. On page 523 of this issue, Hilger et al. ( 3 ) report a cryo-EM structure of a GCGR-G s complex and reveal the effect of conformational changes on GCGR signaling properties. These studies support a common mechanism for class B receptor activation.

Mass spectrometry (MS) binding assays are a label-free alternative to radioligand or fluorescence binding assays, so the readout is based on direct mass spectrometric detection of the test ligand. The study presented here describes the development and validation of a highly sensitive, rapid, and robust MS binding assay for the quantification of the binding of the metabotropic glutamate 5 (mGlu5) negative allosteric modulator (NAM), MPEP (2-methyl-6-phenylethynylpyridine) at the mGlu5 allosteric binding site. The LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem mass spectrometric) analytical method was established and validated with a deuterated analogue of MPEP as an internal standard. The developed MS binding assay described here allowed for the determination of MS binding affinity estimates that were in agreement with affinity estimates obtained from a tritiated MPEP radioligand saturation binding assay, indicating the suitability of this methodology for determining affinity estimates for compounds that target mGlu5 allosteric binding sites.

The metabotropic glutamate (mGlu) receptors are class C G protein-coupled receptors (GPCRs) that modulate synaptic activity and plasticity throughout the mammalian brain. Signal transduction is initiated by glutamate binding to the venus flytrap domains (VFT), which initiates a conformational change that is transmitted to the conserved heptahelical domains (7TM) and results ultimately in the activation of intracellular G proteins. While both mGlu 1 and mGlu 5 activate Gα q G-proteins, they also increase intracellular cAMP concentration through an unknown mechanism. To study directly the G protein coupling properties of the human mGlu 5 receptor homodimer, we purified the full-length receptor, which required careful optimisation of the expression, N-glycosylation and purification. We successfully purified functional mGlu 5 that activated the heterotrimeric G protein Gq. The high-affinity agonist-PAM VU0424465 also activated the purified receptor in the absence of an orthosteric agonist. In addition, it was found that purified mGlu 5 was capable of activating the G protein Gs either upon stimulation with VU0424465 or glutamate, although the later induced a much weaker response. Our findings provide important mechanistic insights into mGlu 5 G protein-dependent activity and selectivity.

Serial macromolecular crystallography has become a powerful method to reveal room temperature structures of biological macromolecules and perform time-resolved studies. ID29, a flagship beamline of the ESRF 4th generation synchrotron, is the first synchrotron beamline in the world capable of delivering high brilliance microsecond X-ray pulses at high repetition rate for the structure determination of biological macromolecules at room temperature. The cardinal combination of microsecond exposure times, innovative beam characteristics and adaptable sample environment provides high quality complete data, even from an exceptionally small amount of crystalline material, enabling what we collectively term serial microsecond crystallography (SµX). After validating the use of different sample delivery methods with various model systems, we applied SµX to an integral membrane receptor, where only a few thousands diffraction images were sufficient to obtain a fully interpretable electron density map for the antagonist istradefylline-bound A 2A receptor conformation, providing access to the antagonist binding mode. SµX, as demonstrated at ID29, will quickly find its broad applicability at upcoming 4th generation synchrotron sources worldwide and opens a new frontier in time-resolved SµX. Serial macromolecular crystallography has become a powerful method to reveal room-temperature structures of biological macromolecules and perform time-resolved studies, however, the experiments remain complex and challenging for broader applications. Here, the authors develop serial microsecond crystallography using high-brilliance, high-repetition-rate X-ray pulses at the newly constructed ID29 beamline of the ESRF-EBS 4th generation synchrotron, featuring microsecond exposure times, innovative beam characteristics, adaptable sample environment, and high-quality complete data.