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An engineered G protein is used to bind to and stabilize the active conformation of the adenosine A 2A receptor, enabling the acquisition of an X-ray crystal structure of this GPCR in an active state. Structure of an active-state GPCR G-protein-coupled receptors (GPCRs) are essential components of signalling networks throughout the body, and about a third of all clinical drugs target GPCRs. The X-ray structures of GPCRs in an active conformation have proved elusive. This paper describes the crystal structure of adenosine A 2A receptor bound to a G protein, which represents the first X-ray structure of the fully active state of the receptor. The trick used here involved engineering a G protein — termed mini-G s — that binds to and stabilizes the active state of the adenosine A 2A receptor. The hope is that this mini-G s will facilitate the crystallization and characterization of other G s -coupled GPCRs in their active states. G-protein-coupled receptors (GPCRs) are essential components of the signalling network throughout the body. To understand the molecular mechanism of G-protein-mediated signalling, solved structures of receptors in inactive conformations and in the active conformation coupled to a G protein are necessary 1 , 2 . Here we present the structure of the adenosine A 2A receptor (A 2A R) bound to an engineered G protein, mini-G s , at 3.4 Å resolution. Mini-G s binds to A 2A R through an extensive interface (1,048 Å 2 ) that is similar, but not identical, to the interface between G s and the β 2 -adrenergic receptor 3 . The transition of the receptor from an agonist-bound active-intermediate state 4 , 5 to an active G-protein-bound state is characterized by a 14 Å shift of the cytoplasmic end of transmembrane helix 6 (H6) away from the receptor core, slight changes in the positions of the cytoplasmic ends of H5 and H7 and rotamer changes of the amino acid side chains Arg 3.50 , Tyr 5.58 and Tyr 7.53 . There are no substantial differences in the extracellular half of the receptor around the ligand binding pocket. The A 2A R–mini-G s structure highlights both the diversity and similarity in G-protein coupling to GPCRs 6 and hints at the potential complexity of the molecular basis for G-protein specificity.

G-protein-coupled receptors (GPCRs) are involved in many physiological processes and are therefore key drug targets 1 . Although detailed structural information is available for GPCRs, the effects of lipids on the receptors, and on downstream coupling of GPCRs to G proteins are largely unknown. Here we use native mass spectrometry to identify endogenous lipids bound to three class A GPCRs. We observed preferential binding of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2 ) over related lipids and confirm that the intracellular surface of the receptors contain hotspots for PtdIns(4,5)P 2 binding. Endogenous lipids were also observed bound directly to the trimeric Gα s βγ protein complex of the adenosine A 2A receptor (A 2A R) in the gas phase. Using engineered Gα subunits (mini-Gα s, mini-Gα i and mini-Gα 12 ) 2 , we demonstrate that the complex of mini-Gα s with the β 1 adrenergic receptor (β 1 AR) is stabilized by the binding of two PtdIns(4,5)P 2 molecules. By contrast, PtdIns(4,5)P 2 does not stabilize coupling between β 1 AR and other Gα subunits (mini-Gα i or mini-Gα 12 ) or a high-affinity nanobody. Other endogenous lipids that bind to these receptors have no effect on coupling, highlighting the specificity of PtdIns(4,5)P 2 . Calculations of potential of mean force and increased GTP turnover by the activated neurotensin receptor when coupled to trimeric Gα i βγ complex in the presence of PtdIns(4,5)P 2 provide further evidence for a specific effect of PtdIns(4,5)P 2 on coupling. We identify key residues on cognate Gα subunits through which PtdIns(4,5)P 2 forms bridging interactions with basic residues on class A GPCRs. These modulating effects of lipids on receptors suggest consequences for understanding function, G-protein selectivity and drug targeting of class A GPCRs. Mass spectrometry-based assays are used to reveal specificity and structural determinants of lipid binding to class A G-protein-coupled receptors, and the effects of specific lipids on receptor coupling to G proteins.

G protein–coupled receptors (GPCRs) in the G protein–coupled active state have higher affinity for agonists as compared with when they are in the inactive state, but the molecular basis for this is unclear. We have determined four active-state structures of the β₁-adrenoceptor (β₁AR) bound to conformation-specific nanobodies in the presence of agonists of varying efficacy. Comparison with inactive-state structures of β₁AR bound to the identical ligands showed a 24 to 42% reduction in the volume of the orthosteric binding site. Potential hydrogen bonds were also shorter, and there was up to a 30% increase in the number of atomic contacts between the receptor and ligand. This explains the increase in agonist affinity of GPCRs in the active state for a wide range of structurally distinct agonists.

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 complex conformational energy landscape determines G-protein-coupled receptor (GPCR) signalling via intracellular binding partners (IBPs), e.g., G s and β-arrestin. Using 13 C methyl methionine NMR for the β 1 -adrenergic receptor, we identify ligand efficacy-dependent equilibria between an inactive and pre-active state and, in complex with G s -mimetic nanobody, between more and less active ternary complexes. Formation of a basal activity complex through ligand-free nanobody–receptor interaction reveals structural differences on the cytoplasmic receptor side compared to the full agonist-bound nanobody-coupled form, suggesting that ligand-induced variations in G-protein interaction underpin partial agonism. Significant differences in receptor dynamics are observed ranging from rigid nanobody-coupled states to extensive μs-to-ms timescale dynamics when bound to a full agonist. We suggest that the mobility of the full agonist-bound form primes the GPCR to couple to IBPs. On formation of the ternary complex, ligand efficacy determines the quality of the interaction between the rigidified receptor and an IBP and consequently the signalling level. β 1 -adrenergic receptors are expressed in cardiac tissue and stimulated by the sympathetic nervous system. Here, the authors use NMR spectroscopy to unravel the conformational diversity upon β 1 -adrenergic receptor activation and provide structural insights into partial agonism and basal activity.

β-adrenergic receptor structures Two papers by Brian Kobilka and colleagues describe the X-ray crystal structure of the human β 2 adrenergic receptor (β 2 AR) bound to various agonists. β 2 AR is a member of the G protein coupled receptor (GPCR) family of membrane-spanning receptors that sense molecules outside the cell and activate internal signalling pathways. With a ubiquitous role in human physiology, GPCRs are prime targets for drug discovery. A third paper by Christopher Tate and his team describes crystal structures of a similar GPCR, the turkey β 1 -adrenergic receptor (β 1 AR), bound to full and partial agonists. Together, these new structures reveal the subtle structural changes that accompany agonist binding, showing how binding events inside and outside the cell membrane stabilize the receptor's active state. Agonist binding to β 1 AR is shown to induce a contraction of the catecholamine-binding pocket relative to the antagonist-bound receptor, and molecular-dynamics simulations of the β 2 AR agonist complex suggest that the agonist-bound active state spontaneously relaxes to an inactive-like state in the absence of a G protein. Here, the X-ray crystal structure of the β 1 adrenergic receptor, a G-protein-coupled receptor, bound to four small molecules that either act as full agonists or partial agonists is solved. The structures show that agonist binding induces a contraction of the catecholamine-binding pocket relative to the antagonist-bound receptor. This work reveals the pharmacological differences between different ligand classes, which should facilitate the structure-based design of new drugs with predictable efficacies. β-adrenergic receptors (βARs) are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins upon binding catecholamine agonist ligands such as adrenaline and noradrenaline 1 , 2 . Synthetic ligands have been developed that either activate or inhibit βARs for the treatment of asthma, hypertension or cardiac dysfunction. These ligands are classified as either full agonists, partial agonists or antagonists, depending on whether the cellular response is similar to that of the native ligand, reduced or inhibited, respectively. However, the structural basis for these different ligand efficacies is unknown. Here we present four crystal structures of the thermostabilized turkey ( Meleagris gallopavo ) β 1 -adrenergic receptor (β 1 AR-m23) bound to the full agonists carmoterol and isoprenaline and the partial agonists salbutamol and dobutamine. In each case, agonist binding induces a 1 Å contraction of the catecholamine-binding pocket relative to the antagonist bound receptor. Full agonists can form hydrogen bonds with two conserved serine residues in transmembrane helix 5 (Ser 5.42 and Ser 5.46 ), but partial agonists only interact with Ser 5.42 (superscripts refer to Ballesteros–Weinstein numbering 3 ). The structures provide an understanding of the pharmacological differences between different ligand classes, illuminating how GPCRs function and providing a solid foundation for the structure-based design of novel ligands with predictable efficacies.

G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 Å resolution crystal structure of a β 1 -adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey ( Meleagris gallopavo ) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane α-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the β 1 -adrenergic receptor and binding of carazolol to the β 2 -adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the β 2 -adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation. G-protein-coupled receptors: Binding commitment The adrenalin stress hormone receptor (β 1 adrenergic receptor or β 1 AR) regulates heart rate and blood pressure and is the target for β-blockers. Like other members of the G-protein-coupled receptor family, it is difficult to purify. But the form of the enzyme found in the turkey is more stable than the human equivalent, and by using that, and mutagenesis to thermostabilize the receptor, β 1 AR has been crystallized bound to the β-blocker cyano-pindolol. The structure reveals insights into the G-protein-binding interface.

The β1-adrenoceptor (β1AR) is a G protein-coupled receptor (GPCR) that is activated by the endogenous agonists adrenaline and noradrenaline. We have determined the structure of an ultra-thermostable β1AR mutant bound to the weak partial agonist cyanopindolol to 2.1 Å resolution. High-quality crystals (100 μm plates) were grown in lipidic cubic phase without the assistance of a T4 lysozyme or BRIL fusion in cytoplasmic loop 3, which is commonly employed for GPCR crystallisation. An intramembrane Na+ ion was identified co-ordinated to Asp872.50, Ser1283.39 and 3 water molecules, which is part of a more extensive network of water molecules in a cavity formed between transmembrane helices 1, 2, 3, 6 and 7. Remarkably, this water network and Na+ ion is highly conserved between β1AR and the adenosine A2A receptor (rmsd of 0.3 Å), despite an overall rmsd of 2.4 Å for all Cα atoms and only 23% amino acid identity in the transmembrane regions. The affinity of agonist binding and nanobody Nb80 binding to β1AR is unaffected by Na+ ions, but the stability of the receptor is decreased by 7.5°C in the absence of Na+. Mutation of amino acid side chains that are involved in the co-ordination of either Na+ or water molecules in the network decreases the stability of β1AR by 5-10°C. The data suggest that the intramembrane Na+ and associated water network stabilise the ligand-free state of β1AR, but still permits the receptor to form the activated state which involves the collapse of the Na+ binding pocket on agonist binding.

The β₁-adrenergic receptor (β₁AR) is a G-protein-coupled receptor whose inactive state structure was determined using a thermostabilized mutant (β₁AR—M23). However, it was not thought to be in a fully inactivated state because there was no salt bridge between Arg139 and Glu285 linking the cytoplasmic ends of transmembrane helices 3 and 6 (the R 3.50 — D/E 6.30 \"ionic lock\"). Here we compare eight new structures of β₁AR—M23, determined from crystallographically independent molecules in four different crystals with three different antagonists bound. These structures are all in the inactive R state and show clear electron density for cytoplasmic loop 3 linking transmembrane helices 5 and 6 that had not been seen previously. Despite significantly different crystal packing interactions, there are only two distinct conformations of the cytoplasmic end of helix 6, bent and straight. In the bent conformation, the Arg139-Glu285 salt bridge is present, as in the crystal structure of dark-state rhodopsin. The straight conformation, observed in previously solved structures of β-receptors, results in the ends of helices 3 and 6 being too far apart for the ionic lock to form. In the bent conformation, the R 3.50 — E 6.30 distance is significantly longer than in rhodopsin, suggesting that the interaction is also weaker, which could explain the high basal activity in β₁AR compared to rhodopsin. Many mutations that increase the constitutive activity of G-protein-coupled receptors are found in the bent region at the cytoplasmic end of helix 6, supporting the idea that this region plays an important role in receptor activation.

Nature 536, 104–107 (2016); doi:10.1038/nature18966 In this Letter, author B.C. (byronc@mrc-lmb.cam.ac.uk) should have also been included as a corresponding author; this has been corrected online.