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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) form the largest family of receptors encoded by the human genome (around 800 genes). They transduce signals by coupling to a small number of heterotrimeric G proteins (16 genes encoding different α-subunits). Each human cell contains several GPCRs and G proteins. The structural determinants of coupling of G s to four different GPCRs have been elucidated 1 – 4 , but the molecular details of how the other G-protein classes couple to GPCRs are unknown. Here we present the cryo-electron microscopy structure of the serotonin 5-HT 1B receptor (5-HT 1B R) bound to the agonist donitriptan and coupled to an engineered G o heterotrimer. In this complex, 5-HT 1B R is in an active state; the intracellular domain of the receptor is in a similar conformation to that observed for the β 2 -adrenoceptor (β 2 AR) 3 or the adenosine A 2A receptor (A 2A R) 1 in complex with G s . In contrast to the complexes with G s , the gap between the receptor and the Gβ-subunit in the G o –5-HT 1B R complex precludes molecular contacts, and the interface between the Gα-subunit of G o and the receptor is considerably smaller. These differences are likely to be caused by the differences in the interactions with the C terminus of the G o α-subunit. The molecular variations between the interfaces of G o and G s in complex with GPCRs may contribute substantially to both the specificity of coupling and the kinetics of signalling. The high-resolution structure of the serotonin 5-HT 1B receptor in complex with the agonist donitriptan and a G o heterotrimer highlights features that may underlie the specificity of receptor–G-protein coupling and kinetics of signalling.

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.

Mini-G proteins are the engineered GTPase domains of Gα subunits. They couple to GPCRs and recapitulate the increase in agonist affinity observed upon coupling of a native heterotrimeric G protein. Given the small size and stability of mini-G proteins, and their ease of expression and purification, they are ideal for biophysical studies of GPCRs in their fully active state. The first mini-G protein developed was mini-Gs. Here we extend the family of mini-G proteins to include mini-Golf, mini-Gi1, mini-Go1 and the chimeras mini-Gs/q and mini-Gs/i. The mini-G proteins were shown to couple to relevant GPCRs and to form stable complexes with purified receptors that could be purified by size exclusion chromatography. Agonist-bound GPCRs coupled to a mini-G protein showed higher thermal stability compared to the agonist-bound receptor alone. Fusion of GFP at the N-terminus of mini-G proteins allowed receptor coupling to be monitored by fluorescence-detection size exclusion chromatography (FSEC) and, in a separate assay, the affinity of mini-G protein binding to detergent-solubilised receptors was determined. This work provides the foundation for the development of any mini-G protein and, ultimately, for the structure determination of GPCRs in a fully active state.

The fungal class D1 G-protein-coupled receptor (GPCR) Ste2 has a different arrangement of transmembrane helices compared with mammalian GPCRs and a distinct mode of coupling to the heterotrimeric G protein Gpa1–Ste2–Ste18 1 . In addition, Ste2 lacks conserved sequence motifs such as DRY, PIF and NPXXY, which are associated with the activation of class A GPCRs 2 . This suggested that the activation mechanism of Ste2 may also differ. Here we determined structures of Saccharomyces cerevisiae Ste2 in the absence of G protein in two different conformations bound to the native agonist α-factor, bound to an antagonist and without ligand. These structures revealed that Ste2 is indeed activated differently from other GPCRs. In the inactive state, the cytoplasmic end of transmembrane helix H7 is unstructured and packs between helices H1–H6, blocking the G protein coupling site. Agonist binding results in the outward movement of the extracellular ends of H6 and H7 by 6 Å. On the intracellular surface, the G protein coupling site is formed by a 20 Å outward movement of the unstructured region in H7 that unblocks the site, and a 12 Å inward movement of H6. This is a distinct mechanism in GPCRs, in which the movement of H6 and H7 upon agonist binding facilitates G protein coupling. Cryo-electron microscopy structures of ligand-free, agonist-bound and antagonist-bound Ste2 show that this class D1 G protein-coupled receptor has a distinct mechanism of activation compared with other receptor classes.

The adenosine A2A receptor (A2AR) is a prototypical G protein-coupled receptor (GPCR) that couples to the heterotrimeric G protein GS. Here, we determine the structure by electron cryo-microscopy (cryo-EM) of A2AR at pH 7.5 bound to the small molecule agonist NECA and coupled to an engineered heterotrimeric G protein, which contains mini-GS, the βγ subunits and nanobody Nb35. Most regions of the complex have a resolution of ~3.8 Å or better. Comparison with the 3.4 Å resolution crystal structure shows that the receptor and mini-GS are virtually identical and that the density of the side chains and ligand are of comparable quality. However, the cryo-EM density map also indicates regions that are flexible in comparison to the crystal structures, which unexpectedly includes regions in the ligand binding pocket. In addition, an interaction between intracellular loop 1 of the receptor and the β subunit of the G protein was observed.

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.

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.

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.

G-protein-coupled receptors (GPCRs) are divided phylogenetically into six classes 1 , 2 , denoted A to F. More than 370 structures of vertebrate GPCRs (belonging to classes A, B, C and F) have been determined, leading to a substantial understanding of their function 3 . By contrast, there are no structures of class D GPCRs, which are found exclusively in fungi where they regulate survival and reproduction. Here we determine the structure of a class D GPCR, the Saccharomyces cerevisiae pheromone receptor Ste2, in an active state coupled to the heterotrimeric G protein Gpa1–Ste4–Ste18. Ste2 was purified as a homodimer coupled to two G proteins. The dimer interface of Ste2 is formed by the N terminus, the transmembrane helices H1, H2 and H7, and the first extracellular loop ECL1. We establish a class D1 generic residue numbering system (CD1) to enable comparisons with orthologues and with other GPCR classes. The structure of Ste2 bears similarities in overall topology to class A GPCRs, but the transmembrane helix H4 is shifted by more than 20 Å and the G-protein-binding site is a shallow groove rather than a cleft. The structure provides a template for the design of novel drugs to target fungal GPCRs, which could be used to treat numerous intractable fungal diseases 4 . A cryo-electron microscopy structure of the yeast pheromone receptor Ste2, a class D G-protein-coupled receptor, in its active state reveals that Ste2 is a homodimer that couples to two G proteins.