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Biased signaling of G protein-coupled receptors describes an ability of different ligands that preferentially activate an alternative downstream signaling pathway. In this work, we identified and characterized different N-terminal truncations of endogenous chemokine CCL15 as balanced or biased agonists targeting CCR1, and presented three cryogenic-electron microscopy structures of the CCR1–Gi complex in the ligand-free form or bound to different CCL15 truncations with a resolution of 2.6–2.9 Å, illustrating the structural basis of natural biased signaling that initiates an inflammation response. Complemented with pharmacological and computational studies, these structures revealed it was the conformational change of Tyr291 (Y2917.43) in CCR1 that triggered its polar network rearrangement in the orthosteric binding pocket and allosterically regulated the activation of β-arrestin signaling. Our structure of CCL15-bound CCR1 also exhibited a critical site for ligand binding distinct from many other chemokine–receptor complexes, providing new insights into the mode of chemokine recognition.Cryo-EM structures of the CCR1–Gi complex with N-terminal truncations of the endogenous chemokine CCL15 revealed that the conformational change of Tyr291 in CCR1 regulates β-arrestin signaling.

The pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global crisis. Replication of SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp) enzyme, a target of the antiviral drug remdesivir. Here we report the cryo–electron microscopy structure of the SARS-CoV-2 RdRp, both in the apo form at 2.8-angstrom resolution and in complex with a 50-base template-primer RNA and remdesivir at 2.5-angstrom resolution. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp, where remdesivir is covalently incorporated into the primer strand at the first replicated base pair, and terminates chain elongation. Our structures provide insights into the mechanism of viral RNA replication and a rational template for drug design to combat the viral infection.

Much effort has been invested in the investigation of the structural basis of G protein-coupled receptors (GPCRs) activation. Inverse agonists, which can inhibit GPCRs with constitutive activity, are considered useful therapeutic agents, but the molecular mechanism of such ligands remains insufficiently understood. Here, we report a crystal structure of the ghrelin receptor bound to the inverse agonist PF-05190457 and a cryo-electron microscopy structure of the active ghrelin receptor-Go complex bound to the endogenous agonist ghrelin. Our structures reveal a distinct binding mode of the inverse agonist PF-05190457 in the ghrelin receptor, different from the binding mode of agonists and neutral antagonists. Combining the structural comparisons and cellular function assays, we find that a polar network and a notable hydrophobic cluster are required for receptor activation and constitutive activity. Together, our study provides insights into the detailed mechanism of ghrelin receptor binding to agonists and inverse agonists, and paves the way to design specific ligands targeting ghrelin receptors. Ghrelin receptor regulates energy homeostasis through constitutive activity or by the ghrelin. Here the authors report two structures of ghrelin receptor bound to agonist and inverse agonist, providing insights into the mechanism of inverse agonism, which is of interest for specific ligand design.

G-protein-coupled receptors (GPCRs) have central roles in intercellular communication 1 , 2 . Structural studies have revealed how GPCRs can activate G proteins. However, whether this mechanism is conserved among all classes of GPCR remains unknown. Here we report the structure of the class-C heterodimeric GABA B receptor, which is activated by the inhibitory transmitter GABA, in its active form complexed with G i1 protein. We found that a single G protein interacts with the GB2 subunit of the GABA B receptor at a site that mainly involves intracellular loop 2 on the side of the transmembrane domain. This is in contrast to the G protein binding in a central cavity, as has been observed with other classes of GPCR. This binding mode results from the active form of the transmembrane domain of this GABA B receptor being different from that of other GPCRs, as it shows no outside movement of transmembrane helix 6. Our work also provides details of the inter- and intra-subunit changes that link agonist binding to G-protein activation in this heterodimeric complex. Cryo-electron microscopy structure of heterodimeric GABA B receptor in complex with G i1 protein reveals that the mode of G-protein binding in this class-C G-protein-coupled receptor differs from that of other classes.

Metabotropic GABA B G protein-coupled receptor functions as a mandatory heterodimer of GB1 and GB2 subunits and mediates inhibitory neurotransmission in the central nervous system. Each subunit is composed of the extracellular Venus flytrap (VFT) domain and transmembrane (TM) domain. Here we present cryo-EM structures of full-length human heterodimeric GABA B receptor in the antagonist-bound inactive state and in the active state complexed with an agonist and a positive allosteric modulator in the presence of G i1 protein at a resolution range of 2.8–3.0 Å. Our structures reveal that agonist binding stabilizes the closure of GB1 VFT, which in turn triggers a rearrangement of TM interfaces between the two subunits from TM3-TM5/TM3-TM5 in the inactive state to TM6/TM6 in the active state and finally induces the opening of intracellular loop 3 and synergistic shifting of TM3, 4 and 5 helices in GB2 TM domain to accommodate the α5-helix of G i1 . We also observed that the positive allosteric modulator anchors at the dimeric interface of TM domains. These results provide a structural framework for understanding class C GPCR activation and a rational template for allosteric modulator design targeting the dimeric interface of GABA B receptor.

The G-protein-coupled bile acid receptor (GPBAR) conveys the cross-membrane signalling of a vast variety of bile acids and is a signalling hub in the liver–bile acid–microbiota–metabolism axis 1 – 3 . Here we report the cryo-electron microscopy structures of GPBAR–G s complexes stabilized by either the high-affinity P395 4 or the semisynthesized bile acid derivative INT-777 1 , 3 at 3 Å resolution. These structures revealed a large oval pocket that contains several polar groups positioned to accommodate the amphipathic cholic core of bile acids, a fingerprint of key residues to recognize diverse bile acids in the orthosteric site, a putative second bile acid-binding site with allosteric properties and structural features that contribute to bias properties. Moreover, GPBAR undertakes an atypical mode of activation and G protein coupling that features a different set of key residues connecting the ligand-binding pocket to the G s -coupling site, and a specific interaction motif that is localized in intracellular loop 3. Overall, our study not only reveals unique structural features of GPBAR that are involved in bile acid recognition and allosteric effects, but also suggests the presence of distinct connecting mechanisms between the ligand-binding pocket and the G-protein-binding site in the G-protein-coupled receptor superfamily. Using cryo-electron microscopy, the authors report the structures of G-protein-coupled bile acid receptor–G s complexes and reveal the structural basis of bile acid recognition.

Adhesion G-protein-coupled receptors (GPCRs) are a major family of GPCRs, but limited knowledge of their ligand regulation or structure is available 1 – 3 . Here we report that glucocorticoid stress hormones activate adhesion G-protein-coupled receptor G3 (ADGRG3; also known as GPR97) 4 – 6 , a prototypical adhesion GPCR. The cryo-electron microscopy structures of GPR97–G o complexes bound to the anti-inflammatory drug beclomethasone or the steroid hormone cortisol revealed that glucocorticoids bind to a pocket within the transmembrane domain. The steroidal core of glucocorticoids is packed against the ‘toggle switch’ residue W 6.53 , which senses the binding of a ligand and induces activation of the receptor. Active GPR97 uses a quaternary core and HLY motif to fasten the seven-transmembrane bundle and to mediate G protein coupling. The cytoplasmic side of GPR97 has an open cavity, where all three intracellular loops interact with the G o protein, contributing to the high basal activity of GRP97. Palmitoylation at the cytosolic tail of the G o protein was found to be essential for efficient engagement with GPR97 but is not observed in other solved GPCR complex structures. Our work provides a structural basis for ligand binding to the seven-transmembrane domain of an adhesion GPCR and subsequent G protein coupling. The authors report on the structure of a glucocorticoid-bound adhesion G-protein-coupled receptor–G protein complex.

Follicle stimulating hormone (FSH) is an essential glycoprotein hormone for human reproduction, which functions are mediated by a G protein-coupled receptor, FSHR. Aberrant FSH-FSHR signaling causes infertility and ovarian hyperstimulation syndrome. Here we report cryo-EM structures of FSHR in both inactive and active states, with the active structure bound to FSH and an allosteric agonist compound 21 f. The structures of FSHR are similar to other glycoprotein hormone receptors, highlighting a conserved activation mechanism of hormone-induced receptor activation. Compound 21 f formed extensive interactions with the TMD to directly activate FSHR. Importantly, the unique residue H615 7.42 in FSHR plays an essential role in determining FSHR selectivity for various allosteric agonists. Together, our structures provide a molecular basis of FSH and small allosteric agonist-mediated FSHR activation, which could inspire the design of FSHR-targeted drugs for the treatment of infertility and controlled ovarian stimulation for in vitro fertilization. Follicle stimulating hormone (FSH) is a glycoprotein hormone the functions of which are mediated by a G protein-coupled receptor, FSHR. Here, Duan et al. report cryo-EM structures of FSHR in active and inactive states, suggesting the molecular basis of FSH and small allosteric agonist-mediated FSHR activation.

Melanocortin-4 receptor (MC4R) plays a central role in the regulation of energy homeostasis. Its high sequence similarity to other MC receptor family members, low agonist selectivity and the lack of structural information concerning MC4R-specific activation have hampered the development of MC4R-seletive therapeutics to treat obesity. Here, we report four high-resolution structures of full-length MC4R in complex with the heterotrimeric G s protein stimulated by the endogenous peptide ligand α-MSH, FDA-approved drugs afamelanotide (Scenesse™) and bremelanotide (Vyleesi™), and a selective small-molecule ligand THIQ, respectively. Together with pharmacological studies, our results reveal the conserved binding mode of peptidic agonists, the distinctive molecular details of small-molecule agonist recognition underlying receptor subtype selectivity, and a distinct activation mechanism for MC4R, thereby offering new insights into G protein coupling. Our work may facilitate the discovery of selective therapeutic agents targeting MC4R.

The parathyroid hormone type 1 receptor (PTH1R), a class B1 G protein-coupled receptor, plays critical roles in bone turnover and Ca 2+ homeostasis. Teriparatide (PTH) and Abaloparatide (ABL) are terms as long-acting and short-acting peptide, respectively, regarding their marked duration distinctions of the downstream signaling. However, the mechanistic details remain obscure. Here, we report the cryo-electron microscopy structures of PTH– and ABL–bound PTH1R-Gs complexes, adapting similar overall conformations yet with notable differences in the receptor ECD regions and the peptide C-terminal portions. 3D variability analysis and site-directed mutagenesis studies uncovered that PTH–bound PTH1R–Gs complexes display less motions and are more tolerant of mutations in affecting the receptor signaling than ABL–bound complexes. Furthermore, we combined the structural analysis and signaling assays to delineate the molecular basis of the differential signaling durations induced by these peptides. Our study deepens the mechanistic understanding of ligand-mediated prolonged or transient signaling. Here the authors report two structures of human PTH1R-Gs complex bound to ligands PTH and ABL, providing insight into the molecular mechanism of ligand duration.