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Cryo-EM structure of the human PAC1 receptor coupled to an engineered heterotrimeric G protein


Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide hormone. The PACAP receptor PAC1R, which belongs to the class B G-protein-coupled receptors (GPCRs), is a drug target for mental disorders and dry eye syndrome. Here, we present a cryo-EM structure of human PAC1R bound to PACAP and an engineered Gs heterotrimer. The structure revealed that transmembrane helix TM1 plays an essential role in PACAP recognition. The extracellular domain (ECD) of PAC1R tilts by ~40° compared with that of the glucagon-like peptide-1 receptor (GLP-1R) and thus does not cover the peptide ligand. A functional analysis demonstrated that the PAC1R ECD functions as an affinity trap and is not required for receptor activation, whereas the GLP-1R ECD plays an indispensable role in receptor activation, illuminating the functional diversity of the ECDs in class B GPCRs. Our structural information will facilitate the design and improvement of better PAC1R agonists for clinical applications.

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Fig. 1: Overall structure of the PAC1R−mini-GSβ1γ2−Nb35 complex.
Fig. 2: PACAP binding site in TMD.
Fig. 3: Mechanism of receptor activation and Gs coupling.
Fig. 4: Structural comparison of PAC1R and GLP-1R.
Fig. 5: Characterization of the truncated analogs of PACAP and GLP-1.

Data Availability

Source data for Figs. 3c and 5b,c,e and Extended Data Fig. 1a are available with the online version of this paper. Values of the fluorescence intensities in Extended Data Fig. 1b,c are not shown, because each data point is shown in the graphs. Atomic coordinates for the PAC1R−mini-Gsβ1γ2−Nb35 complex have been deposited in the Protein Data Bank under PDB 6LPB. The associated electron microscopy data have been deposited in the Electron Microscopy Data Bank and Electron Microscopy Public Image Archive, under accession codes EMD-0940 and EMPIAR-10351.


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We thank R. Danev and M. Kikkawa for setting up the cryo-EM infrastructure, K. Ogomori for technical assistance and K. Yamashita for model building. We also thank A. Inoue (Tohoku University, Japan) for technical assistance. This work was supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from the Japan Agency for Medical Research and Development (AMED) under grant no. JP19am01011115 (support no. 1109), AMED grants: the PRIME JP18gm5910013 (A.I.) and the LEAP JP18gm0010004 (A.I. and J.A.), JSPS KAKENHI grant nos. 16H06294 (O.N.), 17J30010 (W.S.), 30809421 (W.S.), 17K08264 (A.I.), 17H05000 (T.N.) and the National Institute of Biomedical Innovation.

Author information

Authors and Affiliations



K.K. expressed and purified the mini-Gs heterotrimer and performed the complex formation, grid preparation and cryo-EM observation. W.S. designed the experiments, purified the receptor, established the preparation method for the mini-Gs heterotrimer and Nb35 and refined the structure. T.N. performed the cryo-EM data collection and single-particle analysis. A.I., F.M.N.K. and J.A. performed and oversaw the cell-based assays. The manuscript was mainly prepared by W.S., K.K. and A.I., with assistance from T.N. and O.N.

Corresponding authors

Correspondence to Wataru Shihoya or Osamu Nureki.

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The authors declare no competing interests.

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Peer review information Katarzyna Marcinkiewicz was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Functional characterization of mutant PAC1 receptors.

a, PACAP-induced Gs activation, measured by the NanoBiT-G-protein dissociation assay. Cells transiently expressing the NanoBiT-Gs, along with the indicated PAC1R construct, were treated with PACAP (1–38) and the change in the luminescent signal was measured. The dashed lines in the panels of ∆C and G389A indicate the Gs dissociation signal of the WT receptor. Concentration-response data are displayed as means ± s.e.m. (standard error of the mean) from four independent experiments. b, Cell surface expression of the PAC1R constructs. Cells transiently expressing the indicated FLAG epitope-tagged PAC1R constructs were labeled with an anti-FLAG tag antibody along with an Alexa488-conjugated secondary antibody, and the fluorescent signals from individual cells were measured by a flow cytometer. For the measurement of the WT-L, the plasmid encoding the WT receptor was reduced by 40-fold. c, Cell surface expression of the GLP-1R constructs, measured as in b. Data for graph in panel a are available as Source data.

Source data

Extended Data Fig. 2 Cryo-EM analysis.

Flow chart of the cryo-EM data processing for the PACAP–PAC1R–mini-Gs complex, including particle projection selection, classification, and 3D density map reconstruction. Details are provided in the Methods section.

Extended Data Fig. 3 Map and model quality.

a, The cryo-EM density map and model are shown for PACAP, including all seven transmembrane α-helices, ECD, and α5 of Gαs. b, Dimer interface. The complexes are shown as ribbon representations, colored as in Fig. 1a. The side chains of V3185.48 and M3225.52 are shown as sticks. Two complexes form an anti-parallel dimer with C2 symmetry in the detergent micelles. This dimer does not reflect the physiological condition but is produced during the sample preparation. The molecular packing of the two complexes in the dimer class is mediated through only a weak hydrophobic contact between V3185.48 and M3225.52. Therefore, the dimerization minimally affects the conformation of the Gs-complexed PAC1R structure.

Extended Data Fig. 4 Comparison of peptide binding modes in class B GPCRs.

a-e, Ligand binding interactions with the TMDs in the class B GPCR structures (a, PAC1R, b, GCGR (5YQZ), c, PTH1R (6NBF), d, GLP-1R (5VAI), and e, CLR (6E3Y)). Hydrogen bonding interactions are indicated by black dashed lines. f-j, Relative positions of the peptide ligands and ECDs in the class B GPCR structures (f, PAC1R, g, GCGR, h, PTH1R, i, GLP-1R, and j, CLR).

Extended Data Fig. 5 G-protein coupling interface.

Complete interactions between PAC1R and the mini-Gs heterotrimer.

Extended Data Fig. 6 Structural comparison of PAC1R and PTH1R.

ad, Surface representations of PAC1R (a) and three conformations of PTH1R (bd) (PDB 6NBF, 6NBH, and 6NBI), viewed from the extracellular side. The receptors are shown as ribbon representations with transparent surfaces. The major conformation of PTH1R is colored orange-red, and the other conformations are colored light orange.

Supplementary information

Supplementary Information

Supplementary Table 1 and Supplementary Fig. 1.

Reporting Summary

Source data

Source Data Fig. 3

Raw data for Fig. 3c

Source Data Fig. 5

Raw data for Fig. 5b,c,e

Source Data Extended Data Fig. 1

Raw data for Extended Data Fig. 1a

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Kobayashi, K., Shihoya, W., Nishizawa, T. et al. Cryo-EM structure of the human PAC1 receptor coupled to an engineered heterotrimeric G protein. Nat Struct Mol Biol 27, 274–280 (2020).

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