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

Kazuhiro Kobayashi; Wataru Shihoya; Tomohiro Nishizawa; Francois Marie Ngako Kadji; Junken Aoki; Asuka Inoue; Osamu Nureki

doi:10.1038/s41594-020-0386-8

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

Nat Struct Mol Biol. 2020 Mar;27(3):274-280. doi: 10.1038/s41594-020-0386-8. Epub 2020 Mar 9.

Authors

Kazuhiro Kobayashi^#¹, Wataru Shihoya^#², Tomohiro Nishizawa^#¹, Francois Marie Ngako Kadji³, Junken Aoki³, Asuka Inoue³, Osamu Nureki⁴

Affiliations

¹ Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo, Tokyo, Japan.
² Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo, Tokyo, Japan. wtrshh9@gmail.com.
³ Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan.
⁴ Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo, Tokyo, Japan. nureki@bs.s.u-tokyo.ac.jp.

^# Contributed equally.

PMID: 32157248
DOI: 10.1038/s41594-020-0386-8

Abstract

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 G_s 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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Baculoviridae / genetics
Baculoviridae / metabolism
Binding Sites
Cloning, Molecular
Cryoelectron Microscopy
Crystallography, X-Ray
Escherichia coli / genetics
Escherichia coli / metabolism
Gene Expression
Genetic Vectors / chemistry
Genetic Vectors / metabolism
Glucagon-Like Peptide-1 Receptor / chemistry*
Glucagon-Like Peptide-1 Receptor / genetics
Glucagon-Like Peptide-1 Receptor / metabolism
Humans
Models, Molecular
Peptides / chemistry*
Peptides / metabolism
Pituitary Adenylate Cyclase-Activating Polypeptide / chemistry*
Pituitary Adenylate Cyclase-Activating Polypeptide / genetics
Pituitary Adenylate Cyclase-Activating Polypeptide / metabolism
Protein Binding
Protein Conformation, alpha-Helical
Protein Conformation, beta-Strand
Protein Engineering
Protein Interaction Domains and Motifs
Protein Multimerization
Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / chemistry*
Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / genetics
Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / metabolism
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Sf9 Cells
Spodoptera
Structural Homology, Protein

Substances

ADCYAP1 protein, human
GLP1R protein, human
Glucagon-Like Peptide-1 Receptor
Peptides
Pituitary Adenylate Cyclase-Activating Polypeptide
Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I
Recombinant Proteins