Cryo-EM structure of cell-free synthesized human histamine 2 receptor/Gs complex in nanodisc environment

Abstract

Here we describe the cryo-electron microscopy structure of the human histamine 2 receptor (H₂R) in an active conformation with bound histamine and in complex with G_s heterotrimeric protein at an overall resolution of 3.4 Å. The complex was generated by cotranslational insertion of the receptor into preformed nanodisc membranes using cell-free synthesis in E. coli lysates. Structural comparison with the inactive conformation of H₂R and the inactive and G_q-coupled active state of H₁R together with structure-guided functional experiments reveal molecular insights into the specificity of ligand binding and G protein coupling for this receptor family. We demonstrate lipid-modulated folding of cell-free synthesized H₂R, its agonist-dependent internalization and its interaction with endogenously synthesized H₁R and H₂R in HEK293 cells by applying a recently developed nanotransfer technique.

Fig. 1. Cryo-EM sample preparation of complexes by CF expression.

H₂R is synthesized by cotranslational insertion into preformed NDs (DOPG) in the presence of histamine and G_s heterotrimer purified from insect cells (Created with BioRender.com).

Fig. 2. Characterization of CF synthesized H₂R after nanotransfer into HEK293 cells.

a Schematic of GPCR nanotransfer. b H₁R-mCherry synthesized after transfection internalizes upon histamine treatment (100 µM histamine for 1 h; +H) and co-localizes with nanotransferred and CF synthesized H₂R-mNG (right panel). White bar = 10 µm. Representative images from three independent experiments. c HEK293 cells transferred for 4 h with 0.5 µM purified H₂R-mNG in NDs (DOPG) and treated with histamine. Left panel: Localization of transferred H₂R-mNG in untreated cells. Right panel: Internalization of transferred H₂R after incubation with 100 µM histamine for 1 h. White bar = 10 µm. Representative images from three independent experiments. d Quantification of cytosolic fluorescence of transferred H₂R-mNG. Data are presented as mean (SD) (n = 3 individual experiments with ≥ six data points each, *P < 0.05, two-sided students t-test). Source data are provided as Source Data file. e Interaction of transferred CF synthesized GPCRs with transfected H₁R and H₂R. Transfected cells expressing Flag-tagged H₁R or H₂R were transferred with CF synthesized Strep-tagged H₁R, H₂R, or FFAR₂ for 4 h. Subsequently, cells were washed, lysed and transferred GPCRs were immobilized by anti-Strep pulldowns. Input: Immunoblots of cell lysates before anti-Strep pulldowns. Pulldown: Immunoblots of immobilized GPCRs after anti-Strep pulldowns. Strep: Anti-Strep antibody; Flag: Anti-Flag antibody; Control: Cells without transfection. The experiment was performed once, uncropped figures are provided in the Source Data File. (Fig. 2a, created with BioRender.com).

Fig. 3. Cryo-EM structure of the histamine-bound H₂R-G_s/Nb35/ND complex.

a Cryo-EM density map of the histamine-bound H₂R/G_s/Nb35/ND complex colored by subunit. Blue, H₂R; wheat, Gα_s; cyan, Gβ; purple, Gγ, grey, Nb35; black silhouettes, ND. b Ribbon model of the H₂R/G_s/Nb35/ND complex bound to histamine (yellow spheres) colored by subunit.

Fig. 4. Histamine-binding to H₂R.

a Comparison of the histamine-binding site in H₂R (blue, receptor; yellow, histamine) and H₁R (green, receptor; purple, histamine) (PDB ID 7DFL). Binding pocket residues are represented as sticks and labeled with residue number and Ballesteros-Weinstein code (superscript) colored by the receptor subtype. H-bond interactions are shown as dotted lines colored by the interacting ligand. b, c Mutagenesis analysis of receptor-specific residues within the histamine-binding pocket in H₂R b and H₁R c using a BRET-based G protein dissociation assay. Values in brackets represent the amount of DNA (ng) of receptor plasmid used to transfect one mL of cells. 500 ng DNA per mL of cells was used for all mutants. Signaling graphs show mean ± s.e.m. of three independent biological replicates with a global fit of the data. Source data are provided as Source Data file.

Fig. 5. H₂R activation by histamine.

a Binding pocket of the histamine-activated H₂R (blue) with the inactive famotidine-bound receptor (gray) (PDB ID 7UL3) overlaid. H-bond interactions are shown as dotted lines colored by the interacting ligand. b Close-up view of the CWxP and PIF motif in the inactive (grey) H₂R and the active (blue) H₂R bound to histamine (yellow spheres). c Comparison of the DRY motif and the NPxxY motif in the inactive (grey) H₂R and active (blue) H₂R structures. Amino acid side chains are represented as sticks and labeled with residue number and Ballesteros-Weinstein code (superscript) colored by the receptor subtype. Arrows indicate conformational changes in the H₂R upon activation.

Fig. 6. Agonist binding selectivity of H₂R and H₁R.

a and b Predicted binding modes for the H₂R selective agonist amthamine (light pink) in a H₂R (blue) and b H₁R (green). The orange dotted lines represent H-bond interactions. The grey dotted line represents a possible cation-aromatic interaction between F432^6.52 in H₁R and the protonated amino group of amthamine. Binding pocket residues are represented as sticks and labeled with residue number and Ballesteros-Weinstein code (superscript). Binding mode predictions were obtained with AutoDock Vina. c, d Mutagenesis analysis of receptor-specific residues within the ligand-binding pocket in c H₂R and d H₁R using a BRET-based G protein dissociation assay in the presence of amthamine. Values in brackets represent the amount of DNA (ng) of receptor plasmid used to transfect one mL of cells. 500 ng DNA per mL of cells was used for all mutants. Signaling graphs show mean ± s.e.m. of three independent biological replicates with a global fit of the data.

Fig. 7. Molecular docking of H₂R specific agonists in the H₂R and the H₁R.

a Docking of dimaprit; b Docking of compound 157. Structural elements and relevant residues of the binding pockets of the H₂R (blue) or the H₁R (green) are shown. The orange dotted lines represent H-bond interactions. Binding pocket residues are represented as sticks and labeled with residue number and Ballesteros-Weinstein number (superscript). Binding mode predictions were obtained with AutoDock Vina.

Fig. 8. Comparison of G protein interfaces between the H₂R-G_s and the H₁R-G_q complex.

a Comparison of the relative receptor-G protein orientation between the H₂R (blue) and the H₁R (green) subtype coupled to Gα_s (wheat) and Gα_q (orange), respectively. b, c Comparison of the interface between b the α5 of Gα_s and H₂R and c the α5 of Gα_q and H₁R. d, e Comparison of the interface between the ICL2 and the G protein subtype in d the H₂R-Gα_s complex and e the H₁R-Gα_q complex. Interface residues are represented as sticks and labeled with residue number and for the receptor residues with the Ballesteros-Weinstein code (superscript). Arrows indicate differences in the relative orientation of the G protein subtypes with respect to the receptors.