Abstract
Cryogenic electron microscopy (cryo-EM) has widened the field of structure-based drug discovery by allowing for routine determination of membrane protein structures previously intractable. Despite representing one of the largest classes of therapeutic targets, most inactive-state G protein-coupled receptors (GPCRs) have remained inaccessible for cryo-EM because their small size and membrane-embedded nature impedes projection alignment for high-resolution map reconstructions. Here we demonstrate that the same single-chain camelid antibody (nanobody) recognizing a grafted intracellular loop can be used to obtain cryo-EM structures of inactive-state GPCRs at resolutions comparable or better than those obtained by X-ray crystallography. Using this approach, we obtained structures of neurotensin 1 receptor bound to antagonist SR48692, μ-opioid receptor bound to alvimopan, apo somatostatin receptor 2 and histamine receptor 2 bound to famotidine. We expect this rapid, straightforward approach to facilitate the broad exploration of GPCR inactive states without the need for extensive engineering and crystallization.
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Data availability
All data generated or analyzed in this study are included in this article, the supplementary information, or the source data files. The cryo-EM density maps and corresponding coordinates have been deposited in the Electron Microscopy Data Bank (EMDB) and the Protein Data Bank (PDB), respectively, under the following accession codes: NTSR1 (EMD-26589, PDB 7UL2), H2R (EMD-26590, PDB 7UL3), MOR (EMD-26591, PDB 7UL4) and SSTR2 (EMD-26592, PDB 7UL5). Raw data have been deposited in EMPIAR under the following accession codes: NTSR1 (EMPIAR-11136), MOR (EMPIAR-11135), SSTR2 (EMPIAR-11134) and H2R (EMPIAR-11137). Source data for raw images of cropped gels and data plotted in graphs are provided with this paper.
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Acknowledgements
We thank the Kobilka lab (Stanford) for providing plasmids of NTSR1 and MOR. We thank the Kossiakoff lab (University of Chicago) for providing the plasmid for NabFab and constructive feedback. Cryo-EM data were collected at the Stanford cryo-EM center (cEMc) with support from E. Montabana. This work was supported, in part, by the Mathers Foundation (to G.S., and training grant No. T32GM089626 to J.G.M.), the National Institutes of Health (grant No. R35GM143061 to T.C.) and used the Extreme Science and Engineering Discovery Environment (XSEDE)59 resource comet-gpu through sdsc-comet allocation (grant No. TG-MCB190153 to G.S.), which is supported by National Science Foundation (grant No. ACI-1548562).
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Contributions
M.J.R. cloned constructs, expressed and purified proteins, processed EM data, built models and ran/analyzed molecular dynamics simulations. M.M.P.-S. expressed and purified proteins and collected cryo-EM data. F.H. cloned constructs and expressed proteins. J.G.M. performed BRET assays. A.B.S. prepared cryo-EM samples and collected cryo-EM data. M.C.P. expressed proteins and purified the nanobody. O.P. prepared cryo-EM samples and collected cryo-EM data. T.C. provided constructs for nanobody expression and BRET assays. M.J.R. and G.S. wrote the manuscript with input from M.M.P.-S., F.H., J.G.M., O.P. and T.C. G.S. supervised the project.
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Extended data
Extended Data Fig. 1 Biochemical characterization of receptor Nb6/Mb6 complexes.
(A) Size exclusion chromatography (SEC) profile of hNTSR1/Nb6 complex; bracket indicates fractions harvested for cryoEM. (B) SDS-PAGE gel of hNTSR1/Nb6 complex. (C) SEC profile of MOR/Mb6 complex; bracket indicates fractions harvested for cryoEM. (D) SDS-PAGE gel of MOR/Mb6 complex. (E) SEC profile of SSTR2/Nb6 complex; bracket indicates fractions harvested for cryoEM. (F) SDS-PAGE gel of SSTR2/Nb6 complex. (G) SEC profile of H2R/Nb6M/NabFab/Anti-Fab Nb (H) SDS-PAGE gel of H2R/Nb6M/NabFab/Anti-Fab Nb. All gels are from a single experiment, uncropped versions are provided as source data.
Extended Data Fig. 2 hNTSR1/Nb6 cryo-EM data collection and processing.
(A) Representative micrograph of hNTSR1/Nb6 complex, one micrograph of 5,818. (B) Example final 2D classes of hNTSR1/Nb6 complex. (C) Cryo-EM data processing workflow. (D) Local resolution of hNTSR1/Nb6 global refinement with FSC curve below. (E) Local resolution of hNTSR1/Nb6 local refinement with FSC curve below.
Extended Data Fig. 3 Comparison between hNTSR1/Nb6 cryoEM structure and rNTSR1H4/DARPin crystal structure.
(A) 2Fo-Fc crystallography map at 2.7 Å of rNTSR-H4bmx contoured at 𝜎=1.0. (B) 2Fo-Fc crystallography map at 2.7 Å of rNTSR-H4bmx bound SR antagonist contoured at 𝜎=1.0. (C) Structure of hNTSR1 (green) in the extracellular ligand binding pocket showing water molecules and corresponding cryoEM map features. (D) Overlay of hNTSR1 (green) and rNTSR1H4 (gray) highlighting the movement of TM1 (E) Overlay of hNTSR1 (green) and rNTSR1H4 (gray) with the cryoEM map for hNTSR1 (green) for ECL1 (F) Overlay of hNTSR1 (green) and rNTSR1H4 (gray) with the rNTSR1H4 crystal structure 2Fo-Fc map contoured at 𝜎 = 1.0. (G) Overlay of hNTSR1 (green), rNTSR1H4 (gray), and MOR PDB 4DKL (blue) showing the position of NPXXY motif Y7.53.
Extended Data Fig. 4 MOR/Mb6 cryo-EM data collection and processing.
(A) Representative micrograph of MOR/Mb6 complex, one micrograph of 14,635. (B) Example final 2D classes of MOR/Mb6 complex. (C) Cryo-EM data processing workflow. (D) Local resolution of MOR/Mb6 global refinement with FSC curve below. (E) Local resolution of MOR/Mb6 local refinement with FSC curve below.
Extended Data Fig. 5 Inactive MOR bound to avlimopan.
(A) overlay of snapshots from Mb6 molecular dynamics simulations aligned on the nanobody portion. (B) Comparison of Inactive MOR (dark blue) bound to alvimopan (magenta) and bound water in density modified map (grey) (C) Comparison of Inactive MOR (dark blue) bound to alvimopan (magenta) and carfentanyl (yellow) docked into the active state MOR receptor (light blue).
Extended Data Fig. 6 SSTR2/Nb6 cryo-EM data collection and processing.
(A) Representative micrograph of SSTR2/Nb6 complex, one micrograph of 6,846. (B) Example final 2D classes of SSTR2/Nb6 complex. (C) Cryo-EM data processing workflow. (D) Local resolution of SSTR2/Nb6 global refinement with FSC curve below.
Extended Data Fig. 7 Comparison of CryoEM Maps and Putative Ion Sites.
(A) Sodium ion site of hNTSR1 with cryoEM map. (B) Probable sodium ion site of MOR with cryoEM map. (C) Probable sodium ion site of SSTR2 with cryoEM map. (D) Sodium ion site of DOR (PDB 4N6H) with 2Fo-Fc map contoured at 𝜎 = 2.0 (E) Overlay of NTSR1 (green) cryoEM structures sodium ion binding site with the DOR sodium coordination site structure (gray, PDB 4N6H).
Extended Data Fig. 8 Inactive H2R/Nb6M/NabFab/Anti-Fab Nb cryoEM data collection and processing.
(A), Representative micrograph of H2R complex, one micrograph of 7,728. (B), Example final 2D classes of H2R complex. (C), CryoEM data processing workflow. (D), Local resolution of H2R global refinement (E), Local resolution of H2R local refinement (F), FSC curve of H2R global refinement (G) FSC curve of H2R local refinement.
Extended Data Fig. 9 Inactive H2R Structure and Comparison to H1R.
(A) Cryo-EM map of H2R with lipid density between TM1 and TM7 colored in orange. (B-D) Chemical structures of doxepin (B), loratadine (C), and cetirizine (D) with protonated amine highlighted and crystal structure of H1R (magenta) bound to doxepin (teal) (PDB 3RZE) loratadine (teal, docked pose) and cetirizine (teal, docked pose). (E-G) Chemical structures of famotidine (E), ranitidine (F), and cimetidine (G) with protonated amines highlighted in dashed (high pKa) and dotted (low pKa) boxes, together with cryoEM structure of H2R (lavender) bound to famotidine (goldenrod, this work), ranitidine (goldenrod, docked pose) and cimetidine (goldenrod, docked pose).
Extended Data Fig. 10 Map-Model Agreements.
(A) Map-model comparison for NTSR1. (B) Map-model comparison for MOR. (C) Map-Model comparison for SSTR2. (D) Map-Model comparison for H2R.
Supplementary information
Source data
Source Data Fig. 1
Data for graph plots in Fig. 1.
Source Data Extended Data Fig. 1
Raw uncropped gels.
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Robertson, M.J., Papasergi-Scott, M.M., He, F. et al. Structure determination of inactive-state GPCRs with a universal nanobody. Nat Struct Mol Biol 29, 1188–1195 (2022). https://doi.org/10.1038/s41594-022-00859-8
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DOI: https://doi.org/10.1038/s41594-022-00859-8
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