Abstract
Our sense of smell enables us to navigate a vast space of chemically diverse odour molecules. This task is accomplished by the combinatorial activation of approximately 400 odorant G protein-coupled receptors encoded in the human genome1,2,3. How odorants are recognized by odorant receptors remains unclear. Here we provide mechanistic insight into how an odorant binds to a human odorant receptor. Using cryo-electron microscopy, we determined the structure of the active human odorant receptor OR51E2 bound to the fatty acid propionate. Propionate is bound within an occluded pocket in OR51E2 and makes specific contacts critical to receptor activation. Mutation of the odorant-binding pocket in OR51E2 alters the recognition spectrum for fatty acids of varying chain length, suggesting that odorant selectivity is controlled by tight packing interactions between an odorant and an odorant receptor. Molecular dynamics simulations demonstrate that propionate-induced conformational changes in extracellular loop 3 activate OR51E2. Together, our studies provide a high-resolution view of chemical recognition of an odorant by a vertebrate odorant receptor, providing insight into how this large family of G protein-coupled receptors enables our olfactory sense.
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Data availability
Coordinates for the propionate OR51E2–Gs complex have been deposited in the RCSB Protein Data Bank under accession code 8F76. EM density maps for OR51E2–Gs and the 7TM domain of OR51E2 have been deposited in the Electron Microscopy Data Bank under accession codes EMD-28896 and EMD-28900, respectively. The MD simulation trajectories for apo OR51E2, OR51E2 bound to propionate, and OR51E2–Q18145×53D mutant have been deposited in the GPCRmd database under accession codes 1244, 1245, and 1246, respectively. This paper makes use of RCSB Protein Data Bank accession codes 3SN6, 4LDO and 6FUF.
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Acknowledgements
We thank D. Toso at Cal-Cryo at QB3-Berkeley for help in microscope operation and data collection; and H.M., C.A.d.M. and J.T. thank M. J. Ni and H.-Y. Lu for their technical support. This work was supported by the US NIH grant R01DC020353 (to H.M., N.V. and A.M.) and K99DC018333 (to C.A.d.M.). Cryo-EM equipment at UCSF is partially supported by NIH grants S10OD020054 and S10OD021741. This project was funded by the UCSF Program for Breakthrough Biomedical Research, funded in part by the Sandler Foundation. A.M. acknowledges support from the Edward Mallinckrodt Jr Foundation and the Vallee Foundation, and is a Chan Zuckerberg Biohub Investigator. H.M. acknowledges support from NSF/CIHR/DFG/FRQ/UKRI-MRC Next Generation Networks for Neuroscience Program (award #2014217).
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Contributions
C.B.B., C.A.d.M., W.J.C.v.d.V., N.V., H.M. and A.M. designed the study. C.B.B. cloned constructs, prepared baculoviruses, expressed and purified G protein-complexing reagents, and optimized large-scale production of OR51E2. C.B.B. worked out conditions to biochemically purify and stabilize the propionate-bound OR51E2–Gs complex, and identified optimal cryo-EM grid preparation procedures following screening, collection and processing of 200-kV cryo-EM data. B.F. and A.M. performed 300-kV cryo-EM data collection. C.B.B. determined high-resolution cryo-EM maps by extensive image processing with input from A.M. A.M. and C.B.B. built, refined models of propionate-bound OR51E2 in complex with Gs and Nb35. C.B.B. and A.M. analysed cryo-EM data and models, and prepared figures and tables. C.A.d.M. and J.T. analysed OR models and sequences to design and clone OR mutants, performed Glosensor signalling experiments for OR functional activity and generated OR cell-surface expression data by flow cytometry with input from H.M. C.A.d.M. and J.T. analysed and prepared figures and tables for signalling and flow cytometry data. C.A.d.M. built the phylogenetic tree of ORs and non-olfactory class A GPCRs. N.M. set up and performed MD simulations and ligand docking, and performed binding pocket volume calculations. W.J.C.v.d.V. analysed simulation trajectories and prepared figures describing simulation data. W.J.C.v.d.V., N.M. and N.V. provided mechanistic insight from simulation data. C.L.d.T. performed bioinformatic analysis of OR and non-olfactory class A GPCR conservation. L.L. and C.B.B. performed pilot GloSensor signalling studies in suspension cells. C.B.B., C.A.d.M. and A.M. wrote an initial draft of the manuscript and generated figures with contributions from all authors. Further edits to the manuscript were provided by W.J.C.v.d.V., N.M., N.V. and H.M. The overall project was supervised by N.V., H.M. and A.M.
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H.M. has received royalties from Chemcom, research grants from Givaudan and consultant fees from Kao.
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Extended data figures and tables
Extended Data Fig. 1 Alignment of OR51E2, rhodopsin and β2 adrenergic receptor (β2AR) amino acid sequences as described in part by de March et al.33 and implemented on GPCRdb75.
Conservation is highlighted from low (white) to high (dark blue) and the consensus amino acid is shown. Transmembrane domains are boxed in yellow. The most conserved residue in class A GPCRs for each transmembrane domain is boxed and labeled in orange. Residues used to align OR and Class A GPCR sequences are highlighted by asterisks, which are colored orange when the residue is common to all Class A GPCRs and black when it is specific to ORs. The most conserved residues used for numbering of the intracellular and extracellular loops are also indicated in italic when available. Generic numbers follow the revised Ballesteros-Weinstein numbering for Class A GPCRs32,34.
Extended Data Fig. 2 Biochemical preparation of OR51E2-Gs complex bound to propionate.
a) Schematic outlining the strategy for stabilization and purification of the activated OR51E2-Gs complex bound to propionate. b) GloSensor cAMP assay demonstrating that fusion of miniGs to OR51E2 blocks activation of endogenous Gs in response to treatment with propionate, suggesting that miniGs couples to the OR51E2 transmembrane core. Data points are the mean of analytical replicates from a representative experiment. Error bars represent the standard deviation between replicates (n = 4). c) Size-exclusion chromatogram of purified OR51E2-Gs-Nb35 complex used for structure determinations shown together with a representative SDS-PAGE gel analysis of the collected fraction containing the OR51E2-Gs-Nb35 complex. We observe two bands for OR51E2, likely due to heterogeneous glycosylation of the receptor N-terminus.
Extended Data Fig. 3 Cryo-EM data processing for OR51E2-Gs.
a) A representative cryo-EM micrograph from the curated OR51E2-Gs dataset (n = 8,010) obtained from a Titan Krios microscope. b) A subset of highly populated, reference-free 2D-class averages are shown. Scale bar is 50 Å. c) Schematic showing the image processing workflow for OR51E1-Gs. Initial processing was performed using UCSF MotionCor2 and cryoSPARC. Particles were then transferred using the pyem script package49 to RELION for alignment-free 3D classification. Finally, particles were processed in cryoSPARC using the non-uniform and local refinement tools. Dashed boxes indicate selected classes, and 3D volumes of classes and refinements are shown along with global Gold-standard Fourier Shell Correlation (GSFSC) resolutions. d,e) Map validation for the OR51E2-Gs (d) globally refined, and (e) locally refined cryo-EM maps. GSFSC curves are calculated in cryoSPARC, and shown together with directional FSC (dFSC) curves generated with dfsc.0.0.1.py as previously described80. Map-model correlations calculated in the Phenix suite are also shown. Arrows indicate map and map-model resolution estimates at 0.143 and 0.5 correlation respectively. Euler angle distributions calculated in cryoSPARC are also provided for each map.
Extended Data Fig. 4 Cryo-EM density and atomic model.
a) Orthogonal views of local resolution for the globally refined map of OR51E2-Gs calculated with the local resolution estimation tool in cryoSPARC. b) Close-up view showing the local resolution of the propionate binding site. c) Representative cryo-EM densities from the 3D reconstruction of OR51E2 from a sharpened, globally refined map of OR51E2-Gs at a map threshold of 0.635. Shown are the transmembrane helices and loop regions of OR51E2 as well as the C-terminal helix of miniGαs. d) Close-up view of cryo-EM density (yellow sticks and density) supporting propionate binding pose using a sharpened map locally refined around only the 7TM domain of OR51E2 at map threshold of 1.0.
Extended Data Fig. 5 Interactions between propionate and OR51E2 in molecular dynamics simulations.
a) Minimum distance plot between R2626×59 and propionate from 5 independent runs at different velocities (top to bottom). Minimum distance was measured between guanidinium nitrogens of R2626×59 and oxygens of propionate. Thick trace represents smoothed values with an averaging window of 8 nanoseconds; thin trace represents unsmoothed values. b) Root-mean-square deviation (RMSD) values of production simulation runs for propionate calculated with reference to the equilibrated structure of OR51E2 prior to 1 µs production simulation from 5 independent runs at different velocities (top to bottom). c) Minimum distances (Ȧ) between ligand heavy atoms and residue side chain heavy atoms (hydrogen bond and van der Waals contacts combined) are shown in gray. Gray dashed arrows highlight the interactions made between a certain receptor residue and ligand atom(s). All distances are shown as means from n = 5 independent runs (at different velocities) each 1 μs long. Standard deviation of measurement for each of the residue-ligand distance are as follows; 0.03 Å (R2626×59), 0.10 Å(S2586×55), 0.16 Å (I2025×43), 0.12 Å (G1985×39), 0.23 Å (Q18145×53), 0.23 Å (H18045×52), 0.25 Å (L1584×60), and 0.14 Å (H1043×33).
Extended Data Fig. 6 Conservation of residues within the odorant binding pocket.
a) View of propionate-contacting residues. Conservation weblogo of key residues in Class I (b) and Class II ORs (c). d) The percentage of receptors harboring a given amino acid at each position are shown for all human Class I and Class II ORs. OR51E2 residues at each position are indicated by a black box.
Extended Data Fig. 7 Analysis of active state structure of OR51E2.
a) Structural comparison of G protein interaction for OR51E2 (green) and β2-adrenergic receptor (β2AR in blue, PDB code: 3SN6). b) Close-up views of intracellular loop 2 (ICL2) interaction with the Gαs subunit shown in surface representation. c) interactions between residues in ICL2 and the αΝ and α5 helices of the Gαs subunit. d) G protein-coupling region of OR51E2 is shown along with a weblogo (right) highlighting conservation of key residues for all human ORs. e) Residues that participate in the extended interaction hydrogen bonding network between TM3, TM4, TM5, and TM6 are conserved in human Class I ORs, but not in Class II ORs. f,g) The percentage of receptors harboring a given amino acid at each position are shown for all human Class I and Class II ORs at the G protein-coupling region and connector regions. OR51E2 residues at each position are indicated by a black box.
Extended Data Fig. 8 OR51E2 molecular dynamics simulation trajectories.
a—c) Simulation trajectories for WT and Q18145×53D OR51E2 are shown in a–c. Five independent runs at different velocities are shown for each condition (top to bottom). a) F2506×47 χ1 angle over replicate simulations. b) Minimum distance between oxygen atoms of the hydroxyl groups in the side chains of S111 and Y2516×48 over replicate simulations. c) Minimum distance between R2626×59 sidechain atoms and G1985×39 mainchain atoms (excluding the hydrogens) for replicate simulations. d) Root-mean-square deviation (RMSD) values for TM backbone atoms in the transmembrane helices (see Methods) calculated with reference to the equilibrated structure of the no ligand and propionate bound OR51E2 simulations, as well as for simulations of Q18145×53D OR51E2 from 5 independent MD simulation replicates (top to bottom). Thick traces represent smoothed values with an averaging window of 8 nanoseconds; thin traces represent unsmoothed values. e–f) Aggregate frequency distributions are shown for F2506×47 χ1 angle (e), minimum distance between heavy atoms of the hydroxyl groups of S1113×40 and Y2516×48 (f), and minimum distance between R2626×59 sidechain heavy atoms and G1985×39 main chain heavy atoms (excluding hydrogens) (g) using all five simulation replicates for each condition.
Extended Data Fig. 9 Molecular dynamics snapshots of OR51E2.
a) Comparison of cryo-EM structure of propionate-bound OR51E2 with representative snapshots from simulations of WT OR51E2 with propionate, WT OR51E2 without ligand, and Q18145×53D OR51E2 without ligand. Notably, OR51E2 does not transition to the inactive conformation in any of these simulations. b) Close-up views of OR51E2 binding site and ECL3 region in the cryo-EM structure and simulations. In propionate-bound MD simulations of WT OR51E2, R2626×59 persistently forms an ionic interaction with propionate. In simulations of WT OR51E2 with propionate removed, R2626×59 is flexible. Introduction of Asp in position 45x53 (Q18145×53D) stabilizes R2626×59 in an active-like state by a direct ionic interaction. c) Close-up views of OR51E2 connector region shows increased flexibility of WT OR51E2 simulated without propionate. This flexibility is decreased for the Q18145×53D mutant. In a-c, displayed snapshots are the last 1000th ns snapshots from each simulation replicate. d,e) Molecular dynamics trajectories from representative simulations to highlight structural organization of connector region. d) Minimum distance between S1113×40 and Y2516×47 hydroxyl groups is comparable for Q18145×53D and propionate-bound WT OR51E2. e) Rotamer angle of F2506×47is comparable for Q18145×53D and propionate-bound WT OR51E2. Simulations were performed with or without propionate over the course of 1000 ns (see Extended Data Fig. 8 for replicates of simulation trajectories). Thick traces represent smoothed values with an averaging window of 8 nanoseconds; thin traces represent unsmoothed values.
Extended Data Fig. 10 AlphaFold2 model of OR51E2.
a) AlphaFold2 predicted structure of OR51E2. The pLDDT confidence metric is shown highlighting relatively high confidence in the transmembrane regions and extracellular loops. b) AlphaFold2 predicted structure of unbound OR51E2 (gray) superimposed onto the experimentally determined structure of propionate-bound OR51E2 in the active state (green cartoon and yellow spheres). In the AlphaFold2 model, TM6 is inwardly displaced compared to the active structure. Closeup views of (c) the Connector region and (d) the G protein-coupling region are provided. e) Slice through surface representation of AlphaFold2 predicted OR51E2, suggests solvent accessibility of the ligand binding site in the inactive state.
Supplementary information
Supplementary Information
This file contains Supplementary Tables 1–8, which includes summary statistics for molecular dynamics simulations and a structural comparison of OR51E2 to other Class A GPCRs. Also included are Supplementary Figs. 1 and 2, which include snapshots of molecular dynamics simulations and an uncropped SDS-PAGE gel for Extended Data Fig. 2, respectively, and Supplementary Fig. 3, which shows the flow cytometry protocol and gating strategy.
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Billesbølle, C.B., de March, C.A., van der Velden, W.J.C. et al. Structural basis of odorant recognition by a human odorant receptor. Nature 615, 742–749 (2023). https://doi.org/10.1038/s41586-023-05798-y
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DOI: https://doi.org/10.1038/s41586-023-05798-y
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