Abstract
The small cyclic neuropeptide hormone oxytocin (OT) and its cognate receptor play a central role in the regulation of social behaviour and sexual reproduction. Here we report the single-particle cryo-electron microscopy structure of the active oxytocin receptor (OTR) in complex with its cognate ligand oxytocin. Our structure provides high-resolution insights into the OT binding mode, the OTR activation mechanism as well as the subtype specificity within the oxytocin/vasopressin receptor family.
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Introduction
The neurohypophyseal hormones oxytocin (OT) and arginine vasopressin (AVP) are cyclic peptides that activate an evolutionary ancient subfamily of class A G protein-coupled receptors (GPCRs)1, comprising the oxytocin receptor (OTR) and the closely related vasopressin receptors (V1AR, V1BR and V2R). This OT/AVP hormone system is highly conserved among many species and exerts a central role in the regulation of social cognition, social behaviour, and sexual reproduction2.
Currently, several clinical trials are evaluating the efficacy of OTR-mediated signalling through administration of OT to treat malfunctions such as autism-spectrum disorders3, anxiety4 and schizophrenia5. While OT itself is an approved peripheral drug in obstetrics6, OT-based treatments of socio-behavioural deficiencies, requiring central administration of the hormone, have yet failed, potentially due to its poor drug-like properties7 and limited penetration through the blood brain barrier8. Despite recent advances in finding a non-peptide agonist that is active in an animal model9, the high demand for such drugs continues. Up to now, the identification and development of OTR-specific molecules with satisfactory pharmacokinetic properties, favourable biodistribution and specificity has been impeded by the lack of structural information on the OTR:OT signalling complex.
Here, we now report the single-particle cryo-electron microscopy (cryo-EM) structure of the OT-bound human OTR in complex with a heterotrimeric G protein at a resolution of 3.2 Å.
Results and discussion
Receptor and G protein engineering
Initially, structural studies of the OTR:OT signalling complex were hampered by the poor biophysical behaviour of the wild-type OTR (wtOTR). To improve expression and purification yields we included a single stabilising mutation (D153Y), which we previously identified by a next-generation sequencing (NGS)-based in-depth analysis of directed evolution experiments10. This mutation enabled a 50-fold increase in the yield of purified functional receptor, with very similar agonist binding and signalling behaviour (Supplementary Fig. 1a, b). As an additional hurdle, complexes of OTR with an otherwise frequently employed engineered mini-Gs11,12 or a Gs/q chimera were not stable and dissociated upon plunge freezing. While Gq-based signalling is the main route of OTR activation, the receptor has been shown to also interact with Go and Gi13, but not with Gs. Therefore, we hypothesised that the observed instability of the OTR:mini-Gs/q complex may be attributed to unfavourable interactions of OTR with the Gs domain, and interactions of the Gq α5 helix are not sufficient to overcome this. Thus, to maximally stabilise the OTR active state, we designed a G protein chimera using mini-Go11 as a basis, substituting the Go α5 helix with the corresponding amino acids of Gq. Additionally, we replaced the N terminus with the respective Gi1 residues to permit binding of the complex-stabilising single-chain variable fragment 16 (scFv16)14. For simplicity, the resulting mini-G protein is denoted Go/q henceforth. These combined engineering efforts finally enabled single-particle cryo-EM analysis of the OTR:OT:Go/q:scFv16 complex at a resolution of 3.2 Å (Fig. 1a, b, Supplementary Figs. 1c, d, 2–4 and Supplementary Table 1), enabling unprecedented insights into OTR activation by OT and the receptor G protein interaction.
OT binding mode
In the orthosteric ligand binding pocket, all nine amino acids of OT participate in OTR binding (Fig. 1c). The cyclic part (residues 1–6) is buried deep inside the pocket while the C-terminal tripeptide (residues 7–9) is facing the extracellular loops. Interestingly, the amidated C terminus of Gly9, known to be important for activation15, is located in proximity of residues E421.35 and D1002.65 (Fig. 1d; numbers in superscripts correspond to Ballesteros-Weinstein numbering16) of transmembrane helices I and II, which are involved in magnesium-dependent modulation of OT binding17. Together with the neighbouring Leu8, the extracellular surface of the orthosteric pocket is lined by Pro7 and Asn5, which pack against extracellular loop 3 (ECL3) and ECL2, respectively. Leu8 is oriented towards the extracellular space, explaining why position 8 is best suited for the attachment of fluorophores in OT18. Gln4 is the only residue pointing out perpendicularly from the ring plane and stabilizes the cyclic ring position through a hydrogen bond to Q2956.55. Ile3 is buried in a hydrophobic pocket formed by side-chain residues of transmembrane helices IV, V, and VI. The critical contribution of this hydrophobic pocket is underlined by the observed reduction in OT potency when mutating the main contact residues I2015.39, I2045.42 and F2916.51 of the receptor to alanine (Fig. 1d, e and Supplementary Table 2). Tyr2 penetrates deep into a crevice at the bottom of the orthosteric pocket formed by residues from helices II, III, VI and VII. While the carboxy group of Tyr2 interacts with Q1714.60, the phenol ring engages in hydrophobic interactions with Q922.57 and F2916.51, and the hydroxyl group forms a hydrogen bond to the backbone amide oxygen of L3167.40. The importance of these interactions explains the loss of potency when either Q1714.60 or F2916.51 are mutated to alanine (Fig. 1d, e). Finally, Cys1, which stabilizes the OT ring conformation through a disulphide with Cys6, contacts with its backbone oxygen a polar cluster consisting of residues Q962.61, K1163.29, and Q1193.32 observed in the OT/AVP family, consistent with earlier mutagenesis studies19 (Fig. 1d, e).
OT mediated receptor activation
A comparison of the OTR active-state structure with the previously reported inactive-state structure of the OTR in complex with the small-molecule antagonist retosiban17 enabled us to identify the molecular changes involved in receptor activation (Figs. 2 and 3). In contrast to OT, retosiban only partially occupies the region of the orthosteric pocket, where the cyclic part of OT binds (Fig. 2b, c). Nonetheless, the OT-induced helical rearrangements in the orthosteric pocket are relatively small, reflected by the subtle change of the pocket volume between the active and inactive state (Fig. 2d, e). OT interacts with residues F2916.51 and F2926.52 at the bottom of the binding pocket. This interaction induces a rearrangement of F2916.51, initiating the large outward movement of helix VI through a series of side-chain reorientations in conserved microswitches, including W2886.48 (CWxP motif), F2846.44 (PIF motif) and Y3297.53 (NPxxY motif) (Fig. 3c, d). These rearrangements ultimately lead to the breakage of the interaction between T2736.33 and R1373.50 of the DRY motif (DRC in OTR), and the reorientation of D1363.49 into a position enabling direct contact with the α5 helix of Go/q (Fig. 3e).
Early functional studies on OT derivatives identified Cys1, Tyr2 and Gln4 as centrally involved ligand residues in receptor activation. For example, alkylation of the Tyr2 hydroxy group led to decreased agonistic activity20, suggesting an important role of this residue, consistent with our structural data. Remarkably, we observe a local unfolding of helix VII at the extracellular receptor side in the region of L3167.40, creating a pronounced kink which is stabilised by a hydrogen bond formed between Tyr2 of OT with the backbone oxygen of L3167.40 (Fig. 3b). Importantly, a similar helix VII conformation was also observed in active-state structures of V2R21 (Supplementary Fig. 5), suggesting that partial helix VII unfolding is a feature of the OT/AVP family receptor activation. Sequence alignments of the four receptors of the OT/AVP family reveal that all receptors share a conserved kink region, with the exception of position 7.42, where both OTR and V2R share an alanine, whereas V1AR and V1BR carry a glycine. To test if a kink region carrying a glycine is compatible with the observed helix VII reorientation, we determined ligand binding affinity and receptor activation of an OTR mutant where we mutated A3187.42 to glycine. We observe only little differences in activity and binding affinity, supporting an unaltered activation mechanism. It appears that glycine might potentially destabilise the local helical conformation and even facilitate ligand binding, as indicated by the slightly improved affinity and potency of OT to A3187.42G. Accordingly, we find that removal of the glycine in V1AR by mutation G3377.42A has the inverse effect (Supplementary Fig. 5, Supplementary Tables 2 and 3).
Oxytocin/Vasorpessin receptor family subtype specificity
OT differs only in two positions from the closely related AVP (Ile3 and Leu8 in OT vs. Phe3 and Arg8 in AVP) (Fig. 4a). While these differences suffice to render OT specific for the OTR over the vasopressin receptors, the OTR itself is not selective between OT and AVP22. To investigate possible contributions to subtype selectivity, we compared the OT-bound OTR structure to the previously published AVP-bound V2R structures21,23,24 (Fig. 4). Both OT and AVP adopt a similar orientation in the orthosteric pocket of their respective receptor with highest similarity observed for the agonist’s cyclic portion, where Phe3 of AVP penetrates only marginally deeper than Ile3 of OT (Fig. 4). The largest structural differences are found for positions eight and nine of the ligand in the tripeptide C terminus. Leu8 and Gly9 in OT are located along the ring plane, with Gly9 facing helix I (Figs. 1d and 4). Conversely, Arg8 and Gly9 in AVP, which in each reported V2R:AVP structure have been modelled differently, are facing away from the ring plane, enabling contacts to residues of ECL1, ECL3 and the N terminus of V2R. In the OTR, helix I adopts a position further away from the central axis of the receptor compared to V2R. This wider opening of the orthosteric pocket in OTR enables binding of the OT C-terminal residues Leu8 and Gly9 in the observed conformation. In V2R, however, OT binding would be sterically compromised due to a clash of Gly9 with helix I. In contrast, the bound conformation of AVP in V2R is compatible with binding to OTR. Therefore, the positioning of helix I might explain why the OTR is not selective between OT and AVP, but OT binding is specific to OTR and it does not bind to V2R. In V2R, helix I is packed in a more compact manner in the helix bundle compared to the OTR, so it would clash with Gly9 if OT were to be expected to adopt the identical binding conformation as observed in complex with OTR.
In variance to long-standing models22,25, we do not observe an interaction of OT with R34N terminus or F103ECL1 of the OTR. This might indicate an allosteric effect, or in the case of R34, which does not show clear density, a more dynamic interaction. We speculate that this could additionally contribute to subtype selectivity in the OT/AVP receptor family.
On the intracellular receptor side, the main conformational differences between OTR and V2R are found in the positioning of the helix VII-VIII transition region and the elongation of helix V of V2R compared to OTR (Supplementary Fig. 6). Both regions contribute to G protein binding, and the respective differences are likely a feature of the diverse binding modes between the receptors and the interacting Gα subunits (V2R:Gs and OTR:Go/q).
G protein interaction
In the signalling complex of the activated OTR, the α5 helix of Go/q is bound in a crevice constituted by helices II, III, V, VI and VII at the receptor intracellular side, where the G protein C-terminal residues E350 and Y351 engage in hydrogen bonding with R732.38 and D1363.49 of OTR (Fig. 5a, d). Compared to reported receptor:Gs/q complexes12,26,27, the Gq α5 helix in OTR:Go/q is rotated away from helix VI (Fig. 5b). This rotation cannot be attributed to differences in chimera design, as there is a perfect structural alignment of the individual Gα subunits (Fig. 5e). Most importantly, the same rotation is also distinct from GPCR:Go strcutures28,29,30 (Fig. 5c). Instead, the orientation of the Gq α5 helix in OTR:Go/q very much resembles that of Gs coupled to V2R, with the latter penetrating less deeply into the V2R intracellular TMD crevice, suggesting the receptor is governing the α5 orientation (Fig. 5f).
In conclusion, we report here the structure of the human OTR:OT signalling complex, providing insights into the subfamily-specific OTR activation mechanism and an unexpected G protein binding mode as well as the detailed OT binding mode, thereby enhancing our understanding of the subtype specificity within the closely related oxytocin and vasopressin receptor family. After the present manuscript had been submitted, a related structure with a different G protein has appeared, reaching similar conclusions31. Together, these findings are expected to greatly facilitate the development of novel therapeutics for the treatment of a variety of OTR-implicated diseases.
Methods
Design of complex constructs
The sequences of scFv1614 and of wild-type human OTR (wtOTR), codon-optimised for expression in Spodoptera frugiperda (Sf9) (C-terminally truncated after residue 359), were cloned into a modified pFL vector (MultiBac system, Geneva Biotech) for Sf9 expression. The resulting expression constructs contained a melittin signal sequence, followed by a FLAG-tag, a His10-tag, and a human rhinovirus 3C protease cleavage site N-terminal to the gene of interest. To increase OTR purification yield, the mutation D153Y was introduced into the truncated wtOTR sequence, as identified previously10. It shows very similar KD and EC50 for oxytocin (OT) (Supplementary Fig. 1). The mutant was generated by sequence- and ligation-independent cloning as previously described in detail32. To generate an Gαo/q subunit that would allow interaction with scFv1630, the N-terminal 18 amino acids of Gi1 were introduced to the engineered mini-Go11211. To generate Gq-like interactions, residues H5.16, H5.17, H5.18, H5.22, H5.23, H5.24, and H5.26 (according to the common Gα numbering)33 in the C-terminal helix were mutated to corresponding amino acids of Gq. Finally, the engineered Gαo/q chimera sequence was cloned into one pFL vector, together with Gβ1 (including an N-terminal non-cleavable His10-tag) and with Gγ2, with each gene under the control of its own polyhedrin promoter.
Expression and purification of OTR
Expression and purification were performed as previously described in detail12,17. In brief, 4 L of Sf9 insect cell culture at a density of 3 × 106 cells/ml were infected with baculovirus stocks at a multiplicity of infection ≥5. Cells were harvested 72 h post-infection and stored at −80 °C. Cells were thawed on ice before purification, lysed, and membranes were isolated by repeated Dounce homogenisation in hypotonic buffer containing 10 mM Hepes (pH 7.5), 20 mM KCl, 10 mM MgCl2, and protease inhibitors (50 μg/ml Pefabloc SC and 1 μg/ml Pepstatin A, both Carl Roth) and then in hypertonic buffer containing 10 mM Hepes (pH 7.5), 20 mM KCl, 10 mM MgCl2, 1.0 M NaCl, 0.1 mg/ml deoxyribonuclease (DNase, MilliporeSigma) and protease inhibitors. Washed membranes were resuspended in hypotonic buffer, and the low-affinity antagonist SSR 149415 (Tocris) was added to a final concentration of 100 μM and the suspension was incubated for 30 min. Then, iodoacetamide (2 mg/ml final concentration; MilliporeSigma) was added to the solution followed by another 30 min of incubation. Subsequently, the receptor was solubilized in buffer containing 30 mM Hepes (pH 7.5), 500 mM NaCl, 10 mM KCl, 5 mM MgCl2, 50 μM SSR 149415, 1% (w/v) n-dodecyl-β-d-maltopyranoside (DDM, Anatrace), and 0.2% (w/v) cholesteryl hemisuccinate (CHS, MilliporeSigma) for 3 h at 4 °C. Insoluble material was removed by ultracentrifugation at 220,000 g, and the supernatant containing the solubilized receptor was incubated with TALON IMAC resin (Cytiva) at 4 °C.
The receptor-bound resin was washed with 20 column volumes (CVs) of wash buffer I containing 50 mM Hepes (pH 7.5), 500 mM NaCl, 10 mM MgCl2, 5 mM imidazole, 10% (v/v) glycerol, 8 mM adenosine triphosphate (ATP, MilliporeSigma), supplemented with 0.5% (w/v) DDM, 0.01% (w/v) CHS, and 10 μM SSR 149415. Subsequently, the detergent was exchanged with 16 CVs of wash buffer I supplemented with 1% (w/v) lauryl maltose neopentyl glycol (LMNG, Anatrace), 0.1% (w/v) CHS, and 10 μM SSR 149415, and the antagonist was replaced with OT (Psyclo Peptide Inc.) with another 16 CVs of wash buffer I supplemented with 0.5% (w/v) LMNG, 0.05% (w/v) CHS, and 100 μM OT. Agonist-bound OTR was finally washed with 25 CVs of wash buffer II containing 50 mM Hepes (pH 7.5), 150 mM KCl, 15 mM imidazole, 10% (v/v) glycerol, 0.05% (w/v) LMNG, 0.005% (w/v) CHS, and 50 μM OT, before eluting the receptor in 4 subsequent fractions of 1 CV of elution buffer containing 50 mM Hepes (pH 7.5), 150 mM KCl, 250 mM imidazole, 10% (v/v) glycerol, 0.01% (w/v) LMNG, 0.001% (w/v) CHS, and 50 μM OT.
Protein-containing fractions were combined and concentrated to 0.5 ml using a 50-kDa molecular weight cutoff (MWCO) Vivaspin 2 concentrator (Sartorius), and desalted by buffer exchange on a PD MiniTrap G-25 column (Cytiva) equilibrated with G25 buffer containing 50 mM Hepes (pH 7.5), 150 mM KCl, 10% (v/v) glycerol, 0.01% (w/v) LMNG, 0.001% (w/v) CHS, and 50 μM OT. To remove the N-terminal affinity tags and to deglycosylate the receptor, OT-bound receptor was treated with His-tagged 3 C protease and His-tagged peptide N-glycosidase (PNGase) F (both prepared in-house) overnight. To collect cleaved receptor, the reaction was incubated with Ni-nitrilotriacetic acid (Ni-NTA) resin (Cytiva) for 1 h, cleaved receptor was collected as the flow-through, then concentrated to ~3 to 5 mg/ml with a 50-kDa MWCO Vivaspin 2 concentrator, and directly used for complex formation. Protein purity and monodispersity were assessed by LDS–polyacrylamide gel electrophoresis and analytical size exclusion chromatography (SEC) using a Nanofilm SEC-250 column (Sepax).
Purification of Go/q
Purification of the engineered heterotrimeric G protein was carried out similarly to receptor purification, with small adaptations. All buffers used were supplemented with 10 μM guanosine diphosphate (GTP, MilliporeSigma) and 100 μM tris(2-carboxyethyl)phosphine) (TCEP, Thermo Fisher Scientific). In contrast to the receptor purification, monovalent cation concentration never exceeded 150 mM, and all buffers were devoid of any receptor ligands and iodoacetamide. Enriched G protein-containing membranes were washed by Dounce homogenisation without high salt concentrations in physiological buffer, containing 10 mM Hepes (pH 7.5), 150 mM NaCl, 20 mM KCl, 10 mM MgCl2, and protease inhibitors. Solubilisation and immobilisation on TALON IMAC resin was performed as described above.
The G protein–bound resin was initially washed with 30 CVs of wash buffer I containing 50 mM Hepes (pH 7.5), 150 mM KCl, 10 mM MgCl2, 5 mM imidazole, 10% (v/v) glycerol, 1% (w/v) DDM, 0.2% (w/v) CHS, 10 μM GTP and 100 μM TCEP followed by a wash with 30 CV of detergent exchange buffer containing 50 mM Hepes (pH 7.5), 150 mM KCl, 1 mM MgCl2, 5 mM imidazole, 10% (v/v) glycerol, 1% (w/v) LMNG, 0. 1% (w/v) CHS 8 mM ATP, 10 μM GTP and 100 μM TCEP. G proteins were finally washed with 30 CVs of wash buffer II containing 50 mM Hepes (pH 7.5), 150 mM KCl, 1 mM MgCl2, 15 mM imidazole, 10% (v/v) glycerol, 0.01% (w/v) LMNG, 0.001% (w/v) CHS, 10 μM GTP and 100 μM TCEP and eluted stepwise in 4 subsequent fractions of 1 CV of elution buffer containing 750 mM imidazole.
Eluted G protein was concentrated using a 100-kDa MWCO Vivaspin 2 concentrator and further purified using a Superdex 200 10/300 (Cytiva) column equilibrated with SEC buffer containing 25 mM Hepes (pH 7.5), 150 mM KCl, 2% (v/v) glycerol, 0.01% (w/v) LMNG, and 0.001% (w/v) CHS. Fractions containing monodisperse G protein were collected, concentrated to 7.1 mg/ml with a 100-kDa MWCO Vivaspin 2 concentrator (Sartorius), frozen in liquid nitrogen, and stored at −80 °C until use.
Purification of scFv16
ScFv16 was expressed and purified as described before12. Briefly, scFv16 was expressed by secretion from baculovirus-infected Sf9 cells for 72 h. ScFv16-containing expression medium was separated from cells by centrifugation, then pH-adjusted by addition of Hepes (pH 7.5). Metal-chelating agents from the cells and medium were quenched by incubation with 1 mM CoCl2 and 5 mM CaCl2 for 1 h at 22 °C. Resulting precipitates were removed by centrifugation, and the filtrated supernatant was loaded onto a Co2+-loaded HiTrap IMAC HP column (Cytiva). The column was washed with 20 CVs of buffer A containing 20 mM Hepes (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 4 mM ATP, and 5 mM imidazole, followed by 20 CVs of buffer B containing 20 mM Hepes (pH 7.5), 150 mM NaCl, and 30 mM imidazole. The protein was eluted with buffer C [20 mM Hepes (pH 7.5), 150 mM NaCl, and 300 mM imidazole]. Monomeric fractions were pooled, concentrated using a 10-kDa MWCO Amicon Ultra concentrator (Merck), and imidazole was removed by applying the concentrate to a PD-10 desalting column (Cytiva) equilibrated with G25-buffer containing 50 mM Hepes (pH 7.5), 150 mM NaCl, 10% (v/v) glycerol, 0.01% (w/v) LMNG, and 0.001% (w/v) CHS. ScFv16 was incubated for 3 h with His-tagged 3 C protease and His-tagged PNGase F for removal of affinity tags and deglycosylation. After incubation with Ni-NTA resin for 1 h, cleaved scFv16 was collected as the flow-through, concentrated with a 10-kDa MWCO Amicon Ultra concentrator, and further purified using a Superdex 200 10/300 column equilibrated with SEC buffer. Monomeric fractions were pooled, concentrated to ~6 to 7 mg/ml, flash-frozen in liquid nitrogen, and stored at −80 °C. Right before complex formation, scFv16 was thawed and the buffer was exchanged on a PD MiniTrapTM G-25 column (Cytiva) equilibrated with G25-buffer.
Complex formation
Purified OTR and engineered heterotrimeric G protein (Gαo/qGβ1Gγ2) were mixed in a molar ratio of 1:1.2 in complexation buffer (25 mM Hepes (pH 7.5), 100 mM KCl, 1 mM MgCl2, 0.01% (w/v) LMNG, 0.001% (w/v) CHS, 100 μM OT, and 100 μm TCEP). After 30 min, apyrase (0.8 U/ml; MilliporeSigma) and λ-phosphatase (1,000 U/ml, New England Biolabs) were added to the mixture. After 2 h, purified scFv16 was added at 4-fold molar excess over receptor, and complex formation was allowed to proceed overnight at 4 °C. Stable complex was isolated by SEC on a Superdex 200 10/300 column (Cytiva) equilibrated with blotting buffer (25 mM Hepes (pH 7.5), 100 mM KCl, 1 mM MgCl2, 100 μM OT, and 100 μm TCEP, 0.001% (w/v) LMNG, and 0.0001% (w/v) CHS). Corresponding peak fractions were concentrated to 1 mg/ml for EM studies, using a 100-kDa MWCO Vivaspin Turbo PES (Sartorius) concentrator.
Single-particle cryo-EM data collection
For cryo-EM grid preparation, 3 µL of purified OTR:OT:Go/q:scFv16 complex in blotting buffer were applied to glow-discharged holey carbon gold grids (Quantifoil R1.2/1.3, 300 mesh), and subsequently vitrified using a Vitrobot Mark IV (Thermo Fisher Scientific) operated at 100% humidity and 4 °C. Cryo-EM images were acquired by a Titan Krios G3i electron microscope (Thermo Fisher Scientific), operated at 300 kV, at a nominal magnification of 130,000 using a K3 direct electron detector (Gatan) in super-resolution mode, corresponding to a pixel size of 0.325 Å. A BioQuantum energy filter (Gatan) was operated in a zero-loss mode, using 20 eV energy slit-width. A total of 6,450 movies were obtained, with a defocus range of −0.8 to −2.4 µm using automatic data acquisition with EPU software (version 2.5;Thermo Fisher Scientific). The total exposure time was 1.79 s with an accumulated dose of ~63.78 electrons/Å2 and a total of 67 frames per micrograph. A second set of 5,217 image stacks were acquired with the same conditions and parameters.
Single-particle cryo-EM data processing
All image stacks were binned to generate a pixel size of 0.65 Å followed by motion-correction and dose-weighting using MotionCor234 (version 1.4). All images were contrast transfer function corrected using Gctf35 (version 1.06), as implemented in cryoSPARC36(version 3.0.1). Subsequent image processing steps were performed in cryoSPARC. Initial particle selection was done using the automated blob picker on 100 micrographs, using a 100 Å minimum and 150 Å maximum particle diameter, to extract a total of 38,166 particles. Next, the extracted particles were subjected to one round of 2D classification, into 200 classes, from which 7 classes were selected and used as a template for the automatic particle picking process. A total of 3,062,337 particles were extracted from the first 6,450 micrographs, followed by a round of 2D classification that resulted in 200 classes. Finally, a round of 3D reconstructions and classification produced 6 classes.
A total of 3,504,800 particles were extracted from the second data-set of image-stacks, followed by a round of 2D classification, split into 200 classes, and a round of 3D reconstructions and classification into 3 classes. The particles from the best classes from both datasets were then joined together and subjected to one ab-initio round of 3D reconstructions split into 6 classes. A final data-set of 392,370 particles from the best 3D classes were subjected to local and global CTF refinements, followed by a 3D non-uniform refinement. The final density map was resolved to 3.25 Å, after map sharpening, as determined by gold-standard Fourier shell correlation using the 0.143 criterion. Local resolution estimation was performed using cryoSPARC.
Model building
An initial model was created by docking the individual complex components into the cryo-EM map using the “fit in map” routine in UCSF Chimera37 (version 1.15). The following structures from the Protein Data Bank (PDB) were used: OTR (PDB ID: 6TPK), Gαo (PDB ID: 6WWZ), Gβ1γ2, scFv16 (PDB ID: 6OIJ), and AVP (PDB ID: 7DW9). All initial model components were manually rebuilt in Coot38 (version 0.9.7), followed by several rounds of manual real-space refinement in Coot and real-space refinement with the software package Phenix.real_space_refine from Phenix39 (version 1.20.1-4487). The quality of the models was assessed using MolProbity40 before PDB deposition. PyMOL (version 2.5) was used for visual inspection, model comparison and figure preparation.
IP1 accumulation assays
Ligand-induced IP1 accumulation and ligand-binding experiments were measured using transiently transfected Human Embryonic Kidney (HEK) 293 T/17 cells (American Type Culture Collection). The cells were cultivated in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific) supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml, MilliporeSigma), and 10% (v/v) foetal calf serum (BioConcept) and maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Transient transfections were performed with TransIT-293 (Mirus Bio) according to the manufacturer’s instructions. Full-length OTR, truncated V1AR (residues 1-378 with C-terminal mutation T378K) and mutants thereof were directly cloned into a mammalian expression vector containing an N-terminal SNAP-tag (pMC08; Cisbio). Cells were transfected and directly seeded at 7500 cells per well in poly-D-lysine-coated white 384-well plates (Greiner).
To compare IP1 accumulation a homogeneous time-resolved fluorescence (HTRF) signalling assay was performed, adapting a previously described protocol41. The cells were washed 48 h after transfection with phosphate-buffered saline (PBS) and stimulation buffer (Cisbio) and subsequently incubated for 1 h at 37 °C with a concentration range of oxytocin (Psyclo Peptide Inc.) diluted in stimulation buffer. The IP1 accumulation was determined using the HTRF IP-One Kit (Cisbio) according to the manufacturer’s protocol. Fluorescence intensities were measured on a Spark fluorescence plate reader (Tecan). To generate concentration-response curves, data were analysed by a three-parameter logistic equation in GraphPad Prism software (version 9.2.0).
Whole-cell ligand binding assays
Ligand-binding experiments were performed on whole cells for comparison of affinities for wild-type and receptor mutants using an HTRF binding assay as previously described17. Forty-eight hours post-transfection the cells were labelled with 50 nM SNAP-Lumi4-Tb (Cisbio) in labelling buffer (20 mM Hepes (pH 7.5), 100 mM NaCl, 3 mM MgCl2, and 0.05% (w/v) bovine serum albumin (BSA)) for 1.5 h at 37 °C. The cells were washed two times with labelling buffer and two times with assay buffer (20 mM Hepes (pH 7.5), 100 mM KCl, 3 mM MgCl2, and 0.05% (w/v) BSA)) and subsequently incubated for 1 h at room temperature with a concentration range of fluorescently labelled peptide HiLyte Fluor 488-Orn8 oxytocin (Eurogentec) in assay buffer. Fluorescence intensities were measured on a Spark fluorescence plate reader with an excitation wavelength of 340 nm and emission wavelengths of 620 nm and 510 nm for Tb3+ and the fluorophore HiLyte Fluor 488, respectively. The ratio of fluorescence resonance energy transfer (FRET) donor and acceptor fluorescence intensities was calculated (F510 nm/F620 nm). Nonspecific binding was determined in the presence of a 1000-fold excess of unlabelled oxytocin. Data were analysed by global fitting to a one-site saturation binding equation with the GraphPad Prism software.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Data availability
Atomic coordinates of the OTR:Go/q:OT:scFv16 complex have been deposited in the PDB under the accession code 7QVM. Cryo-EM maps used have been deposited in the EMDB found under code EMD-14180. Source data for ligand binding and receptor activation are provided with this paper.
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Acknowledgements
We thank the staff, in particular S. Sorrentino, of the Centre for Microscopy and Image Analysis at the University of Zurich for their support in the initial phases of the project. We further thank O. Eggenberger for assistance in protein production and I. Berger for providing us with baculovirus transfer vectors. This work was supported by Schweizerischer Nationalfonds Grant 31003A_182334 (to A.P), and by the European Research Council (810057-HighResCells to A.P. and O.M.).
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The project was outlined by Y.W. and A.P. Y.W. and J.E. designed the G protein and scFv constructs. Y.W. purified all proteins and prepared the complexes. Y.W, J.E. and S.V. vitrified the samples on cryo-EM grids. Y.W. and J.E. collected cryo-EM data with the help of S.V. Y.W. and S.V. processed data and refined the cryo-EM density map. J.E. and Y.W. built and refined the structure model. C.T. designed and performed the functional assays. Project management was carried out by Y.W, J.E., C.T., O.M. and A.P. The manuscript was prepared by Y.W., C.T., S.V., J.E., O.M. and A.P. All authors contributed to the final editing and approval of the manuscript.
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Waltenspühl, Y., Ehrenmann, J., Vacca, S. et al. Structural basis for the activation and ligand recognition of the human oxytocin receptor. Nat Commun 13, 4153 (2022). https://doi.org/10.1038/s41467-022-31325-0
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DOI: https://doi.org/10.1038/s41467-022-31325-0
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