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The oxytocin signaling complex reveals a molecular switch for cation dependence

Nature Structural & Molecular Biology volume 29, pages 274–281 (2022)Cite this article

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Abstract

Oxytocin (OT) and vasopressin (AVP) are conserved peptide signaling hormones that are critical for diverse processes including osmotic homeostasis, reproduction, lactation and social interaction. OT acts through the oxytocin receptor (OTR), a magnesium-dependent G protein-coupled receptor that is a therapeutic target for treatment of postpartum hemorrhage, dysfunctional labor and autism. However, the molecular mechanisms that underlie OTR activation by OT and the dependence on magnesium remain unknown. Here we present the wild-type active-state structure of human OTR bound to OT and miniGq/i determined by cryo-EM. The structure reveals a unique activation mechanism adopted by OTR involving both the formation of a Mg2+ coordination complex between OT and the receptor, and disruption of transmembrane helix 7 (TM7) by OT. Our functional assays demonstrate the role of TM7 disruption and provide the mechanism of full agonism by OT and partial agonism by OT analogs. Furthermore, we find that the identity of a single cation-coordinating residue across vasopressin family receptors determines whether the receptor is cation-dependent. Collectively, these results demonstrate how the Mg2+-dependent OTR is activated by OT, provide essential information for structure-based drug discovery efforts and shed light on the molecular determinants of cation dependence of vasopressin family receptors throughout the animal kingdom.

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Fig. 1: Structure of the OTR–miniGq complex bound to oxytocin.
Fig. 2: OT binding mode and G protein interaction.
Fig. 3: Disruption of TM7 helix underlies receptor activation.
Fig. 4: Architecture of the magnesium cofactor coordination site.
Fig. 5: An aspartate to lysine transition reverses magnesium dependence in the OT/AVP receptor family.

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Data availability

All data generated or analyzed during this study are included in the Article and its Supplementary Information. Cryo-EM maps of the OT–OTR–Gq complex have been deposited in the Electron Microscopy Data Bank under accession code EMD-24733, including the composite map, global non-uniform refinement, local refinement of TMs with deepEMhancer, and local refinement of G protein. The original micrographs have been deposited in the Electron Microscopy Public Image Archive under accession code EMPIAR-10936. The atomic coordinates have been deposited in the Protein Data Bank under the accession code 7RYC. All other relevant data is available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank C. Theophanopoulou and E. Jarvis at The Rockefeller University for their advice and discussions, E. Montabana for support in data collection and the Stanford cryo-Electron Microscopy center (cEMc). We thank R. Hibbs for gifting the pEZT vector. This work was supported, in part, by National Institute of Health T32-GM089626 (J.G.M.), and the Extreme Science and Engineering Discovery Environment (XSEDE)37 resource comet-gpu and expanse through sdsc-comet/sdsc-expanse allocation TG-MCB190153, which is supported by National Science Foundation grant number ACI-1548562.

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  1. These authors contributed equally: Justin G. Meyerowitz, Michael J. Robertson.

Authors and Affiliations

  1. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA

    Justin G. Meyerowitz, Michael J. Robertson, Ximena Barros-Álvarez, Ouliana Panova, Robert M. Nwokonko, Yang Gao & Georgios Skiniotis

  2. Department of Anesthesiology, Perioperative and Pain Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Justin G. Meyerowitz

  3. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA

    Justin G. Meyerowitz, Michael J. Robertson, Ximena Barros-Álvarez, Ouliana Panova, Robert M. Nwokonko, Yang Gao & Georgios Skiniotis

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Contributions

J.G.M. expressed and purified proteins, prepared cryo-EM samples, processed cryo-EM data, built and refined the structural model, designed and generated the expression and mutagenesis constructs, and performed biochemical assays and analyzed the data. M.J.R. performed molecular dynamics simulations and free energy calculations, model refinement, generation of expression constructs, and protein purification. X.B.-Á. expressed and purified miniGqi. O.P. collected cryo-EM data. R.M.N. assisted with cell surface expression experiments. Y.G. prepared cryo-EM grids. J.G.M. and G.S. wrote the manuscript with input from M.J.R., X.B.-Á., O.P., R.M.N. and Y.G. G.S. supervised the project.

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Correspondence to Georgios Skiniotis.

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Extended data

Extended Data Fig. 1 Oxytocin receptor signaling and purification.

a, Dose−response curves of G protein-specific activation pathways of the oxytocin receptor by oxytocin (orange squares), neurotensin 1 receptor by neurotensin (teal circles), or beta-2 adrenergic receptor by isoproterenol (teal triangles) from simultaneous curve fitting of 3 independent biological replicates with Hill Slope constrained to 1. Error bars are S.E.M. b, Gel filtration chromatography profile and c, protein gel of oxytocin receptor with GFP tag. d, Gel filtration chromatography profile and e, protein gel of oxytocin receptor complex from the protein preparation used for data collection.

Source data

Extended Data Fig. 2 Oxytocin-Oxytocin Receptor-MiniGqi-scFv16 cryoEM data collection and processing.

a, Representative micrograph of OTR complex from 7,727 micrographs. b, Example final 2D classes of OTR complex (128 classes). c, CryoEM data processing workflow. d, Gold-standard FSC curves of global non-uniform refinement of complex, local refinement of transmembrane helices, and local refinement of G protein heterotrimer. e, Overall combined map from two local (TMs and G protein) and global (scFv16) refinements.

Extended Data Fig. 3 Map/model fit and signaling complex architecture.

Fit into cryo-EM map of a, oxytocin, b, the N-terminal domain (NTD), c, extracellular loop 3 (ECL3) and extracellular loop 2 (ECL2), d, cation binding site at high (purple) threshold and low (transparent) threshold. e, the C-terminal alpha-helical domain (α-C) of miniGqi, f, transmembrane (TM) helices 1-7. g, Representative cell cytometry data of surface expression levels of wild-type OTR (WT), OTRR34A, OTRF103A, and OTRR104A detected by Alexa Fluor-647 (AF-647) corresponding to transient transfections used in BRET assays.

Extended Data Fig. 4 Oxytocin receptor ligand and G protein interactions.

a, View from TM3 of oxytocin (OT) in the oxytocin receptor binding pocket with overlay of cryoEM map at threshold level 0.105 in ChimeraX; receptor in blue, oxytocin in orange, and Mg2+ in green. Superscript is Ballesteros-Weinstein notation. b, Overlay of OT (orange) bound to OTR (blue) and arginine vasopressin (AVP; purple) bound to V2R (tan; PDB: 7KH0). c, Overlay of active state structures of vasopressin 2 receptor-Gs (V2R-Gs; PDB: 7KH0), serotonin receptor 2A-miniGq/i (HTR2A-miniGq; PDB: 5WHA) aligned on TM bundle (transparent) showing placement of G protein N-terminal alpha helical domain (α-N). d, Overlay of same alignment showing slight difference in angular orientation of G protein C terminal helix (α5) between receptors. e, Comparison of G protein engagement between OTR-miniGq (blue) with V2R-Gs (salmon; PDB: 7KH0) aligned on TM bundle.

Extended Data Fig. 5 Activation mechanism of the oxytocin receptor.

a, Overlay of TM7 from OT-bound cryoEM structure (blue) and antagonist-bound crystal structure (mustard) showing broken helix in active state and interaction between Tyr2 of OT (orange) and the backbone carbonyl of L3167.40. b, Overlay of OTR-bound OT with both the L-state and T-state poses (AVP-L and AVP-T) of AVP-bound V2R-Gs (PDB: 7BB6, 7BB7). c, Overlay of OT with OTR-bound retosiban. d, Overlay of TM6 and TM7 from OT-bound (blue) and antagonist-bound (mustard) OTR showing stabilization of TM7 by hydrogen bond between N3217.45 and N325K7.49 in inactive structure, and the rotation of Y3297.53 and displacement of TM6 in the OT-bound structure. Backbone of full transmembrane helix 7 with helical hydrogen bonds and ligand pose for the active-state structures of e, OTR bound to OT and f, HTR2A bound to 25-CN-NBOH (PDB: 6WHA) aligned on TMs and viewed from identical perspective, with receptor TM helices in transparent cartoon.

Extended Data Fig. 6 Oxytocin receptor partial agonists.

a, Gq, G11, and G15 signaling measured by G protein BRET-based biosensors and b, recruitment of β-arrestin 1 and β-arrestin 2 to C-terminal rLuc8-tagged OTR. c, Dose-response curve of 2-(O-methyltyrosine)-oxytocin (meOT) demonstrating antagonism of OTR-dependent Gq signaling in the presence of 100 nM OT, with net BRET normalized to BRET ratio in presence of 100 nM OT. d, Arginine vasopressin (AVP) or Phe2-AVP activation of V1aR or V1bR measured by Gq BRET biosensor, or V2R measured by Gs BRET biosensor. Data points are represented as mean values + /- SEM and curves are simultaneously fit to n = 3 biologically independent replicates.

Extended Data Fig. 7 Magnesium dependence of oxytocin and vasopressin receptors.

a, Recruitment of beta-arrestin 1 (β1) or beta-arrestin 2 (β2) and b, activation of Gq-based BRET biosensor by oxytocin (OT)- or vasopressin (AVP)-stimulated oxytocin receptor (OTR) or vasopressin receptor 1a (V1aR) and mutants in the presence of 2 mM Mg2+ or 0.5 mM EDTA. Data points are represented as mean values + /− SEM and curves are simultaneously fit to n = 3 biologically independent replicates. c, Sequence alignment of cation-coordinating residues in vasopressin 2 receptor (V2R; VTR2C) in primates, with protein accession codes.

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Meyerowitz, J.G., Robertson, M.J., Barros-Álvarez, X. et al. The oxytocin signaling complex reveals a molecular switch for cation dependence. Nat Struct Mol Biol 29, 274–281 (2022). https://doi.org/10.1038/s41594-022-00728-4

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