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Autoantibody mimicry of hormone action at the thyrotropin receptor

Abstract

Thyroid hormones are vital in metabolism, growth and development1. Thyroid hormone synthesis is controlled by thyrotropin (TSH), which acts at the thyrotropin receptor (TSHR)2. In patients with Graves’ disease, autoantibodies that activate the TSHR pathologically increase thyroid hormone activity3. How autoantibodies mimic thyrotropin function remains unclear. Here we determined cryo-electron microscopy structures of active and inactive TSHR. In inactive TSHR, the extracellular domain lies close to the membrane bilayer. Thyrotropin selects an upright orientation of the extracellular domain owing to steric clashes between a conserved hormone glycan and the membrane bilayer. An activating autoantibody from a patient with Graves’ disease selects a similar upright orientation of the extracellular domain. Reorientation of the extracellular domain transduces a conformational change in the seven-transmembrane-segment domain via a conserved hinge domain, a tethered peptide agonist and a phospholipid that binds within the seven-transmembrane-segment domain. Rotation of the TSHR extracellular domain relative to the membrane bilayer is sufficient for receptor activation, revealing a shared mechanism for other glycoprotein hormone receptors that may also extend to other G-protein-coupled receptors with large extracellular domains.

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Fig. 1: Cryo-EM structures of native human TSH and TR1402 bound to active TSHR complexed with heterotrimeric Gs.
Fig. 2: The activation mechanism of the TSHR revealed by the inactive structure bound to the inverse agonist CS-17.
Fig. 3: Activation of the TSHR by a Graves’ disease autoantibody.
Fig. 4: Membrane bilayer interactions are critical for TSHR activation.
Fig. 5: Model for TSHR activity.

Data availability

Coordinates for TSH-bound TSHR–Gs, TR1402-bound TSHR–Gs, M22-bound TSHR–Gs and CS-17-bound TSHR have been deposited in the PDB under accession codes 7T9I, 7UTZ, 7T9N and 7T9M, respectively. Sharpened and unsharpened cryo-EM density maps for TSH-bound TSHR–Gs (composite), TR1402-bound TSHR–Gs (composite), M22-bound TSHR–Gs (composite), CS-17-bound TSHR, and Org 2274179-0-bound TSHR have been deposited in the Electron Microscopy Data Bank under accession codes EMD-25758, EMD-26795, EMD-25763, EMD-25762 and EMD-27640, respectively. Sharpened maps, unsharpened maps, half-maps and masks for each composite map component (ligand-bound ECD or 7TM–G protein), for TSH-bound TSHR–Gs, TR1402-bound TSHR–Gs and M22-bound TSHR-Gs have been deposited in the Electron Microscopy Data Bank under accession codes EMD-27649 (TSH-bound ECD), EMD-27650 (TSH-bound 7TM–Gs), EMD-27647 (TR1402-bound ECD), EMD-27648 (TR1402 7TM–Gs), EMD-27651 (M22-bound ECD) and EMD-27652 (M22-bound 7TM–Gs). Final particle stacks and .star files for TSH-bound TSHR–Gs, TR1402-bound TSHR–Gs and M22-bound TSHR–Gs and CS-17-bound TSHR-containing particle shift and pose assignments have been uploaded to the Electron Microscopy Public Image Archive under the accession codes EMPIAR-11120. Structure files for OPM-based modelling of inactive and active TSHR are available in the Supplementary Information.

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Acknowledgements

We thank R. Yan and D. Toso for help in microscope operation and data collection; C. Bracken for mass spectrometry troubleshooting; and H. Shan for sample preparation for lipid mass spectrometry. This work was supported by National Institutes of Health (NIH) grants DP5OD023048 (A.M.), 1R35GM140847 (Y.C.), P30CA014195, R01GM102491 (A.S.), R01GM127359 (R.O.D.), 5R44CA224376 (M.W.S.) and a Human Frontier Science Program Long-Term Fellowship LT000916/2018-L (C.-M.S.). Cryo-EM equipment at UCSF is partially supported by NIH grants S10OD020054 and S10OD021741. Some of this work was performed at the Stanford-SLAC Cryo-EM Center (S2C2), which is supported by the National Institutes of Health Common Fund Transformative High-Resolution Cryo-Electron Microscopy program (U24 GM129541). This research was, in part, supported by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract HSSN261200800001E. Some of this work was supported by the Mass Spectrometry Core of the Salk Institute with funding from NIH-NCI CCSG: P30CA014195. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. A.M. acknowledges support from the Pew Charitable Trusts, the Esther A. & Joseph Klingenstein Fund, the Searle Scholars Program, the Edward Mallinckrodt Jr Foundation, and the Vallee Foundation. Y.C. is an Investigator of Howard Hughes Medical Institute. A.M. is a Chan Zuckerberg Biohub investigator.

Author information

Authors and Affiliations

Authors

Contributions

B.F. cloned, expressed, and biochemically optimized the purification of all TSHR constructs for structural studies. B.F. expressed and purified Gβ1γ2, Nb35, M22 Fab, CS-17 IgG and K1-70 IgGs, and performed enzymatic digestion and further purification to form CS-17 and K1-70 Fab fragments. B.F. performed complexing and identified optimal cryo-EM grid preparation procedures, screened samples, and collected 300 keV datasets. B.F. determined high-resolution cryo-EM maps by extensive image processing under the guidance of A.M. and Y.C. B.F. and A.M built and refined models of TSHR complexes. B.F. and C.B.B. generated receptor constructs and determined expression levels by flow cytometry and performed signalling studies and analysed the data. C.B.B. assisted with cloning of TSHR constructs, expression, purification and labelling of CS-17 and M22 Fab fragments, and cloning and generation of baculoviruses for expression of Gβ1γ2. C.-M.S. performed and analysed molecular dynamics simulations under the supervision of R.O.D. I.S. performed initial biochemical optimization of TSHR complexes with M22 Fab and worked with K.Z. to collect negative stain and cryo-EM data. N.H. prepared samples for, performed, and analysed native mass spectrometry experiments and prepared control samples for lipid identification experiments. J.K.D. analysed native human TSH glycosylation by mass spectrometry. Y.M. generated TSHR and M22 Fab expression constructs and performed pilot biochemical purification of TSHR complexes. A.F.M.P. performed and analysed data from the lipid identification experiments with guidance from A.S. M.W.S. led production of highly pure TR1402 agonist. Figures were generated and the manuscript was written by B.F., C.B.B. and A.M., with edits from Y.C., R.O.D., C.-M.S., and M.W.S. and with approval from all authors. The overall project was supervised by Y.C. and A.M.

Corresponding authors

Correspondence to Yifan Cheng or Aashish Manglik.

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Competing interests

A.M. and R.O.D. are consultants for and stockholders in Septerna Inc. Y.C. is a consultant and advisor of Shuimu BioScience Ltd.

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Nature thanks Marvin Gershengorn, Patrick M. Sexton and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Signaling studies for TSHR.

a, b) DNA titrations of TSHR mutants tested in cAMP production assays. Median fluorescence intensity ± SD (n = 3) of anti-FLAG-A647 staining is plotted. Filled in bars represent the DNA concentration used for co-transfection experiments with pGlo cAMP biosensor plasmid. Horizontal red line indicates the mean wild-type (WT) TSHR fluorescence intensity. Flow cytometry experiments were performed in triplicate, with identical gating across all cell lines tested. c) cAMP production of TSHR cysteine mutants comparing the basal level (-) to 100 nM of TSH, M22, or CS-17 for untreated cells (left panel) and cells treated with 500 µM TCEP (right panel). Data points are means ± SD from three biological replicates. d, e) cAMP production curves comparing TSH and M22-mediated activation of WT and C-peptide deleted TSHR constructs. f) cAMP production curves for TSH and TR1402-mediated WT TSHR activation. g) cAMP production curves for TSH-mediated WT and TSHR mutant cell line (Y385F, Y385A) activation. h) cAMP production curves for M22-mediated WT and lipid-displacing TSHR mutant cell line (A644K, A647K) activation. Plotted data points in panels c-g are means of triplicate measurements ± SD from a representative experiment of n = 3 biological replicates.

Extended Data Fig. 2 Cryo-EM data processing for TSH-bound TSHR-Gs complex.

a) Representative image from 15,345 micrographs. Scale bar, 50 nm. b) Selected 2D class averages. c) Processing approach used for reconstruction of TSH-bound TSHR-Gs complex. A local resolution map was calculated from cryoSPARC using masks from indicated local refinement, then visualized with the composite map in the same scale. A viewing distribution plot was generated using scripts from the pyEM software suite83 and visualized in ChimeraX. GS-FSC and Directional FSC (dFSC, shown as purple lines) curves were generated in cryoSPARC and as previously described in Dang, S. et al. Nature 552, 426–429 (2017).

Extended Data Fig. 3 Model map correlation coefficients for cryo-EM structures.

Correlation values for resolved residues in each modeled chain are shown. Low values indicate regions that are poorly resolved, e.g. the 7TM domain of TSHR in CS-17-TSHR complex or the constant domains of the Fab fragments for CS-17 and M22.

Extended Data Fig. 4 Cryo-EM data processing for TR1402-bound TSHR-Gs complex.

a) Representative image from 14,277 micrographs. Scale bar, 50 nm. b) Selected 2D class averages from final reconstruction. c) Processing approach used for reconstruction of TR1402-bound TSHR-Gs complex. A local resolution map was calculated from cryoSPARC using masks from indicated local refinement, then visualized with the composite map in the same scale. A viewing distribution plot was generated using scripts from the pyEM software suite and visualized in ChimeraX. GS-FSC and dFSC curves were generated in cryoSPARC and as previously described in Dang, S. et al. Nature 552, 426–429 (2017).

Extended Data Fig. 5 Glycosylation status of native human TSH.

a) Cryo-EM density map of TSH-bound TSHR showing resolved glycan density for Asn52. b) Peptide coverage of Asn52 from native human TSH GPHα chain in mass spectrometry experiments. c) Peptide spectra match (PSM) of glycans detected on Asn52 of GPHα chain. d,e) Representative MS/MS spectra showing Asn52 with various length glycans.

Extended Data Fig. 6 Receptor:hormone interaction comparisons across the glycoprotein hormone receptor family.

a) Comparison of TSHR, FSHR (PDB: 4AY9), and LH/CGR ECD:hormone (PDB: 7FIH) interactions shows the comparable concave binding-interface and lateral-ECD contacts made by GPHα and receptor-specific β subunits. b) Alignment of the GPHR ECDs reveals the variability in modeled poses of hinge-hormone contacts observed in prior GPHR:hormone structures. c) The N-terminal hinge-hormone disulfide-linked alpha helix presents a hydrophobic face to the α-L1 and α-L3 loops of the TR1402 α-chain and is stabilized by multiple phenylalanine contacts in the GPHα and TSHβ chains. C-terminal hinge-region α helix hidden for visualization. d) Comparison of the TSHR and LHCGR (PDB: 7FIH) transmembrane pockets highlights the similarity in the DPPC and the allosteric agonist Org43554 binding sites. TM4/5 and ECL2 not shown. e) Lipid:protein molar ratio comparison between the M22:TSHR:Gs complex and a non-GPHR Class A receptor control. Data points represent individual measurements from technical replicates (n = 3 for non-GPHR control and n = 4 for M22:TSHR:Gs complex) of the ratio of pmol of lipid DPPC per pmol of protein. **P = 0.0088; Unpaired two-tailed t test was used to calculate statistical differences in lipid:protein molar ratios.

Extended Data Fig. 7 Cryo-EM data processing for Org 274179-0 bound TSHR.

a) Representative image from 10,003 micrographs. Scale bar, 50 nm. b) Selected 2D class averages generated from curated particles. c) Processing approach used for low resolution reconstruction. Despite starting with a similar or larger sized dataset as for other TSHR samples, the TSHR–Org 274179-0 complex did not yield high resolution reconstruction of the 7TM domain. A low-resolution reconstruction of the TSHR ECD was observed. This suggests potential flexibility between the TSHR 7TM domain and the ECD.

Extended Data Fig. 8 Cryo EM data processing for the CS-17 bound TSHR:Org 274179-0 complex.

a) Representative image from 4,952 micrographs. Scale bar, 50 nm. b) Selected 2D class averages generated from the final reconstruction. c) Processing approach used for reconstruction of the complex. A viewing distribution plot was generated using scripts from the pyEM software suite and visualized in ChimeraX. Local resolution map generated from non-uniform refinement mask in cryoSPARC. GS-FSC and dFSC curves were generated in cryoSPARC and as previously described in Dang, S. et al. Nature 552, 426–429 (2017).

Extended Data Fig. 9 Comparison of Org 274179-0 bound- and CS-17 bound TSHR EM maps.

a) Org 274179-0 bound-TSHR EM density map (dark) fit into the CS-17-bound TSHR map (colored by TSHR-CS-17 model). b) CS-17-bound TSHR model fit into Org 274179-0 bound-TSHR map, suggesting that inactive state TSHR ECD orientations are similar between CS-17 and Org 274179-0-bound states.

Extended Data Fig. 10 Comparison of TSHR activation with other GPCRs.

a) Ribbon diagram of TSHR in active and inactive conformations. b) Ribbon diagram of LH/CGR in active (PDB:7FIH) and inactive (PDB:7FIJ) conformations. A similar reorientation of the ECD is shared between TSHR and LH/CGR upon activation. c) Comparison of active TSHR to active β2-adrenoceptor (β2AR, PDB:3SN6). d) Comparison of inactive TSHR to inactive β2AR, PDB:5JQH). e) Comparison of active conformations of TSHR and LH/CGR reveals similar overall structures of the 7TM domain and the p10 peptide.

Extended Data Fig. 11 Cryo-EM data processing for M22-bound TSHR-Gs complex.

a) Representative image from 25,030 micrographs. Scale bar, 50 nm. b) Selected 2D class averages from final reconstruction. c) Processing approach used for reconstruction of M22-bound TSHR-Gs complex. A local resolution map was calculated from cryoSPARC using masks from indicated local refinement, then visualized with the composite map in the same scale. A viewing distribution plot was generated using scripts from the pyEM software suite and visualized in ChimeraX. GS-FSC and dFSC curves were generated in cryoSPARC and as previously described in Dang, S. et al. Nature 552, 426–j429 (2017).

Extended Data Fig. 12 Extent of ECD activation for all individual simulations across four simulation conditions.

Dashed lines indicate the projection metric values (see Methods) in the inactive (0.0 Å) and active (33.5 Å) state cryo-EM structures. Thick traces indicate the moving average smoothed over a 25-ns window, and thin traces represent unsmoothed data. Each column represents a distinct simulation condition: started from the active structure, with M22 bound (brown traces, first column); started from the active structure, with M22 removed (grey traces, second column); started from the inactive structure, with CS-17 bound (blue traces, third column); started from the inactive structure, with CS-17 removed (grey traces, fourth column).

Extended Data Fig. 13 Glycosylation of engineered K1-70glyco construct.

a) Native mass spectrum (nMS) of K1-70, K1-70glyco, and K1-70glyco(N16Q) Fab fragments. b) Accurate mass assignment of Fab fragments. nMS demonstrates ~1.7 kDa increased mass for K1-70glyco and more heterogeneity consistent with N-linked glycosylation. The smaller mass and increased homogeneity of the K1-70glyco(N16Q) construct further supports glycosylation at the engineered N16 position. c) Crystal structure of K1-70 TSHR-ECD complex (PDB: 2XWT) aligned to CS-17 bound TSHR. The membrane-proximal N229 residue (of the “GlycoB” glycosylation motif) is highlighted in red. d) cAMP production comparing K1-70 WT, K1-70 GlycoB and M22 Fab fragment-mediated activation of TSHR. Plotted data points are means of triplicate measurements ± SD from one of three biological replicates.

Extended Data Table 1 Cryo-EM data collection, refinement, and validation statistics

Supplementary information

Supplementary Table 1

Summary of TSHR signalling studies. Values are expressed as mean pEC50 or mean Emax ± s.e.m. from (n) biological replicates. ND, Not determined.

Reporting Summary

Supplementary Data 1

Structure files for static modelling of TSHR in active and inactive conformations bound to TSH, M22 CS-17, and K1-70.

Peer Review File

Supplementary Video 1

3D variability analysis of TR1402-bound TSHR–Gs complex.

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Faust, B., Billesbølle, C.B., Suomivuori, CM. et al. Autoantibody mimicry of hormone action at the thyrotropin receptor. Nature 609, 846–853 (2022). https://doi.org/10.1038/s41586-022-05159-1

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