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Cited by 147Thyroid Disorders and TreatmentsEnzyme Structure and FunctionProtein Structure and Dynamics

The structure of human thyroglobulin

Open access
Coscia, Francesca, Taler-Verčič, Ajda, Chang, Veronica T., Sinn, Ludwig, O’Reilly, Francis J., Izoré, Thierry, Renko, Miha, Berger, Imre +3 more·2020·Nature
DOI 10.1038/s41586-020-1995-4PMC PMC7170718
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Abstract

Thyroglobulin (TG) is the protein precursor of thyroid hormones, which are essential for growth, development and the control of metabolism in vertebrates1,2. Hormone synthesis from TG occurs in the thyroid gland via the iodination and coupling of pairs of tyrosines, and is completed by TG proteolysis3. Tyrosine proximity within TG is thought to enable the coupling reaction but hormonogenic tyrosines have not been clearly identified, and the lack of a three-dimensional structure of TG has prevented mechanistic understanding4. Here we present the structure of full-length human thyroglobulin at a resolution of approximately 3.5 Å, determined by cryo-electron microscopy. We identified all of the hormonogenic tyrosine pairs in the structure, and verified them using site-directed mutagenesis and in vitro hormone-production assays using human TG expressed in HEK293T cells. Our analysis revealed that the proximity, flexibility and solvent exposure of the tyrosines are the key characteristics of hormonogenic sites. We transferred the reaction sites from TG to an engineered tyrosine donor–acceptor pair in the unrelated bacterial maltose-binding protein (MBP), which yielded hormone production with an efficiency comparable to that of TG. Our study provides a framework to further understand the production and regulation of thyroid hormones. The cryo-electron microscopy structure of human thyroglobulin reveals that proximity, flexibility and solvent exposure are key characteristics of its hormonogenic tyrosine pairs, and provides a framework for understanding the formation of thyroid hormones.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper reports the cryo-EM structure of full-length human thyroglobulin at ~3.5 Å and identifies/validates its four hormonogenic sites and five donor tyrosines. It shows that proximity, flexibility, solvent exposure, and roughly antiparallel orientation enable T4 synthesis, and transfers a functional hormonogenic site into engineered maltose binding protein.

Key findings

  • The human thyroglobulin dimer structure was determined at ~3.5 Å resolution (eTG C2 map 3.39 Å; rTG expanded map 3.67 Å), with a bilobed shape of 120 × 235 Å, a dimer interface of 29,350 Ų, and an atomic model covering 93% of 2,749 amino acids (2,569 protein residues; PDB 6SCJ).
  • Four hormonogenic acceptor tyrosines were identified—Y24 (Site A), Y2573 (Site B), Y2766 (Site C), and Y1310 (Site D)—with five donor tyrosines: Y234 and Y149 for Site A, Y2540 for Site B, Y2766 for Site C, and Y108 for Site D. Mutating all acceptors abolished T4 synthesis; mutating all five donors gave no significant T4 formation, and Site A was confirmed to have two donors.
  • TG synthesises seven T4 molecules per dimer: two each of Sites A, B, and D, and one Site C. Key requirements for hormonogenesis are solvent exposure, proximity, roughly antiparallel orientation, and high mobility; a conserved acidic residue precedes the acceptor, and D1309 was essential for Site D while K1415 was not.
  • An engineered MBP variant with a C-terminal SGSDYS insert (C-ins2) produced T4 at levels comparable to a single TG site. Crosslinking mass spectrometry validated the architecture, no inter-subunit disulfides were found, and 12 previously predicted N-linked GlcNAc plus four new sites (N110, N484, N1869, N2122) were resolved; glycan N2013 mediates NTD–CTD contact.

Methods

  • Recombinant full-length human TG was expressed in HEK293T cells; endogenous TG was purified from goitrous human thyroid glands. Cryo-EM used a Titan Krios G2 with K2 Summit detector, C2 and symmetry-expanded refinements, and model building combined de novo and homology modelling.
  • Site-directed mutagenesis replaced acceptor or donor tyrosines with phenylalanines. In vitro iodination used KI, glucose/glucose oxidase, and lactoperoxidase or thyroid peroxidase; T4 and T3 were measured by commercial ELISA. MBP variants were engineered and assayed similarly.
  • Cryo-EM datasets: eTG 422,087 initial and 181,830 final particles; rTG 240,194 initial and 151,601 final particles. Crosslinking mass spectrometry used BS3, tryptic digestion, SEC enrichment, and LC-MS/MS with Xi search at 2% residue-pair FDR.

Limitations

  • Stated: map resolution is non-uniform, with central ChEL regions at ~3 Å but peripheral Arm regions at ~6 Å; the model has variable local quality. The NTD is partially flexible, Y24 is partially disordered, residues 24–29 were built tentatively as poly-alanine, and Y2766 was not resolved in the maps. Only T4, not T3, was detected under the in vitro conditions.
  • Stated: endogenous TG was partly iodinated and partially degraded; recombinant TG was non-iodinated. The in vitro assay relies on LPO/TPO and ELISA detection rather than the native thyroid environment.
  • Evident from the text: the atomic model covers 93% of the 2,749 amino acids, leaving some regions unmodelled; side-chain placements at hormonogenic sites are difficult due to conformational disorder; and the MBP engineering test is limited to a small set of designed variants.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41586-020-1995-4

Cites

50 of 50 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

12 of 27 cited sources checked · 5 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    The lack of a three-dimensional structure of TG has prevented mechanistic understanding.

    “hormonogenic tyrosines have not been clearly identified and the lack of a three-dimensional structure of TG has prevented mechanistic understanding4.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [4] Cahnmann, H. J., Pommier, J. & Nunez, J. Spatial requirement for coupling of iodotyrosine residues to form thyroid hormones. Proc. Natl Acad. Sci. USA 74, 5333–5335 (1977). source

      The introductory passage (page unavailable) says thyroglobulin’s primary sequence was unknown and the molecule was too complex to study the factors governing spatial alignment, prompting the authors to use simpler models. It does not specifically say that the lack of a three-dimensional structure prevented mechanistic understanding more broadly. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Interpretationcitation

    In the context of the thyroid gland and the iodine cycle in vertebrates, TG's structure effectively combines hormonogenesis with iodination of many other solvent exposed tyrosine residues for iodine storage.

    “However, in the context of the thyroid gland and the iodine cycle in vertebrates, TG's structure effectively combines hormonogenesis with iodination of many other solvent exposed tyrosine residues for iodine storage21,13.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [21] Lamas, L., Anderson, P. C., Foxy, J. W. & Dunn, J. T. Consensus sequences for early iodination and hormonogenesis in human thyroglobulin. J. Biol. Chem. 264, 13541–13545 (1989). source
    • cites [13] Dedieu, A., Gaillard, J.-C., Pourcher, T., Darrouzet, E. & Armengaud, J. Revisiting iodination sites in thyroglobulin with an organ-oriented shotgun strategy. J. Biol. Chem. 286, 259–269 (2011). source

      The Abstract describes Tg as both a scaffold for thyroid hormonogenesis and an iodine reservoir. The Discussion reports many iodinated tyrosines but only a few hormonogenic sites, and suggests that the additional iodination probably stores iodine. The paper does not establish that those other residues are solvent-exposed; its direct findings are from mouse Tg rather than vertebrates generally. Page numbers are unavailable in our copy. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Interpretationcitation

    At Site C, Y2766, not resolved in the maps, probably acts both as donor and acceptor across the C2 dimer axis.

    “At Site C, Y2766, not resolved in our maps, probably acts both as donor and acceptor across the C2 dimer axis19.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [19] Citterio, C. E., Morishita, Y., Dakka, N., Veluswamy, B. & Arvan, P. Relationship between the dimerization of thyroglobulin and its ability to form triiodothyronine. J. Biol. Chem. 293, 4860–4869 (2018). source
  • Premisecitation

    TG is a non-covalent dimer.

    “Most of TG's disulfide bonds show clear EM density in our maps (Extended Data Figure 5a) and we found no inter-subunit disulfide bonds, in agreement with previous observations that TG is a non-covalent dimer19.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [19] Citterio, C. E., Morishita, Y., Dakka, N., Veluswamy, B. & Arvan, P. Relationship between the dimerization of thyroglobulin and its ability to form triiodothyronine. J. Biol. Chem. 293, 4860–4869 (2018). source
  • Findingown data

    Only T4 was produced in detectable amounts in the in vitro TH production assay.

    “Only T4 was produced in detectable amounts in our in vitro TH production assay.”
    Quote in the paperNot a substantial claim
  • Classified as probably not a substantial claim. 21
  • May combine two claims. 39

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    With the ELISA, modest T4 synthesis could also be obtained from random tyrosine copolymers, as previously reported.

    “Finally, with our ELISA we could also obtain modest T4 synthesis from random tyrosine copolymers, as previously reported4 (Extended Data Figure 6e).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [4] Cahnmann, H. J., Pommier, J. & Nunez, J. Spatial requirement for coupling of iodotyrosine residues to form thyroid hormones. Proc. Natl Acad. Sci. USA 74, 5333–5335 (1977). source
    • Rests on an assumption: The in vitro LPO/TPO iodination assay reproduces the hormonogenic reaction of TG in vivo.

  • May combine two claims.

    Premisecitation

    TG is a protein dimer of 600 kDa with an unusually high number of ~60 disulfide bonds per monomer and 17 glycosylation sites, which confer remarkable stability and solubility to the protein.

    “TG, a protein dimer of 600 kDa, has an unusually high number of ~ 60 disulfide bonds per monomer and 17 glycosylation sites, which confer remarkable stability and solubility to the protein1,2,9.”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [1] Di Jeso, B. & Arvan, P. Thyroglobulin from molecular and cellular biology to clinical endocrinology. Endocr. Rev. 37, 2–36 (2016). source

      The Introduction reports about 60 disulfide bonds per Tg monomer, and section V identifies Tg as a homodimer, but gives its mass as 660 kDa rather than 600 kDa. The glycan-dependent oxidoreductase section reports 10–15 N-linked oligosaccharides per monomer, not 17; section VI says glycans generally increase protein solubility, but does not establish that the claimed structural features confer remarkable stability and solubility on Tg. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • cites [2] Citterio, C. E., Targovnik, H. M. & Arvan, P. The role of thyroglobulin in thyroid hormonogenesis. Nat. Rev. Endocrinol. 15, 323–338 (2019). source

      A Results passage in the indexed copy describes TG as a homodimeric glycoprotein, supporting the dimer description. The passages do not give a 600 kDa mass, approximately 60 disulfide bonds per monomer, 17 glycosylation sites, or their proposed effect on stability and solubility. The indexed text also appears to contain a different article from the cited review, so those details cannot be verified against it. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [9] Heidelberger, M. The molecular weight of thyroglobulin. Science 80, 414 (1934). source

  • Classified as probably not a substantial claim.

    Premisecitation

    Hormonogenic tyrosines in thyroglobulin have not been clearly identified.

    “hormonogenic tyrosines have not been clearly identified and the lack of a three-dimensional structure of TG has prevented mechanistic understanding4.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [4] Cahnmann, H. J., Pommier, J. & Nunez, J. Spatial requirement for coupling of iodotyrosine residues to form thyroid hormones. Proc. Natl Acad. Sci. USA 74, 5333–5335 (1977). source

      The introductory passage (page not supplied) says thyroglobulin’s primary sequence was unknown and that its complexity prevented study of factors governing coupling. This is consistent with hormonogenic tyrosines not being clearly identified, but the paper does not specifically attribute the mechanistic limitation to a lack of three-dimensional structure. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The molecular events behind thyroid hormone synthesis are yet to be completely understood.

    “Approximately 5% of the world's human population suffer from thyroid diseases, but the molecular events behind TH synthesis are yet to be completely understood6.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [6] Taylor, P. N. et al. Global epidemiology of hyperthyroidism and hypothyroidism. Nat. Rev. Endocrinol. 14, 301–316 (2018). source

      The Introduction discusses thyroid dysfunction and hormone measurement, but not whether the molecular events of thyroid hormone synthesis are fully understood. A search for synthesis found a reference to a drug inhibiting synthesis, not a statement about gaps in understanding; searches for biosynthesis and molecular mechanisms likewise did not establish the claim. The indexed passages provide no page numbers. (LLM, conf=0.74)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    When all acceptors (Y24, Y2573, Y2766 and Y1310) were mutated, no T4 synthesis was observed, demonstrating that there are no more than four significant hormonogenic sites in TG.

    “As shown in Figure 2c, when we mutated all acceptors (Y24, Y2573, Y2766 and Y1310)21,13 we observed no T4 synthesis, demonstrating that there are no more than four significant hormonogenic sites in TG.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [21] Lamas, L., Anderson, P. C., Foxy, J. W. & Dunn, J. T. Consensus sequences for early iodination and hormonogenesis in human thyroglobulin. J. Biol. Chem. 264, 13541–13545 (1989). source
    • cites [13] Dedieu, A., Gaillard, J.-C., Pourcher, T., Darrouzet, E. & Armengaud, J. Revisiting iodination sites in thyroglobulin with an organ-oriented shotgun strategy. J. Biol. Chem. 286, 259–269 (2011). source
    • Rests on an assumption: Mutating a tyrosine to phenylalanine completely abolishes its ability to act as a hormonogenic donor or acceptor.
    • Rests on an assumption: The in vitro LPO/TPO iodination assay reproduces the hormonogenic reaction of TG in vivo.

  • May combine two claims.

    Findingown data

    Most of TG's disulfide bonds show clear EM density in the maps, and no inter-subunit disulfide bonds were found.

    “Most of TG's disulfide bonds show clear EM density in our maps (Extended Data Figure 5a) and we found no inter-subunit disulfide bonds, in agreement with previous observations that TG is a non-covalent dimer19.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [19] Citterio, C. E., Morishita, Y., Dakka, N., Veluswamy, B. & Arvan, P. Relationship between the dimerization of thyroglobulin and its ability to form triiodothyronine. J. Biol. Chem. 293, 4860–4869 (2018). source

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Mass spectrometry analyses of thyroid-extracted TG have identified four or more acceptor tyrosines, but the position of the donors has remained unclear.

    “Mass spectrometry analyses of thyroid-extracted TG have identified four or more acceptor tyrosines, but the position of the donors has remained unclear13.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [13] Dedieu, A., Gaillard, J.-C., Pourcher, T., Darrouzet, E. & Armengaud, J. Revisiting iodination sites in thyroglobulin with an organ-oriented shotgun strategy. J. Biol. Chem. 286, 259–269 (2011). source

      The Identification of Iodination Sites section identifies four distinct T3 or T4 acceptor positions: Tyr 25, 993, 1310, and 2572. But the Fate of the Lost Side Chain section identifies two donor positions, Tyr 259 and Tyr 2539, so the claim that donor positions remained unclear does not describe this study's findings. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Resolved Sites A, B and D do not show obvious structural similarity, and active tyrosines are in solvent accessible and flexible regions.

    “Resolved Sites A, B and D do not show obvious structural similarity and active tyrosines are in solvent accessible and flexible regions (Figure 2a, Extended Data Figure 7a,c).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    All acceptor mutants with one hormonogenic site active at a time contribute to hormone formation, and the sum of the concentrations measured individually recapitulates the total T4 amount produced by unmodified rTG.

    “Furthermore, we showed that all acceptor mutants with one hormonogenic site active at a time (Figure 3a) contribute to hormone formation and the sum of the concentrations measured individually recapitulates the total T4 amount produced by unmodified rTG.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The four TG hormonogenic sites contribute independently to total T4 production, so their individual activities can be summed.
    • Rests on an assumption: The in vitro LPO/TPO iodination assay reproduces the hormonogenic reaction of TG in vivo.

  • May combine two claims.

    Findingown data

    When all five proposed donors were mutated, no significant T4 formation could be detected, confirming that Site A indeed has two donor tyrosines: Y234 and Y149.

    “When all five proposed donors were mutated, no significant T4 formation could be detected, confirming that Site A indeed has two donor tyrosines: Y234 and Y149.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Mutating a tyrosine to phenylalanine completely abolishes its ability to act as a hormonogenic donor or acceptor.
    • Rests on an assumption: The in vitro LPO/TPO iodination assay reproduces the hormonogenic reaction of TG in vivo.

  • May combine two claims.

    Findingown data

    In the TG monomer, the globular NTD is connected to the Core region via a linker (residues 610-620), crossing the central dimer interface, and it is partially flexible.

    “In the TG monomer, the globular NTD is connected to the Core region via a linker (residues 610-620), crossing the central dimer interface and it is partially flexible (Extended Data Figure 2f).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingnone given

    The Core contains two triplets of type-1 repeats (domain H with a very large insertion), separated by domain I, located near the C2 axis.

    “The Core contains two triplets of type-1 repeats (domain H with a very large insertion), separated by domain I, located near the C2 axis.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Lysozyme, SaFtsZ, and T3 produce no T4 signal.

    “Lysozyme (some tyrosines), SaFtsZ (no tyrosines) and T3 produce no T4 signal.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Using the reaction conditions, only significant production of T4, but not T3, could be detected.

    “Using our reaction conditions, we could only detect significant production of T4, but not of T3 (Extended Data Figure 6f-g).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The in vitro LPO/TPO iodination assay reproduces the hormonogenic reaction of TG in vivo.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The heme occupancy of TPO was estimated by measuring the ratio of absorbance at 412 nm and 280 nm, which was 0.2, roughly equating to 20% occupancy.

    “The heme occupancy of TPO was estimated by measuring the ratio of absorbance at 412 nm and 280 nm which was 0.2, which roughly equates to 20% occupancy30.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [30] Guo, J., McLachlan, S. M., Hutchison, S. & Rapoport, B. The greater glycan content of recombinant human thyroid peroxidase of mammalian than of insect cell origin facilitates purification to homogeneity of enzymatically protein remaining soluble at high concentration. Endocrinology 139, 999–1005 (1998). source

  • May combine two claims.

    Findingnone given

    In Sites A, B and D the donor backbone conformation is constrained within secondary structure elements, whereas the acceptor is in more flexible regions.

    “Because in Sites A, B and D the donor backbone conformation is constrained within secondary structure elements, whereas the acceptor is in more flexible regions, the acidic Asp or Glu residues (pointing towards the solvent) could play a role in orienting the following acceptor towards the more rigid donor.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    The Flap extends along the minor axis of the molecule with the M domain protruding at the opposite side of the NTD and folding back onto the Arm region.

    “The Flap extends along the minor axis of the molecule with the M domain protruding at the opposite side of the NTD and folding back onto the Arm region.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The central regions of the maps, corresponding to the C-terminal ChEL domains, were well defined (~3 Å in resolution), whereas peripheral regions corresponding to the Arms were noisy and at lower resolutions (~6 Å).

    “The central regions of the maps, corresponding to the C-terminal ChEL domains, were well defined (~3 Å in resolution), whereas peripheral regions corresponding to the Arms were noisy and at lower resolutions (~ 6 Å).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Twelve previously predicted N-linked GlcNac were resolved in the maps, and four that had not been identified were found at N110, N484, N1869 and N2122.

    “We resolved 12 previously predicted N-linked GlcNac in our maps, and four that had not been identified, at N110, N484, N1869 and N212220 (Extended Data Table 3).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [20] Yang, S.-X., Pollock, H. G. & Rawitch, A. B. Glycosylation in human thyroglobulin: location of the N-linked oligosaccharide units and comparison with bovine thyroglobulin. Arch. Biochem. Biophys. 327, 61–70 (1996). source

  • May combine two claims.

    Findingown data

    Within the overall TG structure, Site A is located in the NTD, Site B in the CTD, Site C is at the C-terminus across the C2 dimer interface and Site D bridges the NTD and Flap (M domain) of the other subunit in the dimer.

    “Within the overall TG structure, Site A is located in the NTD, Site B in the CTD, Site C is at the C-terminus across the C2 dimer interface and Site D bridges the NTD and Flap (M domain) of the other subunit in the dimer (Figure 2b).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingown data

    Five TG regions were defined: NTD, Core, Flap, Arm and CTD.

    “Following the domain arrangement in the context of TG's 3D structure, we defined five TG regions: NTD (N-terminal domain), Core, Flap, Arm and CTD (C-terminal domain), as indicated in Figures 1a-b.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    Endogenous TG from human thyroid glands contains some T4, as measured by the T4-ELISA, but not T3.

    “We found it contains some T4, as measured by using our T4-ELISA, but not T3 (Extended Data Figure 6).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The in vitro LPO/TPO iodination assay reproduces the hormonogenic reaction of TG in vivo.

  • May combine two claims.

    Findingreasoning

    The resolution of the cryo-EM map is non-uniform, and consequently the model has variable quality depending on the map region.

    “The resolution of the map is non-uniform and consequently the model has variable quality depending on the map region.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In cryo-EM, TG has a bilobed shape with average dimensions of 120 x 235 Å.

    “In cryo-EM, TG has a bilobed shape with average dimensions of 120 x 235 Å, which correlate well with previously reported negative staining EM data15.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [15] Berg, G., Björkman, U. & Ekholm, R. The structure of newly synthesized intracellular thyroglobulin molecules. Mol. Cell. Endocrinol. 20, 87–98 (1980). source

  • May combine two claims.

    Findingreasoning

    There is no preceding acidic residue in the C-terminal Site C, where both acceptor and donor (Y2766 from the two chains of the dimer) are in unstructured regions with higher intrinsic flexibility.

    “There is no preceding acidic residue in the C-terminal Site C, where both acceptor and donor (Y2766 from the two chains of the dimer) are in unstructured regions with higher intrinsic flexibility.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Overall, TG's structure appears entangled and revolves around the central ChEL dimer that interacts with different regions of the Arm and the Core of the same chain and, via the E domain, with the NTD of the other chain.

    “Overall, TG's structure appears entangled and revolves around the central ChEL dimer that interacts with different regions of the Arm and the Core of the same chain and, via the E domain, with the NTD of the other chain.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The Core is connected to the Flap region, which is composed of two Ig-like domains, M and N.

    “The Core is then connected to the Flap region, which is composed of two Ig-like domains, M and N.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Endogenous TG is partially degraded, as shown by SDS-PAGE.

    “It is also partially degraded, as shown by SDS-PAGE (Extended Data Figure 2a).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    By mutagenesis of Site D, K1415 is not relevant, but D1309 is essential for hormonogenesis.

    “By mutagenesis of Site D, we showed that K1415 is not relevant, but D1309 is essential for hormonogenesis (Extended Data Figure 6d).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Mutating a tyrosine to phenylalanine completely abolishes its ability to act as a hormonogenic donor or acceptor.
    • Rests on an assumption: The in vitro LPO/TPO iodination assay reproduces the hormonogenic reaction of TG in vivo.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    LPO is as active as TPO, taking the reduced 20% heme content in our TPO into account.

    “LPO is as active as TPO, taking the reduced 20% heme content in our TPO into account.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The best final maps used for model building were the C2 map from the eTG dataset (overall 3.39 Å resolution) and the expanded map from the rTG dataset (overall 3.67 Å resolution).

    “The best final maps we used for model building were the C2 map from the eTG dataset (overall 3.39 Å resolution) and the expanded map from the rTG dataset (overall 3.67 Å resolution).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The hormonogenic sites show conformational disorder, making precise side chain placements difficult, but the backbone positions are resolved in the rTG map.

    “showing conformational disorder within the hormonogenic sites, making precise side chain placements difficult, but the backbone positions are resolved (rTG map).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    The T4 ELISA calibration curves under modified conditions were similar to the manufacturer's curves and showed good dynamic range.

    “This yielded calibration curves similar to the ones provided by the manufacturer, and also showed good dynamic range (Extended Data Figure 6).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The glycans linked to N2013 mediate the contact between the NTD and CTD and might contribute to the dimer stability.

    “Notably, the glycans linked to N2013 mediate the contact between the NTD and CTD and might contribute to the dimer stability (Extended Data Figure 5b & f, Supplementary Video 1).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationreasoning

    The C-ins1 variant, producing a putative tyrosine pair with Y341, also showed low activity, possibly because the added linker was designed to be too short for the two tyrosines to come into proximity.

    “The C-ins1 variant, producing a putative tyrosine pair with Y341, also showed low activity, possibly because the added linker was designed to be too short for the two tyrosines to come into proximity.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The in vitro LPO/TPO iodination assay reproduces the hormonogenic reaction of TG in vivo.

  • May combine two claims.

    Interpretationreasoning

    TG likely contains four hormonogenic acceptor tyrosines and five donor tyrosines.

    “Therefore, TG likely contains four hormonogenic acceptor tyrosines and five donor tyrosines.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    The involvement of glycans in TG dimer formation as revealed by the structure might explain the poor expression yields of thyroglobulin when produced in expression systems with reduced or altered glycosylation.

    “We note that the involvement of glycans in TG dimer formation as revealed by the structure might explain the poor expression yields of thyroglobulin when produced in expression systems with reduced/altered glycosylation, such as insect cells, HEK293S GnT1-/- cells or HEK293T cells in the presence of kifunensine (data not shown).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    The solvent exposure of tyrosines is presumably compensated by the exceptional solubility and stability that allows TG to persist at high concentrations in the harsh environment of the colloid.

    “The solvent exposure of tyrosines is presumably compensated by the exceptional solubility and stability that allows TG to persist at high concentrations in the harsh environment of the colloid.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationcitation

    The complexity of the TG molecule might fulfil further important roles in endocytosis, regulation of the T3/T4 ratio, TG proteolytic processing and in the trafficking to lysosomes.

    “Moreover, the complexity of the TG molecule might fulfil further important roles in endocytosis, regulation of the T3/T4 ratio, TG proteolytic processing and in the trafficking to lysosomes25,26.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [25] Botta, R. et al. Sortilin is a putative postendocytic receptor of thyroglobulin. Endocrinology 150, 509–518 (2009). source
    • cites [26] Weber, J. et al. Interdependence of thyroglobulin processing and thyroid hormone export in the mouse thyroid gland. Eur. J. Cell Biol. 96, 440–456 (2017). source

  • May combine two claims.

    Study design and conductown data

    Symmetry expansion and re-centring improved the local resolution and continuity of the density in the Arm region.

    “Although the overall resolution of the expanded and re-centred map was very similar to the C2 counterpart, the local resolution and continuity of the density in the Arm region was significantly improved (Extended Data Figure 2e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    TG synthesises seven molecules of T4 per dimer, since each TG dimer contains two sites (one per monomer) of A, B and D and only one Site C.

    “We conclude that TG synthesises seven molecules of T4 per dimer, since each TG dimer contains two sites (one per monomer) of A, B and D and only one Site C.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The four TG hormonogenic sites contribute independently to total T4 production, so their individual activities can be summed.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The final model has a Molprobity score of 1.85 (83rd percentile).

    “Molprobity score (percentile) | 1.85 (83rd)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationnone given

    Nature has selected the complex and large scaffold of the TG dimer to synthesise only seven molecules of hormones, using a chemical reaction involving radicals that could be carried out by unfolded peptides or other less complex proteins.

    “Nature has selected the complex and large scaffold of the TG dimer to synthesise only seven molecules of hormones, using a chemical reaction involving radicals that could be carried out by unfolded peptides or other less complex proteins.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Four features seem to be crucial for hormonogenesis: tyrosine pairs must be solvent exposed to be iodinated, in proximity and in roughly antiparallel orientation, and in highly mobile regions of the protein in order to allow the significant bond rearrangement resulting in T4 synthesis.

    “Although the reaction mechanism of T4 synthesis from polypeptide chains remains to be established in more detail, four features seem to be crucial for hormonogenesis: tyrosine pairs must be solvent exposed to be iodinated, in proximity and in roughly antiparallel orientation, and in highly mobile regions of the protein in order to allow the significant bond rearrangement resulting in T4 synthesis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The basal T4 activity of unmodified MBP can be attributed to the Y171/Y176 pair since it could be suppressed by introducing a Y171A mutation.

    “We attribute this basal activity to the Y171/Y176 pair since it could be suppressed by introducing a Y171A mutation.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The in vitro LPO/TPO iodination assay reproduces the hormonogenic reaction of TG in vivo.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The correct assignment of the polypeptide chain register was often helped by the presence of large aromatic side chains and disulfide bond pairs, as well as glycosylation sites at Asn residues.

    “The correct assignment of the polypeptide chain register was often helped by the presence of large aromatic side chains and disulfide bond pairs, as well as the presence of glycosylation sites at Asn residues”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    eTG presumably contains already reacted tyrosine side chains.

    “eTG presumably contains already reacted tyrosine side chains.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Residues 24 to 29 were built tentatively into a lower-resolution filtered map, and their coordinates are only indicative and included as poly-alanine.

    “Residues 24 (acceptor Site A) to 29 were built tentatively into a map filtered to lower resolution using MAIN score map density modification procedure39, however their coordinates are only indicative and hence are included in the model as poly-alanine”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The final model has 0% Ramachandran disallowed.

    “Disallowed (%) | 0”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Hormone synthesis from thyroglobulin occurs in the thyroid gland via the iodination and coupling of pairs of tyrosines and is completed by TG proteolysis.

    “Hormone synthesis from thyroglobulin (TG) occurs in the thyroid gland via the iodination and coupling of pairs of tyrosines and is completed by TG proteolysis3.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [3] Carvalho, D. P. & Dupuy, C. Thyroid hormone biosynthesis and release. Mol. Cell. Endocrinol. 458, 6–15 (2017). source

  • May combine two claims.

    Interpretationown data

    The low level of T4 production can be attributed to a lack of flexibility of the backbone near Y171 and Y176, and to their relative orientations, which is different to any of the sites in TG.

    “The low level of T4 production can be attributed to a lack of flexibility of the backbone near Y171 and Y176, and to their relative orientations, which is different to any of the sites in TG.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The final model has a clashscore of 4.88.

    “Clashscore | 4.88”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    A conserved lysine is found in proximity of the donor, and a conserved acidic residue (Glu or Asp) always precedes the acceptor.

    “A conserved lysine is found in proximity of the donor, and a conserved acidic residue (Glu or Asp) always precedes the acceptor 21.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [21] Lamas, L., Anderson, P. C., Foxy, J. W. & Dunn, J. T. Consensus sequences for early iodination and hormonogenesis in human thyroglobulin. J. Biol. Chem. 264, 13541–13545 (1989). source

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Identified crosslinked peptide candidates were filtered to a 2% FDR on residue-pair level using XiFDR.

    “Identified crosslinked peptide candidates were filtered to an FDR of 2% on residue-pair-level using XiFDR47.”
    Quote in the paperSubstantial claim
54 of 54

  • The paper relies on this without support.

    Mutating a tyrosine to phenylalanine completely abolishes its ability to act as a hormonogenic donor or acceptor.

    “rTG variants where acceptor or donor tyrosines were mutated to phenylalanines, abolishing hormone formation”

  • The paper relies on this without support.

    The four TG hormonogenic sites contribute independently to total T4 production, so their individual activities can be summed.

    “the sum of the concentrations measured individually recapitulates the total T4 amount produced by unmodified rTG”

  • The paper relies on this without support.

    The in vitro LPO/TPO iodination assay reproduces the hormonogenic reaction of TG in vivo.

    “TH synthesis was performed by rTG in vitro iodination, and the T4 or T3 concentrations were measured by adapting a commercial ELISA assay”

  • The paper relies on this without support.

    TG has 3-4 hormonogenic sites.

    “Considering that TG has 3-4 sites, 0.1 μM was determined to be the optimal starting concentration for the assay.”
4 of 4

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Recombinant TG frame dose does not match the reported total

    Minor

    The stated 52 frames at 0.91 electrons per Å2 per frame give 47.32 electrons per Å2, not 36.3. Extended Data Table 1 lists a third value, 40, for the same dataset.

    Reported 36.3 · recomputed 47.32

  • Endogenous TG electron exposure differs between methods and table

    Minor

    The methods report a total accumulated dose of 47.2 electrons per Å2 for eTG, whereas Extended Data Table 1 reports 45.

2 of 2

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Authors
Coscia, F., Taler-Verčič, A., Chang, V. T., Sinn, L., O’Reilly, F. J., Izoré, T., Renko, M., Berger, I., Rappsilber, J., Turk, D., & Löwe, J.
Affiliations
  • MRC Laboratory of Molecular Biology (GB)
  • Technische Universität Berlin (DE)
  • Jožef Stefan Institute (SI)
  • University of Bristol (GB)
  • Wellcome Centre for Cell Biology (GB)
Corresponding author
  • Turk, Dušan
  • Löwe, Jan
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2020-02-05
Volume
578
Issue
7796
Pages
627-630
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/s41586-020-1995-4
PMC
PMC7170718
OpenAlex
W3004797639
Semantic Scholar
ddcfe1716cc392aaecc3f5bf1709ab4f690dbaa0
Cited by
147
References
49
Citations by year
2026: 21 · 2025: 14 · 2024: 32 · 2023: 29 · 2022: 22 · 2021: 22 · 2020: 7
FWCI
15.01
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature (publishedVersion)
  • PubMed (publishedVersion)
  • Edinburgh Research Explorer (University of Edinburgh) (publishedVersion, other-oa)
  • Bristol Research (University of Bristol) (publishedVersion, other-oa)
  • PubMed Central (acceptedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • Explore Bristol Research (submittedVersion, other-oa)
Funders
Wellcome Trust (103139, 103139/Z/13/Z, 202754/Z/16/Z, 202754, 203149)
Topics
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Thyroid Disorders and Treatments
  • Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
Keywords
thyroglobulin, thyroid hormones, hormone biosynthesis, protein structure, solvent exposure, maltose-binding protein, site-directed mutagenesis
MeSH terms
Bacterial Proteins, Humans, Models, Molecular, Mutation, Solvents, Thyroglobulin, Thyroid Hormones, Tyrosine, Reproducibility of Results, Cryoelectron Microscopy, HEK293 Cells, Maltose-Binding Proteins

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-03.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-03.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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