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. 2020 Feb;578(7796):627-630.
doi: 10.1038/s41586-020-1995-4. Epub 2020 Feb 5.

The structure of human thyroglobulin

Francesca Coscia  1 , Ajda Taler-Verčič  2   3 , Veronica T Chang  1 , Ludwig Sinn  4 , Francis J O'Reilly  4 , Thierry Izoré  1 , Miha Renko  2 , Imre Berger  5 , Juri Rappsilber  4   6 , Dušan Turk  7   8 , Jan Löwe  9
Affiliations

The structure of human thyroglobulin

Francesca Coscia et al. Nature. 2020 Feb.

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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Extended Data Figure 1
Extended Data Figure 1. The iodine cycle in the thyroid gland and the chemistry of thyroid hormone formation.
a) Iodide is extracted from the blood vessels and into the thyroid cells via the Na/I symporter (NIS). TSH binds TSH receptor (TSHR) to induce the expression of TG. TG is secreted into the extracellular lumen of follicular cells (colloid). DUOX and TPO catalyse the iodination of TG, therefore T4 (or T3) hormones are formed on the TG polypeptide chain. After hormonogenesis, TG is reimported and proteolysed in lysosomes to release T4/T3 into the blood. DEHAL1 deiodinates iodo-tyrosines to recycle iodide in thyroid cells. b) T4 (or T3) synthesis from thyroglobulin (TG) in the thyroid gland.
Extended Data Figure 2
Extended Data Figure 2. Cryo-EM reconstruction of endogenous and recombinant thyroglobulin (eTG, rTG).
a) SDS-PAGE of endogenous eTG from goitrous thyroid extracts and recombinant rTG expressed in HEK293T cells. b) Cryo-EM micrograph of eTG with calculated reference-free 2D class averages below. Scale bar 200 Å. c) Cryo-EM micrograph of recombinant rTG with 2D class averages, showing the two proteins to be structurally identical at this level of analysis. Scale bar 200 Å. d) Schematic illustrating the C2 symmetry-expansion and re-centring procedure, which was used to enhance TG map quality in peripheral regions. For a detailed procedure see the Methods section ‘Cryo-EM image processing’. e) Local resolution of the C2 and symmetry-expanded and re-centred eTG maps. f) Flexibility of N-terminal domain (NTD) resulting in varying map quality and occupancy of this region in a number of 3D class averages (calculated in RELION). g) Fourier shell correlation (FSC) between RELION 'gold standard' half-maps and between the final eTG and rTG maps, showing their strong similarity.
Extended Data Figure 3
Extended Data Figure 3. Local properties of the atomic TG model.
a) Per-residue atomic B-factor and cross correlation with the rTG map, plotted per residue number. b) Local B-factor colour-coded onto the surface of the TG structure. c) FSC between the map and model calculated for rTG. FSC 0.5 is indicated.
Extended Data Figure 4
Extended Data Figure 4. Validation of TG's three-dimensional architecture by MS crosslinking.
a) Size-exclusion chromatograms of rTG before and after BS3 crosslinking and subsequent SDS-PAGE (Coomassie stained). b) Negative staining micrograph of crosslinked rTG, showing the absence of higher-order structures caused by unwanted inter-dimer crosslinks. c) Plot representing experimental crosslinks (circles) overlapping with predicted crosslinks, calculated from the structure determined here. d) - f) Detail of key TG interfaces confirmed by the crosslinking.
Extended Data Figure 5
Extended Data Figure 5. TG cryo-EM map details.
a) All disulfide bonds in TG included in the model (yellow spheres). b) Glycans detected in the cryo-EM maps and included in the TG atomic model (green spheres). c) Close-up view of a typical alpha helix in the TG cryo-EM map (part of the Core region). d) Close-up of a beta sheet in TG (part of the ChEL domain). e) Close-up of the disulfide bond C900-C921 (Core region). f) Map details of N2013 and N-linked GlcNAc between two TG subunits. g) Close-up views showing conformational disorder within the hormonogenic sites, making precise side chain placements difficult, but the backbone positions are resolved (rTG map).
Extended Data Figure 6
Extended Data Figure 6. Quantitative TH ELISA assays.
a) Schematic summarising in vitro TH synthesis and quantification via ELISA assays. b) T4 assay calibration curves with added T4 under manufacturer-recommended and modified (T4 synthesis as performed here) conditions. c) Validation of the T4 ELISA assay. eTG presumably contains already reacted tyrosine side chains. rTG produces T4. Addition of iodide is required for the reaction to occur. LPO is as active as TPO, taking the reduced 20% heme content in our TPO into account. Lysozyme (some tyrosines), SaFtsZ (no tyrosines) and T3 produce no T4 signal. d) Mutating residues in hormonogenic Site D in a version of TG that is only active in Site D shows that a conserved lysine residue is not important for the reaction. Adding an extra Ser-Asp before Y1310 has no effect, but the mutation D1309S abolished activity. e) Synthesis of T4 from tyrosine copolymers as measured by the T4 ELISA assay. Only a polymer where tyrosines are spaced apart and preceded by Lys-Asp produce some T4. Note that activity is lower than in a single site of TG (or MBP, compare with Figure 4). f) T3 assay calibration curves with added T3 under recommended and modified (as for T4) conditions. g) No significant T3 production was detected from iodinated rTG or eTG from goiter.
Extended Data Figure 7
Extended Data Figure 7. Tyrosine pair proximity plots for TG and MBP.
a & b) Proximity plots of tyrosine residues closer than 15 Å to each other, calculated from TG a) and MBP b) atomic models (TG: this study; MBP PDB ID: 1ANF). The coordinates of each point in the plot represent a tyrosine pair position (residue number). For the TG dimer, the distance between tyrosines from the same or the other subunit in the dimer are shown in grey or black, respectively. In TG there are no more than five pairs that are exposed and in < 15 Å proximity at the same time, predicting the absence of other significant hormonogenic sites. In MBP only one pair closer than 15 Å is sufficiently exposed to be a candidate for hormonogenesis. c) & d) Ribbon diagram of TG and MBP where tyrosine residues are represented as spheres and coloured by B-factor, which largely indicates solvent exposed residues.
Figure 1
Figure 1. The structure of human TG by cryo-EM
a) Domain assignment of human thyroglobulin. Five regions (NTD, Core, Flap, Arm and CTD) contain domains of type-1 to type-3 TG repeats, as well as the choline esterase-like domain (ChEL), labelled as A to V. b) Structural gallery of all resolved TG domains. c) TG cryo-EM map where individual subunits are coloured blue and grey. The NTD crosses the major C2 interface. d) Ribbon diagram of panel c), coloured as in panel a). e) Schematic representation of TG structure in the same colour scheme as in a) and d).
Figure 2
Figure 2. Identification and validation of hormonogenic donor-acceptor tyrosine pairs in TG
a) Close-up view of the T4 hormonogenic sites resolved in the cryo-EM map, donor and acceptor tyrosines are highlighted in yellow. Site A suggests two donors, Y234 and Y149. b) Location of the four hormonogenic Sites A to D in the TG structure. c) T4 ELISA after in vitro iodination in triplicate. Bar plot and error bars indicate average and standard deviation. Replacing all acceptor tyrosines (Y) with phenylalanines (F) prevents hormone formation (4 Acc: 24, 2573, 2766, 1310). Replacing all five donors suppresses T4 synthesis (4 Donors (1): 2540, 2766, 108, 234; 4 Donors (2): 2540, 2766, 108, 149; 5 Donors: 2540, 2766, 108, 234, 149). Replacing other tyrosines at the surface (258, 704, 1467, 1782) has no effect (4eY).
Figure 3
Figure 3. Engineering T4 hormone synthesis by bacterial maltose binding protein (MBP)
a) rTG mutants with only one site active at a time. T4 release measured with the same T4 ELISA as in Figure 2c. The sum of T4 produced by the individual sites recapitulates the T4 produced by WT. b) T4 ELISA after in vitro iodination of engineered MBP to reconstruct TG's hormonogenic sites. See text and Supplementary Video 2 for details. An MBP version adding a flexible SGSDYS tail to the C-terminus shows activity comparable to a single site on TG as shown in panel a). All measurements done in triplicate. Bar plot and error bars indicate average and standard deviation.
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