Cryo-EM structure of native human thyroglobulin

Abstract

The thyroglobulin (TG) protein is essential to thyroid hormone synthesis, plays a vital role in the regulation of metabolism, development and growth and serves as intraglandular iodine storage. Its architecture is conserved among vertebrates. Synthesis of triiodothyronine (T₃) and thyroxine (T₄) hormones depends on the conformation, iodination and post-translational modification of TG. Although structural information is available on recombinant and deglycosylated endogenous human thyroglobulin (hTG) from patients with goiters, the structure of native, fully glycosylated hTG remained unknown. Here, we present the cryo-electron microscopy structure of native and fully glycosylated hTG from healthy thyroid glands to 3.2 Å resolution. The structure provides detailed information on hormonogenic and glycosylation sites. We employ liquid chromatography-mass spectrometry (LC-MS) to validate these findings as well as other post-translational modifications and proteolytic cleavage sites. Our results offer insights into thyroid hormonogenesis of native hTG and provide a fundamental understanding of clinically relevant mutations.

Fig. 1. The cryo-EM map of hTG at 3.2 Å.

(Right) Density map of hTG with one monomer in white and corresponding atomic model (left). Map and model are colored as in the bottom linear diagram of the primary structure of hTG.

Fig. 2. Disposition of the different type 1 repeats in the hTG structure.

Surface representation of one hTG monomer in the background-colored charcoal. Ribbon representation of the second hTG monomer in the foreground with each type 1 repeat colored as in the scheme on the right: non-type 1 repeats are colored in light gray.

Fig. 3. Location of type 1 repeat NHIs and proteolysis clusters.

(Top) Surface representation of one hTG monomer (in the background) colored charcoal. Ribbon representation of the second hTG monomer in the foreground with each NHI colored differently: orange—repeat 1.3 NHIs; purple—repeat 1.5 NHI; blue—repeat 1.8 NHI; wine—repeat 1.7 NHI. Detailed clipped views of each insertion are displayed in the dashed boxes; box B view direction is the same as the top left image while boxes A and C were reoriented for better depiction. (Bottom) Histogram of the non-tryptic cleavage sites detected by MS with major sectors depicting the approximate position of previously reported (yellow, see text for references) and herein experimentally determined (gray) cleavage clusters.

Fig. 4. Type 2 repeats.

(Top) Representation and location of the type 2 repeats as pink, light, and dark purple ribbon within the hTG structure and independent repeat structures. (Bottom) Ribbon representation of the type 2 repeats as disposed in the context of the hTG structure. Cysteine residues are represented in sticks with DSBs and sulfur atoms colored yellow.

Fig. 5. Type 3 repeats.

(Top) Surface representation of the hTG dimer with type 3 repeats colored as represented in the bottom panel; the other individual hTG structures within a monomer are highlighted charcoal and light gray. (Bottom) Ribbon representation of the aligned type 3 repeats.

Fig. 6. Location of hormonogenic sites in hTG structure.

(Top left) hTG surface representation with one monomer in charcoal and one monomer in white. Acceptor tyrosine residues, Y2573 and Y1310 in ~15 Å proximity at each canonical site (A to D) are marked in red, for which at Y2573, additionally MIT and DIT were detected in MS experiments. The insets show sites B and D in detail with the respective cryo-EM density and its corresponding threshold level to aid confidence assessment.

Fig. 7. Location of nonsense and missense mutations in hTG responsible for congenital hypothyroidism.

(Top) hTG linear diagram depicting the location of nonsense mutations in black squares and missense mutations in pink circles; domain color code is the same as Fig. 1. Dashed boxes represent the 5 mutation clusters (MC1 to MC5) with the highest density of mutations. Bar diagram visualizing the number of mutations per 50 residues. (Bottom) Location of the same mutations in the ribbon representation of hTG; nonsense mutations in black spheres and missense mutations in pink spheres.