12-Feb-2021

Francesca Coscia (MRC Laboratory of Molecular Biology, Cambridge, UK)

“The structure of human thyroglobulin”

Thyroglobulin is the protein precursor of thyroid hormones, which are essential for growth, development and control of metabolism in vertebrates. Hormone synthesis from thy-roglobulin (TG) occurs in the thyroid gland via the iodination and coupling of pairs of tyrosines and is completed by TG proteolysis. 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 understand-ing. Here we present the de novostructure of full-length human thyroglobulin at ~3.5 Å resolution determined by electron cryomicroscopy (cryo-EM). To obtain images amenable to high-resolution analysis of heterogeneous and diluted TG extracted from thyroid glands, we combined deglycosylation and the use of graphene-oxide supported EM grids. Moreo-ver, to address the anisotropic resolution of theTG flexible dimer we used symmetry ex-pansion and focused refinement approaches. From TG structure we identified all hormon-ogenic tyrosine pairs in the structure and verified them via site-directed mutagenesis and in vitro hormone production assays using human TG expressed in HEK cells. Analysis revealed that proximity, flexibility and solvent exposure of the tyrosines are the key char-acteristics of hormonogenic sites. To support the validity of our insights, we transferred the reaction site properties from TG to an engineered tyrosine donor-acceptor pair in the unre-lated bacterial maltose binding protein (MBP). Strikingly, this yielded hormone production with efficiency comparable to TG. This structure provides an essential framework to fur-ther understand the production and regulation of thyroid hormones.