Cryo-EM: A new dawn in thyroid biology

Abstract

The thyroid gland accumulates the rare dietary element iodine and incorporates it into iodinated thyroid hormones, utilising several tightly regulated reactions and molecular mechanisms. Thyroid hormones are essential in vertebrates and play a central role in many biological processes, such as development, thermogenesis and growth. The control of these functions is exerted through the binding of hormones to nuclear thyroid hormone receptors that rule the transcription of numerous metabolic genes. Over the last 50 years, thyroid biology has been studied extensively at the cellular and organismal levels, revealing its multiple clinical implications, yet, a complete molecular understanding is still lacking. This includes the atomic structures of crucial pathway components that would be needed to elucidate molecular mechanisms. Here we review the currently known protein structures involved in thyroid hormone synthesis, regulation, transport, and actions. We also highlight targets for future investigations that will significantly benefit from recent advances in macromolecular structure determination by electron cryo-microscopy (cryo-EM). As an example, we demonstrate how cryo-EM was crucial to obtain the structure of the large thyroid hormone precursor protein, thyroglobulin. We discuss modern cryo-EM compared to other structure determination methods and how an integrated structural and cell biological approach will help filling the molecular knowledge gap in our understanding of thyroid hormone metabolism. Together with clinical, cellular and high-throughput 'omics' studies, atomic structures of thyroid components will provide an important framework to map disease mutations and to interpret and predict thyroid phenotypes.

Keywords: Cryo-EM; Electron microscopy; Iodine; Structural biology; Thyroglobulin; Thyroid hormones.

Fig. 1

Schematic overview of the thyroid hormone cycle. a) Thyroid-pituitary axis and general thyroid hormone pathway in vertebrates. TSH: thyroid-stimulating hormone, TSHR: TSH receptor, TG: thyroglobulin, T4: 3,5,3′,5′-tetraiodo-L-thyronine, T3: 3,5,3′-triiodo-L-thyronine, THR: thyroid hormone receptors, rT3: 3,3′,5′-triiodo-L-thyronine, T2: 3,3′-diiodo-L-thyronine, TAM: thyronamine derivatives. b) Molecular structure of the thyroid hormones (TH): T4, T3, rT3 and T2.

Fig. 2

Overview of the main proteins involved in the thyroid hormone pathway and their atomic structures. Proteins involved in the synthesis (a), transport (b) and regulation/action (c) of thyroid hormones (TH). Protein Data Bank (PDB) identifiers are provided in red. White squares indicate unresolved protein moieties. Question marks indicate poorly understood mechanisms or structures. Inset in a): Schematic representation of a thyroid follicle. TSHR: TSH receptor, NIS: sodium/iodide symporter, Pnd: pendrin, TG: thyroglobulin, DUOX: dual oxidases, TPO: thyroperoxidase, Dehal1: iodo-tyrosine deiodinase, MCT8: monocarboxylate transporter 8, TBG: thyroxine-binding globulin, TTR: transthyretin, DIO: deiodinase, THR: thyroid hormone receptors.

Fig. 3

Schematic of the single-particle cryo-EM methodology. Simplified overview of single-particle cryo-EM sample preparation (a) and transmission electron microscopy (TEM) image analysis, resulting in an atomic model (b). a) EM support samples are metal mesh grids covered with a thin film containing tiny holes of about 0.6–2 μm. An approximately 3 μL drop is applied to the grid, and excess liquid is blotted away to obtain a very thin layer (about 30 nm thick) of solution in the holes. The grid is then vitrified in liquid ethane and inserted into the transmission electron microscope for imaging. b) The resulting micrographs are 2D projections of the 3D objects in the thin vitrified layer. Single objects within the image representing macromolecules are selected (red boxes) and averaged first in two dimensions and then combined to reconstruct a 3D density map of the imaged molecular solution. Finally, this map is used to build and refine the atomic model of the molecule.