Cryo-EM structure of native human thyroglobulin.
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ABSTRACT:
SUBMITTER: Adaixo R
PROVIDER: S-EPMC8748809 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The Tthyroglobulin (Tg) protein is the essential fundament toto thyroid hormone synthesis, a key player playing a vital role in the regulation of metabolism, development and growth and serving as intraglandular iodine storage. Its structure is conserved among vertebrates. Tg is delivered through the secretory pathway of the thyroid follicular unit to the central colloid depository, where it is iodinated at specific tyrosine sites to form mono- or diiodotyrosine, which combine to produce triiodothyronine (T3) and thyroxine (T4), respectively. Synthesis of these hormones depends on the precise 3D structure, iodination capacity and post-translational modification of Tg. The Tg structure is overall conserved among vertebrates and , which and structural information has long been remained unknown missing despite decades of research until recently structural information has been made available on human thyroglobulin (hTg) originating from recombinant hTg and endogenous hTg from patients with goitres. Here, we present the cryo-electron microscopy structure of native and fully functional human thyroglobulin (hTg) originating from healthy human thyroid glands, to a global resolution of 3.2 Å. We used LC-MS experiments to The structure provides detailed information on the location of the hTg hormonogenic sites by revealing native iodination, and reveals the position as well as the role of many of its glycosylation sites and other post-translational modifications. Our results offer structural insights into thyroid hormonogenesis of natively functional hTg and provide a fundamental understanding of clinically relevant hTg mutations, which can improve treatment of thyroid diseases.
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Project description:Imaging of rod photoreceptor outer-segment disc membranes by atomic force microscopy and cryo-electron tomography has revealed that the visual pigment rhodopsin, a prototypical class A G protein-coupled receptor (GPCR), can organize as rows of dimers. GPCR dimerization and oligomerization offer possibilities for allosteric regulation of GPCR activity, but the detailed structures and mechanism remain elusive. In this investigation, we made use of the high rhodopsin density in the native disc membranes and of a bifunctional cross-linker that preserves the native rhodopsin arrangement by covalently tethering rhodopsins via Lys residue side chains. We purified cross-linked rhodopsin dimers and reconstituted them into nanodiscs for cryo-EM analysis. We present cryo-EM structures of the cross-linked rhodopsin dimer as well as a rhodopsin dimer reconstituted into nanodiscs from purified monomers. We demonstrate the presence of a preferential 2-fold symmetrical dimerization interface mediated by transmembrane helix 1 and the cytoplasmic helix 8 of rhodopsin. We confirmed this dimer interface by double electron-electron resonance measurements of spin-labeled rhodopsin. We propose that this interface and the arrangement of two protomers is a prerequisite for the formation of the observed rows of dimers. We anticipate that the approach outlined here could be extended to other GPCRs or membrane receptors to better understand specific receptor dimerization mechanisms.
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