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Ricardo Núñez Miguel FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff, UK

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Paul Sanders FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff, UK

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Lloyd Allen FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff, UK

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Michele Evans FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff, UK

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Matthew Holly FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff, UK

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William Johnson FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff, UK

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Andrew Sullivan FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff, UK

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Jane Sanders FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff, UK

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Jadwiga Furmaniak FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff, UK

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Bernard Rees Smith FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff, UK

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Determination of the full-length thyroid-stimulating hormone receptor (TSHR) structure by cryo-electron microscopy (cryo-EM) is described. The TSHR complexed with human monoclonal TSHR autoantibody K1-70™ (a powerful inhibitor of TSH action) was detergent solubilised, purified to homogeneity and analysed by cryo-EM. The structure (global resolution 3.3 Å) is a monomer with all three domains visible: leucine-rich domain (LRD), hinge region (HR) and transmembrane domain (TMD). The TSHR extracellular domain (ECD, composed of the LRD and HR) is positioned on top of the TMD extracellular surface. Extensive interactions between the TMD and ECD are observed in the structure, and their analysis provides an explanation of the effects of various TSHR mutations on TSHR constitutive activity and on ligand-induced activation. K1-70™ is seen to be well clear of the lipid bilayer. However, superimposition of M22™ (a human monoclonal TSHR autoantibody which is a powerful stimulator of the TSHR) on the cryo-EM structure shows that it would clash with the bilayer unless the TSHR HR rotates upwards as part of the M22™ binding process. This rotation could have an important role in TSHR stimulation by M22™ and as such provides an explanation as to why K1-70™ blocks the binding of TSH and M22™ without activating the receptor itself.

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Ricardo Núñez Miguel FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Jane Sanders FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Paul Sanders FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Stuart Young FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Jill Clark FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Katarzyna Kabelis FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Jane Wilmot FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Michele Evans FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Emma Roberts FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Xiaoling Hu FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Jadwiga Furmaniak FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Bernard Rees Smith FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Binding of a new thyroid-stimulating human monoclonal autoantibody (MAb) K1–18 to the TSH receptor (TSHR) leucine-rich domain (LRD) was predicted using charge–charge interaction mapping based on unique complementarities between the TSHR in interactions with the thyroid-stimulating human MAb M22 or the thyroid-blocking human MAb K1–70. The interactions of K1–18 with the TSHR LRD were compared with the interactions in the crystal structures of the M22–TSHR LRD and K1–70–TSHR LRD complexes. Furthermore, the predicted position of K1–18 on the TSHR was validated by the effects of TSHR mutations on the stimulating activity of K1–18. A similar approach was adopted for predicting binding of a mouse thyroid-blocking MAb RSR-B2 to the TSHR. K1–18 is predicted to bind to the TSHR LRD in a similar way as TSH and M22. The binding analysis suggests that K1–18 light chain (LC) mimics binding of the TSH-α chain and the heavy chain (HC) mimics binding of the TSH-β chain. By contrast, M22 HC mimics the interactions of TSH-α while M22 LC mimics TSH-β in interactions with the TSHR. The observed interactions in the M22–TSHR LRD and K1–70–TSHR LRD complexes (crystal structures) with TSH–TSHR LRD (comparative model) and K1–18–TSHR LRD (predictive binding) suggest that K1–18 and M22 interactions with the receptor may reflect interaction of thyroid-stimulating autoantibodies in general. Furthermore, K1–70 and RSR-B2 interactions with the TSHR LRD may reflect binding of TSHR-blocking autoantibodies in general. Interactions involving the C-terminal part of the TSHR LRD may be important for receptor activation by autoantibodies.

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Paul Sanders FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Stuart Young FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Jane Sanders FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Katarzyna Kabelis FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Stuart Baker FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Andrew Sullivan FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Michele Evans FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Jill Clark FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Jane Wilmot FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Xiaoling Hu FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Emma Roberts FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Michael Powell FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Ricardo Núñez Miguel FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Jadwiga Furmaniak FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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Bernard Rees Smith FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK

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A complex of the TSH receptor extracellular domain (amino acids 22–260; TSHR260) bound to a blocking-type human monoclonal autoantibody (K1-70) was purified, crystallised and the structure solved at 1.9 Å resolution. K1-70 Fab binds to the concave surface of the TSHR leucine-rich domain (LRD) forming a large interface (2565 Å2) with an extensive network of ionic, polar and hydrophobic interactions. Mutation of TSHR or K1-70 residues showing strong interactions in the solved structure influenced the activity of K1-70, indicating that the binding detail observed in the complex reflects interactions of K1-70 with intact, functionally active TSHR. Unbound K1-70 Fab was prepared and crystallised to 2.22 Å resolution. Virtually no movement was observed in the atoms of K1-70 residues on the binding interface compared with unbound K1-70, consistent with ‘lock and key’ binding. The binding arrangements in the TSHR260–K1-70 Fab complex are similar to previously observed for the TSHR260–M22 Fab complex; however, K1-70 clasps the concave surface of the TSHR LRD in approximately the opposite orientation (rotated 155°) to M22. The blocking autoantibody K1-70 binds more N-terminally on the TSHR concave surface than either the stimulating autoantibody M22 or the hormone TSH, and this may reflect its different functional activity. The structure of TSHR260 in the TSHR260–K1-70 and TSHR260–M22 complexes show a root mean square deviation on all Cα atoms of only 0.51 Å. These high-resolution crystal structures provide a foundation for developing new strategies to understand and control TSHR activation and the autoimmune response to the TSHR.

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Jennifer Miller-Gallacher FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Paul Sanders FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Stuart Young FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Andrew Sullivan FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Stuart Baker FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Samuel C Reddington FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Matthew Clue FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Katarzyna Kabelis FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Jill Clark FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Jane Wilmot FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Daniel Thomas FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Monika Chlebowska FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Francesca Cole FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Emily Pearson FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Emma Roberts FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Matthew Holly FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Michele Evans FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Ricardo Núñez Miguel FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Michael Powell FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Jane Sanders FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Jadwiga Furmaniak FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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Bernard Rees Smith FIRS Laboratories, RSR Ltd, Cardiff, CF14 5DUUK

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The crystal structures of the thyroid-stimulating hormone receptor (TSHR) leucine-rich repeat domain (amino acids 22–260; TSHR260) in complex with a stimulating human monoclonal autoantibody (M22TM) and in complex with a blocking human autoantibody (K1-70™) have been solved. However, attempts to purify and crystallise free TSHR260, that is not bound to an autoantibody, have been unsuccessful due to the poor stability of free TSHR260. We now describe a TSHR260 mutant that has been stabilised by the introduction of six mutations (H63C, R112P, D143P, D151E, V169R and I253R) to form TSHR260-JMG55TM, which is approximately 900 times more thermostable than wild-type TSHR260. These six mutations did not affect the binding of human TSHR monoclonal autoantibodies or patient serum TSHR autoantibodies to the TSHR260. Furthermore, the response of full-length TSHR to stimulation by TSH or human TSHR monoclonal autoantibodies was not affected by the six mutations. Thermostable TSHR260-JMG55TM has been purified and crystallised without ligand and the structure solved at 2.83 Å resolution. This is the first reported structure of a glycoprotein hormone receptor crystallised without ligand. The unbound TSHR260-JMG55TM structure and the M22 and K1-70 bound TSHR260 structures are remarkably similar except for small changes in side chain conformations. This suggests that neither the mutations nor the binding of M22TM or K1-70TM change the rigid leucine-rich repeat domain structure of TSHR260. The solved TSHR260-JMG55TM structure provides a rationale as to why the six mutations have a thermostabilising effect and provides helpful guidelines for thermostabilisation strategies of other soluble protein domains.

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