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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Jennifer Miller-Gallacher, Paul Sanders, Stuart Young, Andrew Sullivan, Stuart Baker, Samuel C Reddington, Matthew Clue, Katarzyna Kabelis, Jill Clark, Jane Wilmot, Daniel Thomas, Monika Chlebowska, Francesca Cole, Emily Pearson, Emma Roberts, Matthew Holly, Michele Evans, Ricardo Núñez Miguel, Michael Powell, Jane Sanders, Jadwiga Furmaniak, and Bernard Rees Smith
Paul Sanders, Stuart Young, Jane Sanders, Katarzyna Kabelis, Stuart Baker, Andrew Sullivan, Michele Evans, Jill Clark, Jane Wilmot, Xiaoling Hu, Emma Roberts, Michael Powell, Ricardo Núñez Miguel, Jadwiga Furmaniak, and Bernard Rees Smith
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.
Stuart Baker, Ricardo Núñez Miguel, Daniel Thomas, Michael Powell, Jadwiga Furmaniak, and Bernard Rees Smith
Determination of the structure of the extracellular domain of human thyroid peroxidase (hTPO) by cryo-electron microscopy (cryo-EM) is described. TPO, purified to homogeneity was complexed with the hTPO monoclonal autoantibody 2G4 Fab and also with a mouse monoclonal TPO antibody 4F5 Fab (which competes with autoantibody binding to TPO). Both complexes were analysed by cryo-EM. The two structures (global resolution 3.92 and 3.4 Å for the 2G4 complex and 4F5 complex, respectively) show TPO as a monomer with four domains; the N-terminal domain, the peroxidase domain (POD), the complement control protein (CCP)-like domain and the epidermal growth factor-like domain which are all visible in the structures. The relative positions of the domains are fixed with a disulphide bond between cysteine residues Cys146 in the POD and Cys756 in the CCP domain preventing significant flexibility of the molecule. The entrance to the enzyme active site, the haem group and the calcium binding site are clearly visible on the opposite side of the TPO molecule from the 2G4 and 4F5 binding sites. Extensive interactions are seen between TPO and the two antibodies which both bind to distinct epitopes on the POD domain, including some residues in the immunodominant region B mainly via different residues. However, the epitopes of the two antibodies contain three shared TPO residues. This is the first high-resolution structure of TPO to be reported and it should help guide the development of new inhibitors of TPO enzyme activity for therapeutic applications.