Endocrine Abstracts

Objectives: The crystal structures of the TSH receptor (TSHR) leucine rich repeat domain (LRD) bound to TSHR stimulating monoclonal autoantibody M22^TM or to TSHR blocking monoclonal autoantibody K1-70^TM and antibody free have been solved previously. Cryo-electron microscopy (cryo-EM) was now used to solve the structure of full length TSHR in complex with K1-70^TM.

Methods: Recombinant human TSHR expressed in CHO cells was incubated with K1-70^TM Fab, the complex solubilised in 10mM Tris pH7.5, 50 mM NaCl, 0.5g/l NaN₃, 2% LMNG, 0,2% CHS and purified to homogeneity by affinity and size exclusion chromatography. Cryo-EM imaging was performed on a Titan Krios 300kV with a K3 Direct Electron Detector.

Results: The cryo-EM TSHR- K1-70™ structure was determined to aglobal resolution of 3.3Æ. A model was built using the solved crystal structure of the TSHR LRD- K1-70 complex and the AlfaFold model of the TSHR. Model rebuilding and refinement were done in COOT v0.9 and Discovery Studio 2021 suite of programs The cryo-EM structure shows full length TSHR in a monomeric state with all three domains; LRD, hinge region (HR) and transmembrane domain (TMD) visible. The binding arrangements of K1-70™ Fab with the LRD are similar to those observed in the crystal structure. The LRD and HR form the TSHR extracellular domain (ECD) in a similar arrangement to that seen in the crystal structure of the FSHR ECD and in the cryo-EM structure of the LH/CGR. The structure shows the TSHR ECD positioned on top of the extracellular surface of the TMD. The HR helix and the HR C-terminus form interactions with the TMD N terminus, extracellular loops 1 and 2 and the extracellular part of helix 7. The relative positioning of the ECD and TMD in the TSHR is similar to that seen in the cryo-EM structure of the LH/CGR inactive state. In particular the TSHR P10 region (amino acids 405-414), highly conserved in glycoprotein hormone receptors, is in a similar conformation to that seen in the LH/CGR inactive state structure. The structure and spacial arrangements of the TMD helices in the two cryo-EM structures are similar except that the TSHR extracellular end of helix 6 is displaced by approximately 6.5Æ compared to the LH/CGR inactive state structure.

Conclusions: Our high resolution structure of full length TSHR in complex with K1-70™ provides an excellent basis for understanding the mechanism of TSHR activation.