Endocrine Abstracts

Objectives: Thyroid peroxidase (TPO) is a key enzyme in the synthesis of thyroid hormones and is a target for autoimmune responses in autoimmune thyroid disease. TPO autoantibody (TPOAb) binding epitopes have been mapped on the peroxidase domain (POD) and the complement control protein like domain (CCP). This study aimed to solve the molecular structures of TPO bound to TPO antibodies.

Methods: An extracellular domain (ECD) of human TPO (amino acids; aa 1-839) was expressed in insect cells. Fabs were prepared from two TPO antibodies; a human monoclonal autoantibody 2G4 and a mouse monoclonal antibody 4F5. The structures of TPO-2G4 and TPO-4F5 complexes were determined by cryo-electron microscopy using a Titan Krios 300kV with a Falcon 3 Direct Detector.

Results: The structure of TPO-2G4 was solved at 3.92Æ and TPO-4F5 at 3.4Æ resolutions. The solved ECD structure comprises the POD, the CCP and an incomplete epidermal growth factor like domain (EGF). The POD Arg396 and Arg491 hold the haem group and form salt bridges with the two carboxylate groups of the haem. His494 acts as the proximal histidine interacting with the iron ion of the haem. The enzyme active site is located at the distal side of the haem and is lined by Gln235, Asp238, His239 and Glu399. A calcium ion is coordinated by Asp240, Thr321, Phe323, Asp325 and Ser327. 2G4 and 4F5 bind to TPO in different orientations. For both antibodies the binding epitopes are located exclusively on the POD. The binding interface for 2G4 is larger (2148Ʋ) than for 4F5 (1959Ʋ). 2G4 interacts with aa 194-277 and 604-628 whereas 4F5 with aa 461-659 with three common residues Glu604, Ala607 and Asp608 for both epitopes. In both complexes the antibody heavy chains make greater contributions to the interface than the light chains. 2G4 and 4F5 binding sites on the POD are distant from the CCP and EGF with no contacts between antibodies and the CCP or EGF. Any conformational movement of the CCP towards antibody epitopes on the POD would be prevented by a disulphide bond between POD Cys768 and CCP Cys 794.

Conclusions: Human TPO molecular structure has now been solved. This should be helpful in assessing the autoimmune responses to TPO in more detail and developing TPO enzyme inhibitors for therapeutic applications.