X-Ray Crystal Structure of a Monoclonal Antibody That Binds to a Major Autoantigenic Epitope on Thyroid Peroxidase

Abstract

Thyroid peroxidase (TPO) catalyzes the production of thyroid hormones and is a major autoantigen in autoimmune thyroid disease (AITD). It is believed that the majority of TPO autoantibodies bind to an immunodominant region consisting of two overlapping domains. Precise location of these domains would help our understanding of the interaction between TPO and TPO autoantibodies. 4F5 is a mouse monoclonal antibody (IgG1, kappa) that reacts with high affinity (2.6 x 10(10) mol/L(-1)) with one of the major autoantigenic regions on TPO. Heavy chain genes of 4F5 were from the VH1 germline gene family, germline genes for the D region could not be assigned and the J region was from the JH2 germline. Light chain genes were from Vkappa4/5 and Jkappa2, germline gene families. The Fab fragment of 4F5 was prepared by papain digestion, purified, crystallized, and the structure solved to 1.9 A using molecular replacement. The refined structure had an R factor of 19.5% and a free R factor of 23.9%. Deduced amino acid sequence and amino acid sequence obtained from diffraction analysis were compared and used to finalize the 4F5 Fab model. Structural analysis indicated that the structure of 4F5 is that of a standard Fab and its combining site is flat and is rich in tyrosine residues. Comparison of the structure of 4F5 with that of a TPO autoantibody Fab, TR1.9 suggests that the two antibodies are unlikely to recognise the same structures on TPO.

Analysis of the Thyrotropin Receptor-Thyrotropin Interaction by Comparative Modeling

“…8A) that interact with the indolic group of Trp258 of the LRD. The prominent presence of aromatic residues in the topography of the mAb combining site is consistent with aromatic residues being a usual feature of antibody binding sites (83,84).…”

Localization of the Discontinuous Immunodominant Region Recognized by Human Anti-thyroperoxidase Autoantibodies in Autoimmune Thyroid Diseases

“…Furthermore, we propose two possibilities to explain this phenomenon: either the MPO-like and CCP-like domains interact directly by interactions such as hydrogen bonds or salt links or the flexibility of the hinge region results in constant rearrangement among the three modules, and the binding of an aAb (like T13) on at least two domains "freezes" the structure into a stable conformation. If the latter alternative is true, this could explain why it is so difficult to obtain high resolution diffracting crystals of the TPO ectodomain (17,18). Co-crystallization of TPO with a specific Fab stabilizing the structure should solve this problem definitively.…”

“…Alignment studies and structural homologies have shown that TPO is formed by three distinct domains: a myeloperoxidase (MPO)-like, a complement control protein (CCP)-like, and an EGF-like domain, from the N-to the Cterminal extremities (13). Although the structure of each domain has been elucidated in part by three-dimensional modeling (13-16), the full three-dimensional structure of TPO remains unknown, even though low resolution crystals have been obtained (17,18). The flexibility observed for the hinge regions probably make difficult the exact positioning of each domain in relation to the others (15).…”

How human IgGs against myelin basic protein (MBP) recognize oligopeptides and MBP

“…Contacts between antibody and vascular endothelial growth factor (VEGF) are formed by van der Waals contact and improved hydrogen bonding . The site of Fab fragments of Abs against major autoantigenic epitope of thyroid peroxidase is rich in tyrosine residues . Thus, depending on the protein variable parts of monoclonal antibodies or Fab fragments recognizing various antigens can contain different AA residues forming with antigen electrostatic, hydrophobic, van der Waals contacts, and hydrogen bonding.…”