Crystal Structure of a TSH Receptor Monoclonal Antibody: Insight Into Graves' Disease Pathogenesis

Abstract

The TSH receptor (TSHR) A-subunit is more effective than the holoreceptor in inducing thyroid-stimulating antibodies (TSAb) that cause Graves' disease. A puzzling phenomenon is that 2 recombinant, eukaryotic forms of A-subunits (residues 22-289), termed active and inactive, are recognized mutually exclusively by pathogenic TSAb and mouse monoclonal antibody 3BD10, respectively. Understanding the structural difference between these TSHR A-subunit forms could provide insight into Graves' disease pathogenesis. The 3-dimensional structure of the active A-subunit (in complex with a human TSAb Fab, M22) is known, but the structural difference with inactive A-subunits is unknown. We solved the 3BD10 Fab 3-dimensional crystal structure. Guided by prior knowledge of a portion of its epitope, 3BD10 docked in silico with the known active TSHR-289 monomeric structure. Because both TSAb and 3BD10 recognize the active TSHR A-subunit monomer, this form of the molecule can be excluded as the basis for the active-inactive dichotomy, suggesting, instead a role for A-subunit quaternary structure. Indeed, in silico analysis revealed that M22, but not 3BD10, bound to a TSHR-289 trimer. In contrast, 3BD10, but not M22, bound to a TSHR-289 dimer. The validity of these models is supported experimentally by the temperature-dependent balance between active and inactive TSHR-289. In summary, we provide evidence for a structural basis to explain the conformational heterogeneity of TSHR A-subunits (TSHR-289). The pathophysiologic importance of these findings is that affinity maturation of pathogenic TSAb in Graves' disease is likely to involve a trimer of the shed TSHR A-subunit.

Deleting the Redundant TSH Receptor C-Peptide Region Permits Generation of the Conformationally Intact Extracellular Domain by Insect Cells

“…To our surprise, purification of the TSHR ECD was more straightforward, with approximately 90% purity obtained by a single pass over a TSHR mAb CS-17 affinity column ( Figure 5). However, in contrast to the TSHR ECD in conditioned medium, TSH binding to the purified ECD was greatly diminished, possibly consequent to the formation of multimers that obscure the ligand binding site (35). Because of this unexpected difficulty and because the crystal structures of the TSHR LRD (14,15), FSHR LRD (16), and FSHR ECD (17) have only been reported in complex with ligands and not as isolated proteins, it is likely that the TSHR ECD will require complexing with a ligand before purification.…”

Critical Differences between Induced and Spontaneous Mouse Models of Graves’ Disease with Implications for Antigen-Specific Immunotherapy in Humans

“…However, the outcome with respect to TSHR antibodies depends on the B cell epitopes recognized. It should be emphasized that the two forms of TSHR A-subunits (‘active’ and ‘inactive’) have the identical amino acid composition and tertiary conformation but differ in their quaternary structure (41). Evidence suggests that the former is a trimer, the latter a dimer, distinguished by differential recognition by pathogenic and non-pathogenic TSHR antibodies (42).…”

Evidence that TSH Receptor A-Subunit Multimers, Not Monomers, Drive Antibody Affinity Maturation in Graves' Disease

“…However, as mentioned above (Introduction), new information from the crystal structure of the 3BD10 Fab reveals that the A-subunit monomer cannot represent both forms (7). This observation enabled us to address the question of whether the shed A-subunit immunogen in Graves' disease is a monomer or a multimer.…”

“…Conversely, mAb 3BD10 only recognizes inactive A-subunits (5). Guided by amino acids contributing to its epitope, the 3BD10 Fab can be docked in silico with the M22/TSHR structure (7). Because both M22 and 3BD10 can simultaneously bind to the TSHR A-subunit monomer, the latter cannot explain the reciprocally exclusive TSAb and 3BD10 binding that is observed experimentally (5).…”

“…Consequently, because TSAb M22 is known to specifically recognize the active A-subunit form (8,9), the monomeric A-subunit cannot explain the reciprocally exclusive TSAb and 3BD10 binding that is observed experimentally (5). Instead, computer analysis suggested an alternative explanation, namely that TSAb and 3BD10 recognize A-subunits multimers of different valency (7).…”