Unraveling antibody-induced structural dynamics in the ADAMTS13 CUB1-2 domains via HDX-MS.

Allosteric regulation of ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type-1 motif, member 13) activity involves an interaction between its spacer (S) and 2 complement C1r/C1s, Uegf and BMP1 (CUB; CUB1-2) domains to keep the enzyme in a closed, latent conformation. Monoclonal antibodies (mAbs) uncouple the S-CUB interaction to open the ADAMTS13 conformation and thereby disrupt the global enzyme latency. The molecular mechanism behind this mAb-induced allostery remains poorly understood. To gain insights in the mAb-induced S-CUB uncoupling and global latency disruption, we combined hydrogen/deuterium exchange mass spectrometry (HDX-MS) with structural analysis of ADAMTS13 CUB1-2 mutants. Thereby, the CUB1 L3 and L9 loops were fine-mapped as the 17G2 mAb binding epitope. Indirect S-CUB uncoupling was observed as mAb binding-induced extensive structural dynamics within both CUB1-2 domains without directly targeting the contiguous CUB1 surface that engages with the ADAMTS13 S domain. HDX-MS analysis revealed the short interdomain linker to structurally cover the central CUB1-2 domain interface, which also showed some protein regions that became more exposed upon mAb binding. Therefore, repositioning of the central CUB1-2 interface appears crucial to transfer structural dynamics between both domains. Nevertheless, mutagenesis of the short linker did not disrupt the ADAMTS13 global latency because its closed conformation was preserved. Presumably, allosteric disruption of the global latency requires a structural impact extending beyond the central interface repositioning. Because anti-ADAMTS13 autoantibodies from patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP) also induce an open ADAMTS13 conformation, our novel insights in the antibody-mediated global latency disruption boost our understanding of the iTTP disease pathology.