Binding epitope of the anti-ADAMTS13 CUB1 mAb 17G2. (A) Section of the CUB1-2 domains presenting the ADAMTS13 CUB1 L3 (in orange), L5, L7, and L9 (in cyan) loops, differences in HDX (ΔHDX) are presented by the deuterium uptake plots for each binding epitope loop. (B) Cartoon representation of the CUB1 domain (light gray) with mutated, single residues presented as sticks. The L3 and L9 loop mutations are colored in red, whereas the control mutations are indicated in green. The CUB1 residues of the contiguous surface that docks on the S domain are colored in violet.8 Of note, the D1204H substitution is located at the backside of the CUB1 domain and is visually covered by the front β-strands of CUB1. (C) Abolished binding of the 17G2 mAb to single residue, full-length ADAMTS13 mutants (Q1210R, R1272K, R1274H, and C1275R) captured on our anti-MP mAb 3H9, confirmed the CUB1 L3 and L9 loops as the HDX-identified mAb epitope, whereas the mAb 17G2 could bind the 3H9-captured ADAMTS13 mutants with control mutations outside the L3 or L9 loops (H1196Y, D1259G, and E1293G) equally well compared to wild-type (WT) ADAMTS13.
