Abstract Number: OC 07.1
Meeting: ISTH 2020 Congress
Background: Most patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP) develop autoantibodies targeting a cryptic epitope, composed of residues R568/F592/R660/Y661/Y665 in the spacer domain of ADAMTS13. Conservative substitutions of these residues resulted in a gain-of-function (GoF; R568K/F592Y/R660K/Y661F/Y665F) ADAMTS13, which exists in an open conformation. It has been suggested that interactions between the C-terminal CUB domains and the spacer domain maintain ADAMTS13 in a closed conformation. A recent study has shown that ADAMTS13 circulates in open conformation during the acute phase of iTTP, suggesting that conformational changes of ADAMTS13 contribute to the pathogenesis of iTTP.
Aims: Our aim is to investigate these conformational changes by modelling the closed conformation of ADAMTS13 in silico, predict the CUB domain residues which keeps ADAMTS13 in closed conformation and open ADAMTS13 in vitro by mutating these predicted residues.
Methods: YASARA Structure tool was used for homology modelling of the C-terminal CUB domains. Domain interactions between the spacer domain and CUB domains were studied by HADDOCK protein-protein docking. WT- ADAMTS13 was subjected to molecular dynamics simulations and binding free energy calculation with AMBER16. Selected residues were further mutated and the impact of these mutations on the conformation was investigated in an ELISA.
Results: Among different possible conformations of the spacer-CUB domains complex, a pose which is in full agreement with the experimental data was obtained (Figure 1). Our model suggested that several CUB1 and CUB2 domain residues interact with spacer domain residues which included residue D1240. To validate our model, we monitored the conformation of an ADAMTS13 variant in which D1240 was replaced by an alanine. The resulting D1240A variant appeared to be present in an open conformation.
Conclusions: Overall, we have established an integrative approach by combining structural bioinformatics and experimental information to discover ADAMTS13-CUB domain residues which are critical for maintaining ADAMTS13 in a closed conformation.
To cite this abstract in AMA style:Ercig B, Graca NAG, Wichapong K, Roose E, Kaijen P, Arfman T, Reutelingsperger C, Vanhoorelbeke K, Nicolaes GAF, Voorberg J. How to Open ADAMTS13: An Integrative Approach to Build ADAMTS13 in Closed Conformation [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/how-to-open-adamts13-an-integrative-approach-to-build-adamts13-in-closed-conformation/. Accessed January 28, 2022.
« Back to ISTH 2020 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/how-to-open-adamts13-an-integrative-approach-to-build-adamts13-in-closed-conformation/