title: Conformational activation of ADAMTS13.
abstract: A disintegrin and metalloprotease with thrombospondin motifs 13 (ADAMTS13) is a metalloprotease that regulates von Willebrand factor (VWF) function. ADAMTS13-mediated proteolysis is determined by conformational changes in VWF, but also may depend on its own conformational activation. Kinetic analysis of WT ADAMTS13 revealed ∼ 2.5-fold reduced activity compared with ADAMTS13 lacking its C-terminal tail (MDTCS) or its CUB1-2 domains (WTΔCUB1-2), suggesting that the CUB domains naturally limit ADAMTS13 function. Consistent with this suggestion, WT ADAMTS13 activity was enhanced ∼ 2.5-fold by preincubation with either an anti-CUB mAb (20E9) or VWF D4CK (the natural binding partner for the CUB domains). Furthermore, the isolated CUB1-2 domains not only bound MDTCS, but also inhibited activity by up to 2.5-fold. Interestingly, a gain-of-function (GoF) ADAMTS13 spacer domain variant (R568K/F592Y/R660K/Y661F/Y665F) was ∼ 2.5-fold more active than WT ADAMTS13, but could not be further activated by 20E9 mAb or VWF D4CK and was unable to bind or to be inhibited by the CUB1-2 domains, suggesting that the inhibitory effects of the CUB domains involve an interaction with the spacer domain that is disrupted in GoF ADAMTS13. Electron microscopy demonstrated a "closed" conformation of WT ADAMTS13 and suggested a more "open" conformation for GoF ADAMTS13. The cryptic spacer domain epitope revealed by conformational unfolding also represents the core antigenic target for autoantibodies in thrombotic thrombocytopenic purpura. We propose that ADAMTS13 circulates in a closed conformation, which is maintained by a CUB-spacer domain binding interaction. ADAMTS13 becomes conformationally activated on demand through interaction of its C-terminal CUB domains with VWF, making it susceptible to immune recognition.
authors.affiliation: Centre for Haematology, Imperial College London, London W12 ONN, United Kingdom; k.south@imperial.ac.uk d.lane@imperial.ac.uk.
authors.forename: Kieron
authors.initials: K
authors.lastname: South
authors.affiliation: Centre for Haematology, Imperial College London, London W12 ONN, United Kingdom;
authors.forename: Brenda M
authors.initials: BM
authors.lastname: Luken
authors.affiliation: Centre for Haematology, Imperial College London, London W12 ONN, United Kingdom;
authors.forename: James T B
authors.initials: JT
authors.lastname: Crawley
authors.affiliation: Centre for Haematology, Imperial College London, London W12 ONN, United Kingdom;
authors.forename: Rebecca
authors.initials: R
authors.lastname: Phillips
authors.affiliation: Centre for Haematology, Imperial College London, London W12 ONN, United Kingdom;
authors.forename: Mari
authors.initials: M
authors.lastname: Thomas
authors.affiliation: Electron Microscopy Facility, Faculty of Life Sciences, University of Manchester, Manchester M13 9PL, United Kingdom; and.
authors.forename: Richard F
authors.initials: RF
authors.lastname: Collins
authors.affiliation: Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven, 3000 Leuven, Belgium.
authors.forename: Louis
authors.initials: L
authors.lastname: Deforche
authors.affiliation: Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven, 3000 Leuven, Belgium.
authors.forename: Karen
authors.initials: K
authors.lastname: Vanhoorelbeke
authors.affiliation: Centre for Haematology, Imperial College London, London W12 ONN, United Kingdom; k.south@imperial.ac.uk d.lane@imperial.ac.uk.
authors.forename: David A
authors.initials: DA
authors.lastname: Lane
mesh_terms: D051722:ADAM Proteins / Q000097:blood / Q000737:chemistry* / Q000235:genetics; D000071120:ADAMTS13 Protein; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry; D004789:Enzyme Activation; D006801:Humans; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D011697:Purpura, Thrombotic Thrombocytopenic / Q000201:enzymology / Q000235:genetics; D017384:Sequence Deletion; D014841:von Willebrand Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human
keywords: ADAMTS13; TTP; VWF; autoantibodies