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Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation
Published on: November 26, 2011
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Conformational activation of ADAMTS13.
Kieron South1, Brenda M Luken2, James T B Crawley2
1Centre for Haematology, Imperial College London, London W12 ONN, United Kingdom; k.south@imperial.ac.uk d.lane@imperial.ac.uk.
Summary
A disintegrin and metalloprotease with thrombospondin motifs 13 (ADAMTS13) circulates in a closed conformation, limiting its activity. Interaction with von Willebrand factor (VWF) opens the conformation, enhancing ADAMTS13 function and susceptibility to immune recognition.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- A disintegrin and metalloprotease with thrombospondin motifs 13 (ADAMTS13) is crucial for regulating von Willebrand factor (VWF) activity.
- ADAMTS13's proteolytic function is influenced by VWF conformation and potentially its own conformational state.
- Understanding ADAMTS13 conformation is key to comprehending its regulation and role in thrombotic thrombocytopenic purpura (TTP).
Purpose of the Study:
- To investigate the conformational dynamics of ADAMTS13 and their impact on its enzymatic activity.
- To elucidate the role of ADAMTS13's C-terminal CUB domains and spacer domain in its conformational regulation.
- To explore the relationship between ADAMTS13 conformation, VWF interaction, and immune recognition.
Main Methods:
- Kinetic analysis of wild-type (WT) ADAMTS13 and its variants (MDTCS, WTΔCUB1-2).
- Activity assays using anti-CUB mAb (20E9) and VWF D4CK.
- Binding and inhibition assays with isolated CUB1-2 domains.
- Electron microscopy to visualize ADAMTS13 conformations.
- Analysis of a gain-of-function (GoF) ADAMTS13 variant.
Main Results:
- WT ADAMTS13 exhibits reduced activity compared to C-terminal tail or CUB1-2 domain deleted variants, indicating CUB domains limit function.
- ADAMTS13 activity is enhanced by anti-CUB mAb or VWF D4CK, and inhibited by isolated CUB1-2 domains, suggesting CUB-spacer domain interaction.
- A GoF ADAMTS13 variant shows increased activity, lacks CUB domain-mediated regulation, and exhibits an 'open' conformation.
- Electron microscopy revealed a 'closed' conformation for WT ADAMTS13 and an 'open' conformation for GoF ADAMTS13.
- The spacer domain's cryptic epitope, revealed upon unfolding, is a target for TTP autoantibodies.
Conclusions:
- ADAMTS13 circulates in an auto-inhibited 'closed' conformation maintained by CUB-spacer domain interaction.
- VWF binding to C-terminal CUB domains induces conformational activation to an 'open' state, enhancing proteolysis.
- This conformational switch is critical for ADAMTS13 function and its recognition by autoantibodies in TTP.
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