These authors contributed equally to this work.
Crystal structure and enzymatic activity of an ADAMTS-13 mutant with the East Asian-specific P475S polymorphism
Article first published online: 15 JUL 2013
© 2013 International Society on Thrombosis and Haemostasis
Journal of Thrombosis and Haemostasis
Volume 11, Issue 7, pages 1399–1406, July 2013
How to Cite
Crystal structure and enzymatic activity of an ADAMTS-13 mutant with the East Asian-specific P475S polymorphism. J Thromb Haemost 2013; 11: 1399–406., , , , , .
Manuscript handled by: D. Lane
Final decision: D. Lane, 5 April 2013
- Issue published online: 15 JUL 2013
- Article first published online: 15 JUL 2013
- Accepted manuscript online: 27 APR 2013 12:32AM EST
- Manuscript Accepted: 5 APR 2013
- Manuscript Received: 11 OCT 2012
- genetic polymorphism;
- thrombotic thrombocytopenic purpura;
- von Willebrand factor
An East Asian-specific P475S polymorphism in the gene encoding ADAMTS-13 causes an approximately 16% reduction in plasma ADAMTS-13 activity.
To demonstrate the impact of this dysfunctional polymorphism by characterizing the structure and activity of the P475S mutant protein.
We determined the crystal structure of the P475S mutant of ADAMTS-13-DTCS (DTCS-P475S, residues 287–685) and compared it with the wild-type structure. We determined the enzymatic parameters of ADAMTS-13-MDTCS (residues 75–685) and MDTCS-P475S, and further examined the effects of denaturants and reaction temperature on their activity. We also examined the cleavage of shear-treated von Willebrand factor (VWF) by MDTCS-P475S.
MDTCS-P475S showed a reaction rate similar to that of wild-type MDTCS, but showed two-fold lower affinity for the peptidyl substrate, indicating that the Pro475-containing V-loop (residues 474–481) in the CA domain is a substrate-binding exosite. Structural analysis showed that the conformation of the V-loop was significantly different in DTCS-P475S and the wild type, where no obvious interactions of Ser475 with other residues were observed. This explains the higher susceptibility of the enzymatic activity of MDTCS-P475S to reaction environments such as denaturants and high temperature. MDTCS-P475S can moderately cleave shear-treated VWF.
We have provided structural evidence that the P475S polymorphism in ADAMTS-13 leads to increased local structural instability, resulting in lowered affinity for the substrate without changing the reaction rate. The moderate activity of ADAMTS-13-P475S for shear-treated VWF is sufficient to prevent thrombotic thrombocytopenic purpura (TTP) onset.