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J Thromb Haemost. 2013 Jul;11(7):1399-406. doi: 10.1111/jth.12279.

Crystal structure and enzymatic activity of an ADAMTS-13 mutant with the East Asian-specific P475S polymorphism.

Author information

1
Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Osaka, Japan. akiyamam@ri.ncvc.go.jp

Abstract

BACKGROUND:

An East Asian-specific P475S polymorphism in the gene encoding ADAMTS-13 causes an approximately 16% reduction in plasma ADAMTS-13 activity.

OBJECTIVES:

To demonstrate the impact of this dysfunctional polymorphism by characterizing the structure and activity of the P475S mutant protein.

METHODS:

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.

RESULTS:

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.

CONCLUSIONS:

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.

KEYWORDS:

ADAMTS-13; crystallography; genetic polymorphism; human; proteins; thrombotic thrombocytopenic purpura; von Willebrand factor

PMID:
23621748
DOI:
10.1111/jth.12279
[Indexed for MEDLINE]
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