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Keywords:

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

Summary

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.