Crystal structure and enzymatic activity of an ADAMTS-13 mutant with the East Asian-specific P475S polymorphism
M Akiyama1, D Nakayama, S Takeda
1Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Osaka, Japan. akiyamam@ri.ncvc.go.jp
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.
Insights
An East Asian genetic variant in ADAMTS-13 reduces enzyme activity by lowering substrate affinity, not reaction rate. This structural change is insufficient to cause thrombotic thrombocytopenic purpura (TTP).
Area of Science:
- Biochemistry
- Structural Biology
- Genetics
Background:
- An East Asian-specific polymorphism, P475S, in the ADAMTS-13 gene reduces plasma ADAMTS-13 activity by approximately 16%.
- ADAMTS-13 plays a crucial role in regulating von Willebrand factor (VWF) cleavage.
Purpose of the Study:
- To structurally and functionally characterize the P475S mutant of ADAMTS-13.
- To elucidate the impact of the P475S polymorphism on ADAMTS-13 activity and its potential link to thrombotic thrombocytopenic purpura (TTP).
Main Methods:
- Determined the crystal structure of the ADAMTS-13 catalytic domain mutant (DTCS-P475S) and compared it to the wild-type.
- Assessed enzymatic parameters and substrate binding affinity of ADAMTS-13 mutants (MDTCS-P475S).
- Investigated the effect of denaturants and temperature on enzyme activity and VWF cleavage.
Main Results:
- The P475S mutation resulted in a two-fold lower affinity for the peptidyl substrate, identifying the V-loop as a substrate-binding exosite.
- Structural analysis revealed significant conformational changes in the V-loop of DTCS-P475S, leading to increased susceptibility to environmental factors.
- ADAMTS-13-P475S exhibited moderate activity in cleaving shear-treated VWF, with a reaction rate similar to wild-type but reduced substrate affinity.
Conclusions:
- The P475S polymorphism causes local structural instability in ADAMTS-13, reducing substrate affinity without altering the reaction rate.
- The residual activity of ADAMTS-13-P475S on VWF is sufficient to prevent the onset of TTP.
