Crystal structure and enzymatic activity of an ADAMTS ‐13 mutant with the E ast A sian‐specific P 475 S polymorphism

Summary Background An E ast A sian‐specific P 475 S 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 P 475 S mutant protein. Methods We determined the crystal structure of the P 475 S 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 ‐ P 475 S , and further examined the effects of denaturants and reaction temperature on their activity. We also examined the cleavage of shear‐treated von W illebrand factor ( VWF ) by MDTCS ‐ P 475 S . Results MDTCS ‐ P 475 S showed a reaction rate similar to that of wild‐type MDTCS , but showed two‐fold lower affinity for the peptidyl substrate, indicating that the P ro475‐containing V ‐loop (residues 474–481) in the C A 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 ‐ P 475 S can moderately cleave shear‐treated VWF . Conclusions We have provided structural evidence that the P 475 S 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.