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.
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.
Authors: Ashish Goel; Banumathi Ramakrishna; Uday Zachariah; K G Sajith; Deepak K Burad; Thomas A Kodiatte; Shyamkumar N Keshava; K A Balasubramanian; Elwyn Elias; C E Eapen Journal: Indian J Med Res Date: 2019-04 Impact factor: 2.375
Authors: Ryan Hunt; Gaya Hettiarachchi; Upendra Katneni; Nancy Hernandez; David Holcomb; Jacob Kames; Redab Alnifaidy; Brian Lin; Nobuko Hamasaki-Katagiri; Aaron Wesley; Tal Kafri; Christina Morris; Laura Bouché; Maria Panico; Tal Schiller; Juan Ibla; Haim Bar; Amra Ismail; Howard Morris; Anton Komar; Chava Kimchi-Sarfaty Journal: Int J Mol Sci Date: 2019-11-15 Impact factor: 5.923