title: The role of the ADAMTS13 cysteine-rich domain in VWF binding and proteolysis.
abstract: ADAMTS13 proteolytically regulates the platelet-tethering function of von Willebrand factor (VWF). ADAMTS13 function is dependent upon multiple exosites that specifically bind the unraveled VWF A2 domain and enable proteolysis. We carried out a comprehensive functional analysis of the ADAMTS13 cysteine-rich (Cys-rich) domain using engineered glycans, sequence swaps, and single point mutations in this domain. Mutagenesis of Cys-rich domain-charged residues had no major effect on ADAMTS13 function, and 5 out of 6 engineered glycans on the Cys-rich domain also had no effect on ADAMTS13 function. However, a glycan attached at position 476 appreciably reduced both VWF binding and proteolysis. Substitution of Cys-rich sequences for the corresponding regions in ADAMTS1 identified a hydrophobic pocket involving residues Gly471-Val474 as being of critical importance for both VWF binding and proteolysis. Substitution of hydrophobic VWF A2 domain residues to serine in a region (residues 1642-1659) previously postulated to interact with the Cys-rich domain revealed the functional importance of VWF residues Ile1642, Trp1644, Ile1649, Leu1650, and Ile1651. Furthermore, the functional deficit of the ADAMTS13 Cys-rich Gly471-Val474 variant was dependent on these same hydrophobic VWF residues, suggesting that these regions form complementary binding sites that directly interact to enhance the efficiency of the proteolytic reaction.
authors.affiliation: Centre for Haematology, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, London, United Kingdom.
authors.forename: Rens
authors.identifier: http://orcid.org/0000-0002-1044-9499
authors.initials: R
authors.lastname: de Groot
authors.affiliation: Centre for Haematology, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, London, United Kingdom.
authors.forename: David A
authors.identifier: http://orcid.org/0000-0003-4283-3695
authors.initials: DA
authors.lastname: Lane
authors.affiliation: Centre for Haematology, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, London, United Kingdom.
authors.forename: James T B
authors.identifier: http://orcid.org/0000-0002-6723-7841
authors.initials: JT
authors.lastname: Crawley
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry / Q000502:physiology*; D000071120:ADAMTS13 Protein; D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017354:Point Mutation; D011134:Polysaccharides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D014841:von Willebrand Factor / Q000737:chemistry*
chemical_list: D011134:Polysaccharides; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human; D003545:Cysteine