title: A functional calcium-binding site in the metalloprotease domain of ADAMTS13.
abstract: ADAMTS13 regulates the multimeric size of von Willebrand factor (VWF). Its function is highly dependent upon Ca(2+) ions. Using the initial rates of substrate (VWF115, VWF residues 1554-1668) proteolysis by ADAMTS13 preincubated with varying Ca(2+) concentrations, a high-affinity functional ADAMTS13 Ca(2+)-binding site was suggested with K(D(app)) of 80 muM (+/- 15 muM) corroborating a previously reported study. When Glu83 or Asp173 (residues involved in a predicted Ca(2+)-binding site in the ADAMTS13 metalloprotease domain) were mutated to alanine, Ca(2+) dependence of proteolysis of the substrate was unaffected. Consequently, we sought and identified a candidate Ca(2+)-binding site in proximity to the ADAMTS13 active site, potentially comprising Glu184, Asp187, and Glu212. Mutagenesis of these residues within this site to alanine dramatically attenuated the K(D(app)) for Ca(2+) of ADAMTS13, and for D187A and E212A also reduced the V(max) to approximately 25% of normal. Kinetic analysis of the Asp187 mutant in the presence of excess Ca(2+) revealed an approximately 13-fold reduction in specificity constant, k(cat)/K(m), contributed by changes in both K(m) and k(cat). These results were corroborated using plasma-purified VWF as a substrate. Together, our results demonstrate that a major influence of Ca(2+) upon ADAMTS13 function is mediated through binding to a high-affinity site adjacent to its active site cleft.
authors.affiliation: Department of Haematology, Imperial College London, London, United Kingdom.
authors.forename: Michelle D
authors.initials: MD
authors.lastname: Gardner
authors.forename: Chan K N K
authors.initials: CK
authors.lastname: Chion
authors.forename: Rens
authors.initials: R
authors.lastname: de Groot
authors.forename: Anuja
authors.initials: A
authors.lastname: Shah
authors.forename: James T B
authors.initials: JT
authors.lastname: Crawley
authors.forename: David A
authors.initials: DA
authors.lastname: Lane
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000071120:ADAMTS13 Protein; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary / Q000502:physiology; D014841:von Willebrand Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human; D002118:Calcium