A functional calcium-binding site in the metalloprotease domain of ADAMTS13

ADAMTS13 regulates the multimeric size of von Willebrand factor (VWF). Its function is highly dependent upon Ca2+ ions. Using the initial rates of substrate (VWF115, VWF residues 1554-1668) proteolysis by ADAMTS13 preincubated with varying Ca2+ concentrations, a high-affinity functional ADAMTS13 Ca2+-binding site was suggested with K-D(app) of 80 mu M (+/- 15 mu M) corroborating a previously reported study. When Glu83 or Asp173 (residues involved in a predicted Ca2+-binding site in the ADAMTS13 metalloprotease domain) were mutated to alanine, Ca2+ dependence of proteolysis of the substrate was unaffected. Consequently, we sought and identified a candidate Ca2+-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 Ca2+ 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 Ca2+ 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 Ca2+ upon ADAMTS13 function is mediated through binding to a high-affinity site adjacent to its active site cleft. (Blood. 2009;113:1149-1157)