The ADAMTS13 metalloprotease domain: roles of subsites in enzyme activity and specificity.

Blood
Rens de GrootJames T B Crawley

Abstract

ADAMTS13 modulates von Willebrand factor (VWF) platelet-tethering function by proteolysis of the Tyr1605-Met1606 bond in the VWF A2 domain. To examine the role of the metalloprotease domain of ADAMTS13 in scissile bond specificity, we identified 3 variable regions (VR1, -2, and -3) in the ADAMTS family metalloprotease domain that flank the active site, which might be important for specificity. Eight composite sequence swaps (to residues in ADAMTS1 or ADAMTS2) and 18 single-point mutants were generated in these VRs and expressed. Swapping VR1 (E184-R193) of ADAMTS13 with that of ADAMTS1 or ADAMTS2 abolished/severely impaired ADAMTS13 function. Kinetic analysis of VR1 point mutants using VWF115 as a short substrate revealed reduced proteolytic function (k(cat)/K(m) reduced by 2- to 10-fold) as a result of D187A, R190A, and R193A substitutions. Analysis of VR2 (F216-V220) revealed a minor importance of this region. Mutants of VR3 (G236-A261) proteolysed wild-type VWF115 normally. However, using either short or full-length VWF substrates containing the P1' M1606A mutation, we identified residues within VR3 (D252-P256) that influence P1' amino acid specificity, we hypothesize, by shaping the S1' pocket. It is concluded that 2 subsit...Continue Reading

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Citations

Jun 28, 2011·Proceedings of the National Academy of Sciences of the United States of America·Yaozu XiangDavid A Lane
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