Antibodies that conformationally activate ADAMTS13 allosterically enhance metalloprotease domain function
Open Access
- 24 March 2020
- journal article
- research article
- Published by American Society of Hematology in Blood Advances
- Vol. 4 (6), 1072-1080
- https://doi.org/10.1182/bloodadvances.2019001375
Abstract
Plasma ADAMTS13 circulates in a folded conformation that is stabilized by an interaction between the central Spacer domain and the C-terminal CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains. Binding of ADAMTS13 to the VWF D4(-CK) domains or to certain activating murine monoclonal antibodies (mAbs) induces a structural change that extends ADAMTS13 into an open conformation that enhances its function. The objective was to characterize the mechanism by which conformational activation enhances ADAMTS13-mediated proteolysis of VWF. The Q activating effects of a novel anti-Spacer (3E4) and the anti-CUB1 (17G2) mAbs on the kinetics of proteolysis of VWF A2 domain fragments by ADAMTS13 were analyzed. mAb-induced conformational changes in ADAMTS13 were investigated by enzyme-linked immunosorbent assay. Both mAbs enhanced ADAMTS13 catalytic efficiency (k(cat)/K-m) by similar to twofold (3E4: 2.0-fold; 17G2: 1.8-fold). Contrary to previous hypotheses, ADAMTS13 activation was not mediated through exposure of the Spacer or cysteine-rich domain exosites. Kinetic analyses revealed that mAb-induced conformational extension of ADAMTS13 enhances the proteolytic function of the metalloprotease domain (k(cat)), rather than augmenting substrate binding (K-m). A conformational effect on the metalloprotease domain was further corroborated by the finding that incubation of ADAMTS13 with either mAb exposed a cryptic epitope in the metalloprotease domain that is normally concealed when ADAMTS13 is in a closed conformation. We show for the first time that the primary mechanism of mAbinduced conformational activation of ADAMTS13 is not a consequence of functional exosite exposure. Rather, our data are consistent with an allosteric activation mechanism on the metalloprotease domain that augments active site function.This publication has 17 references indexed in Scilit:
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