Clifford-Jacobs Forging Company v. Capital Engineering & (and) Manufacturing Co.: The Continuing Problem within U.C.C. Section 2-207, 16 J. Marshall L. Rev. 589 (1983)

status: accepte

The allosteric activation of ADAMTS13

ADAMTS13 regulates the multimeric size of Von Willebrand Factor (VWF) in plasma by specific, shear-dependent proteolysis. Despite circulating in a seemingly proteolytically competent form, and with a very long active plasma half-life, ADAMTS13 is specific for VWF, and is resistant to plasma inhibitors. This unprecedented specificity has been attributed to exosite interactions between the ADAMTS13 Spacer, Cysteine-rich, Disintegrin-like and Metalloprotease domains and the unraveled VWF A2 domain, which serve to direct proteolysis. I hypothesized that one or more exosite interactions allosterically activate ADAMTS13 by modulating the conformation of its active site, specifically shaping it to accommodate the VWF scissile bond. My first aim was to characterise the independent contribution of each ADAMTS13 exosite to VWF binding and proteolysis. I generated a new VWF A2 domain fragment (VWF96) as a substrate for ADAMTS13, with/without mutations that ablate each exosite interaction separately. I developed a novel ELISA that specifically detects VWF96, and optimized protocols for monitoring the kinetics of proteolysis. My data show that disruption of the Spacer and Cysteine-rich domain exosite interactions reduces proteolysis by ~15-to-20-fold. These deficits were due to reductions in VWF binding. Intriguingly, a remarkable role in proteolysis was revealed for the Disintegrin-like domain exosite interaction; when ablated, this exosite caused a ~780-fold reduction in proteolysis, due to a ~15-fold reduction in binding (Km), and a ~56-fold reduction in substrate turnover (kcat), consistent with the existence of an allosteric link between the Disintegrin-like domain exosite and the active site in the Metalloprotease domain. ADAMTS13 was previously shown to adopt a folded conformation, maintained by inter-domain interactions. My second aim was to characterise the mechanism by which anti-Spacer/anti-CUB domain monoclonal antibodies unfold ADAMTS13, enhancing proteolysis. I showed that the antibody-induced unfolding primarily enhances the substrate turnover (kcat), rather than substrate binding (Km), which would be consistent with an allosteric link between the non-catalytic domains and the active site. Taken together, these findings provide insights into the molecular mechanisms by which the non-catalytic domains of ADAMTS13 influence the active site and confer specificity to VWF proteolysis, and highlight the importance of conformational changes in ADAMTS13 to its enzymatic function.Open Acces

Anti-CUB1 or anti-spacer antibodies that increase ADAMTS13 activity act by allosterically enhancing metalloprotease domain function

status: accepte

Antibodies that conformationally activate ADAMTS13 allosterically enhance metalloprotease domain function

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 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 (kcat/Km) by ∼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 (kcat), rather than augmenting substrate binding (Km). 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 mAb-induced 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.status: publishe