Anti-CUB1 or Anti-Spacer Antibodies That Increase ADAMTS13 Activity Act By Allosterically Enhancing Metalloprotease Domain Function

Background

ADAMTS13 circulates in a folded conformation, which is mediated by interactions between the C-terminal CUB domains and its central Spacer domain. Binding of ADAMTS13 to the VWF D4-CK domains disrupts the CUB-Spacer interaction, inducing a structural change that extends ADAMTS13 into an open conformation that enhances catalytic efficiency ~2-fold. This mechanism supports a model in which ADAMTS13 unfolding induces exposure of an exosite in the Spacer domain that interacts with the VWF A2 domain, increasing the affinity between the two molecules, and, therefore, the rate of proteolysis. The D4-CK-mediated conformational activation of ADAMTS13 can be mimicked in vitro with the use of antibodies that disrupt the CUB-Spacer interaction, such as the previously published anti-CUB antibody, Ab17G2. We recently generated a novel, activating antibody against the Spacer domain (Ab3E4).

Aim

To characterize the mechanism by which the Ab17G2 and Ab3E4 enhance the catalytic efficiency of ADAMTS13.

Methods

The effects of the Ab17G2 and Ab3E4 on the activity of ADAMTS13 were studied using FRETS-VWF73. The effects of the Ab17G2 and Ab3E4 on the kinetics of VWF96 (VWF G1573-R1668) proteolysis were characterized using an in-house assay. ELISA was used to investigate conformational changes in ADAMTS13 induced by the Ab17G2 and Ab3E4.

Results

Both Ab17G2 and Ab3E4 enhanced FRETS-VWF73 proteolysis by ~1.7-fold. This result was reproduced using the VWF96 substrate; the Ab17G2 and Ab3E4 enhanced the catalytic efficiency (k_cat/K_m) of ADAMTS13 by ~1.8- and ~2.0-fold, respectively. The activation was dependent on the conformational extension of ADAMTS13, since the antibodies could not enhance the activity of an ADAMTS13 variant that lacks the TSP2-CUB2 domains (MDTCS). Surprisingly, ADAMTS13 activation was not mediated through exposure of the Spacer or Cys-rich domain exosites as previously proposed, as the Ab17G2 and Ab3E4 efficiently enhanced proteolysis of VWF96 variants in which the Spacer/Cys-rich exosite binding sites were disrupted. Kinetic analysis of VWF96 proteolysis showed that the Ab17G2- and Ab3E4-induced activation of ADAMTS13 is primarily manifest through a ~1.5- to ~2-fold increase in enzyme turnover (k_cat). Thus, contrary to the current model, this suggests that the conformational extension of ADAMTS13 influences the functionality of the active site, and not substrate binding affinity (K_m). Incubating ADAMTS13 with either Ab17G2 or Ab3E4 exposed a cryptic epitope in the metalloprotease domain that was specifically detected by ELISA, further corroborating that the antibodies induce a conformational change in ADAMTS13 affecting the M domain.

Conclusion

Antibodies can be used as tools for understanding the structure/function of enzymes. Using activating antibodies against the Spacer and CUB1 domains of ADAMTS13, we show for the first time that the activation of ADAMTS13 following its unfolding is not a result of exposure of a functional exosite in Spacer/Cys-rich domain that increases affinity to VWF. Rather, our data are consistent with an allosteric activation mechanism upon the metalloprotease domain. We propose that ADAMTS13 unfolding causes a conformational change in the active site that further activates the enzyme. We are currently investigating whether the D4-CK-induced enhancement of ADAMTS13 proteolytic activity is also mediated by conformational changes in the active site.