Conformational activation of ADAMTS13
Proceedings of the National Academy of Sciences of the United States of America
A disintegrin and metalloprotease with thrombospondin motifs 13 (ADAMTS13) is a metalloprotease that regulates von Willebrand factor (VWF) function. ADAMTS13-mediated proteolysis is determined by conformational changes in VWF, but also may depend on its own conformational activation. Kinetic analysis of WT ADAMTS13 revealed ∼2.5-fold reduced activity compared with ADAMTS13 lacking its C-terminal tail (MDTCS) or its CUB1-2 domains (WTΔCUB1-2), suggesting that the CUB domains naturally limit
... S13 function. Consistent with this suggestion, WT ADAMTS13 activity was enhanced ∼2.5-fold by preincubation with either an anti-CUB mAb (20E9) or VWF D4CK (the natural binding partner for the CUB domains). Furthermore, the isolated CUB1-2 domains not only bound MDTCS, but also inhibited activity by up to 2.5-fold. Interestingly, a gain-of-function (GoF) ADAMTS13 spacer domain variant (R568K/ F592Y/R660K/Y661F/Y665F) was ∼2.5-fold more active than WT ADAMTS13, but could not be further activated by 20E9 mAb or VWF D4CK and was unable to bind or to be inhibited by the CUB1-2 domains, suggesting that the inhibitory effects of the CUB domains involve an interaction with the spacer domain that is disrupted in GoF ADAMTS13. Electron microscopy demonstrated a "closed" conformation of WT ADAMTS13 and suggested a more "open" conformation for GoF ADAMTS13. The cryptic spacer domain epitope revealed by conformational unfolding also represents the core antigenic target for autoantibodies in thrombotic thrombocytopenic purpura. We propose that ADAMTS13 circulates in a closed conformation, which is maintained by a CUB-spacer domain binding interaction. ADAMTS13 becomes conformationally activated on demand through interaction of its C-terminal CUB domains with VWF, making it susceptible to immune recognition. ADAMTS13 | VWF | TTP | autoantibodies V on Willebrand factor (VWF) is a large, multidomain glycoprotein that recognizes vascular damage by binding to exposed collagen through its A3 domain (1-3). VWF tethered to collagen responds to shear forces by adapting its conformation (4). Under conditions of low shear, it is thought to adopt a globular conformation, whereas at high shear, it unfolds and reveals its binding site for the platelet GpIbα receptor on its A1 domain, thereby facilitating platelet recruitment to the site of vascular injury. VWF is stored before release into the plasma as multimers that can be as large as 20-40 mers (5-8). On release from the cell, the highest molecular weight multimers are the most hemostatically active. Indeed, "ultra-large" multimers present a potential hazard if their function is unregulated, because they can predispose to the formation of VWF-platelet microthrombi that can occlude small blood vessels, resulting in thrombotic thrombocytopenic purpura (TTP) (9). The metalloprotease ADAMTS13 is able to cleave the VWF A2 domain, dramatically reducing the multimeric size of VWF and its propensity to form platelet microthrombi (10, 11). Cleavage of VWF by ADAMTS13 is a multistep process. An initial positioning interaction occurs between the D4CK domains of globular VWF and ADAMTS13 (12, 13). As unfolding occurs, exposure of the VWF scissile bond, Y1605-M1606, within the A2 domain is controlled by structural elements contained within this domain (14-17). Progressive unfolding allows distinct functional exosites within its A2 domain to be exposed and engaged by complementary binding sites on the protease, spacer, and disintegrin-like domains of ADAMTS13 (18-22) . Ultimately, docking of VWF scissile bond P1′, P1, and P3 residues into subsites on the protease position the scissile bond for cleavage (23). Thus, conformational changes in VWF are essential for its efficient cleavage by ADAMTS13. To explore the possible role of conformation on ADAMTS13 function (24, 25), we studied a recently described gain-of-function (GoF) variant of ADAMTS13 (26) . Jian et al. (26) concluded that an ADAMTS13 GoF variant comprising composite spacer domain substitutions R568K/F592Y/R660K/Y661F/Y665F (hereinafter, GoF) had an approximate fourfold increased ability to cleave VWF substrates. A similar increase in activity on removal of the C-terminal domains from ADAMTS13 has been reported by others (27) . Based on our investigation of the properties of ADAMTS13, the GoF variant, and their derivatives reported herein, we propose that ADAMTS13 normally adopts a globular conformation determined by interaction of its spacer and CUB domains and is unfolded during conformational activation. Results Increased Substrate Recognition Leads to Hyperactivity in GoF ADAMTS13. Our kinetic analysis of VWF115 cleavage suggested an approximate twofold difference between WT ADAMTS13 and the GoF variant (26) (Fig. 1A and k cat /K m values in Table 1 ). There was no difference in k cat values, however, with the enhanced activity instead related to an increased affinity for the substrate, as demonstrated by the K m values of 1.54 μM for WT and 0.86 μM for GoF ADAMTS13. The K D(APP) values determined by equilibrium plate binding assays suggest a twofold increased affinity of GoF ADAMTS13 for VWF (Fig. 1B) . This finding was confirmed by both steady-state and global fit surface Significance We show that a CUB-spacer domain interaction impedes exposure of the ADAMTS13 spacer functional exosite, preventing ADAMTS13 from interacting effectively with its complementary binding site in the VWF A2 domain. This CUB-spacer interaction is disrupted by interaction with the C-terminal domains of VWF, leading to conformational activation of ADAMTS13. Our findings also suggest that activation of ADAMTS13 reveals a cryptic epitope in the spacer domain that constitutes the autoantigenic core in patients with acquired TTP. These antibodies inhibit ADAMTS13, causing deposition of VWF and platelet-rich microthrombi in small blood vessels, resulting in organ damage. Thus, this study provides insight into the complexity of both normal haemostatic control and the pathogenesis of autoimmunity.