Allosteric activation of ADAMTS13 by von Willebrand factor

Abstract

The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) within endovascular platelet aggregates, and ADAMTS13 deficiency causes fatal microvascular thrombosis. The proximal metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains of ADAMTS13 recognize a cryptic site in VWF that is exposed by tensile force. Another seven T and two complement C1r/C1s, sea urchin epidermal growth factor, and bone morphogenetic protein (CUB) domains of uncertain function are C-terminal to the MDTCS domains. We find that the distal T8-CUB2 domains markedly inhibit substrate cleavage, and binding of VWF or monoclonal antibodies to distal ADAMTS13 domains relieves this autoinhibition. Small angle X-ray scattering data indicate that distal T-CUB domains interact with proximal MDTCS domains. Thus, ADAMTS13 is regulated by substrate-induced allosteric activation, which may optimize VWF cleavage under fluid shear stress in vivo. Distal domains of other ADAMTS proteases may have similar allosteric properties. 1A) (1-5), a metalloprotease that severs VWF and releases adherent platelets. Deficiency of ADAMTS13 disrupts this feedback regulatory mechanism and causes thrombotic thrombocytopenic purpura (TTP), which is characterized by life-threatening microvascular thrombosis (3, 6, 7).The recognition and cleavage of VWF is a formidable challenge. VWF and ADAMTS13 occur at ∼10 μg/mL and ∼1 μg/mL, respectively, compared with total plasma protein of ∼80,000 μg/mL. ADAMTS13 is constitutively active and has no known inhibitors in vivo. Nevertheless, VWF is the only identified ADAMTS13 substrate, and VWF is resistant to cleavage until subjected to fluid shear stress (8), adsorbed on a surface (9), or treated with denaturants (8, 10). This specificity depends on structural features of both ADAMTS13 and VWF that have not been characterized fully.The proximal metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains domains of ADAMTS13 bind to cryptic sites that are uncovered by unfolding VWF domain A2 (11-15) (Fig. 1B), and these interactions are required for efficient cleavage of VWF or peptide substrates. More distal ADAMTS13 domains bind to sites in or near VWF domain D4 that are always available (16-18). Deletion of distal ADAMTS13 domains impairs the cleavage of VWF multimers in vitro (16,19) and increases VWF-dependent microvascular thrombosis in vivo (20) but accelerates the cleavage of peptide substrates (12, 13). In addition, ADAMTS13 cleaves guanidine hydrochloride-treated VWF multimers with an apparent K m of ∼15 nM (21), which is 100-fold lower than the K m of ∼1.6-1.7 μM for peptide substrates that are based on the sequence of VWF domain A2 (12,14). These striking differences suggest that distal T or complement c1r/c1s, sea urchin epidermal growth factor, and bone morphogenetic protein (CUB) domains regulate ADAMTS13 activity. We have now shown that these distal domains inhibit ADAMTS13, and binding to VWF relieves this autoinhibition. Result...

The role of the ADAMTS13 cysteine-rich domain in VWF binding and proteolysis

“…Whether the C-terminal domains also interfere with binding of the Cys-rich domain to VWF is unknown. 33,34 In summary, our results demonstrate an important role for the Cys-rich domain in VWF proteolysis and identify hydrophobic residues in both ADAMTS13 and VWF that are critical for efficient proteolysis. These findings are in agreement with previous findings that suggested the presence of extensive contacts between exosites in ADAMTS13 and VWF, involving multiple domains.…”

Structural Architecture of Prothrombin in Solution Revealed by Single Molecule Spectroscopy

“…Binding of kringles to fibrin clots and cell surface receptors is assumed to induce a transition to an open form that can be cleaved and converted to plasmin by physiological activators. The metalloprotease ADAMTS13 has its distal domains closed on the catalytic site, and the autoinhibition is removed upon binding of its physiological substrate von Willebrand factor (43).…”

Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity

“…The first of these properties has, until recently, been explained by the dependence of ADAMTS‐13 proteolytic function on VWF conformation and the exposure of its complementary binding sites. However, it has recently been shown that ADAMTS‐13 undergoes its own conformational change in order to attain a fully active state 22, 23. In this new model of ADAMTS‐13 function the enzyme circulates in a ‘closed’ conformation mediated by binding between its spacer and CUB domains 22.…”