title: A model for the conformational activation of the structurally quiescent metalloprotease ADAMTS13 by von Willebrand factor.
abstract: Blood loss is prevented by the multidomain glycoprotein von Willebrand factor (VWF), which binds exposed collagen at damaged vessels and captures platelets. VWF is regulated by the metalloprotease ADAMTS13, which in turn is conformationally activated by VWF. To delineate the structural requirements for VWF-mediated conformational activation of ADAMTS13, we performed binding and functional studies with a panel of truncated ADAMTS13 variants. We demonstrate that both the isolated CUB1 and CUB2 domains in ADAMTS13 bind to the spacer domain exosite of a truncated ADAMTS13 variant, MDTCS (KDof 135 ± 1 0.1 nm and 86.9 ± 9.0 nm, respectively). However, only the CUB1 domain inhibited proteolytic activity of MDTCS. Moreover, ADAMTS13ΔCUB2, unlike ADAMTS13ΔCUB1-2, exhibited activity similar to wild-type ADAMTS13 and could be activated by VWF D4-CK. The CUB2 domain is, therefore, not essential for maintaining the inactive conformation of ADAMTS13. Both CUB domains could bind to the VWF D4-CK domain fragment (KDof 53.7 ± 2.1 nm and 84.3 ± 2.0 nm, respectively). However, deletion of both CUB domains did not prevent VWF D4-CK binding, suggesting that competition for CUB-domain binding to the spacer domain is not the dominant mechanism behind the conformational activation. ADAMTS13ΔTSP8-CUB2 could no longer bind to VWF D4-CK, and deletion of TSP8 abrogated ADAMTS13 conformational activation. These findings support an ADAMTS13 activation model in which VWF D4-CK engages the TSP8-CUB2 domains, inducing the conformational change that disrupts the CUB1-spacer domain interaction and thereby activates ADAMTS13.
authors.affiliation: From the Centre for Haematology, Imperial College London, London W12 ONN, United Kingdom ksouth@imperial.ac.uk.
authors.forename: Kieron
authors.initials: K
authors.lastname: South
authors.affiliation: From the Centre for Haematology, Imperial College London, London W12 ONN, United Kingdom.
authors.forename: Marta O
authors.initials: MO
authors.lastname: Freitas
authors.affiliation: From the Centre for Haematology, Imperial College London, London W12 ONN, United Kingdom d.lane@imperial.ac.uk.
authors.forename: David A
authors.initials: DA
authors.lastname: Lane
mesh_terms: D000071120:ADAMTS13 Protein / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008956:Models, Chemical; D011485:Protein Binding / Q000502:physiology; D000072417:Protein Domains; D014841:von Willebrand Factor / Q000737:chemistry* / Q000378:metabolism
chemical_list: D014841:von Willebrand Factor; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human
keywords: ADAMTS13; allosteric regulation; hemostasis; protein conformation; von Willebrand factor