Exosite interactions contribute to tension-induced cleavage of von Willebrand factor by the antithrombotic ADAMTS13 metalloprotease.
Von Willebrand factor (VWF) is a multimeric protein that mediates platelet adhesion at sites of vascular injury, and ADAMTS13 (a disintegrin and metalloprotease with thrombospondin)is a multidomain metalloprotease that limits platelet adhesion by a feedback mechanism in which fluid shear stress induces proteolysis of VWF and prevents disseminated microvascular thrombosis. Cleavage of the Tyr(1605)-Met(1606) scissile bond in the VWF A2 domain depends on a Glu(1660)-Arg(1668) segment in the same domain and on the noncatalytic spacer domain of ADAMTS13, suggesting that extensive enzyme-substrate interactions facilitate substrate recognition. Based on mutagenesis and kinetic analysis, we find that the ADAMTS13 spacer domain binds to an exosite near the C terminus of the VWF A2 domain. Deleting the spacer domain from ADAMTS13 or deleting the exosite from the VWF substrate reduced the rate of cleavage approximately 20-fold. A cleavage product containing the exosite was a hyperbolic mixed-type inhibitor of ADAMTS13 proteolysis of either VWF multimers or model peptide substrates but only if the ADAMTS13 enzyme contained the spacer domain. The specificity of this unique mechanism depends on tension-induced unfolding of the VWF A2 domain, which exposes the scissile bond and exosite for interaction with complementary sites on ADAMTS13.
Gao
Weiqiang
W
Howard Hughes Medical Institute and Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
Anderson
Patricia J
PJ
Majerus
Elaine M
EM
Tuley
Elodee A
EA
Sadler
J Evan
JE
D002460:Cell Line; D014644:Genetic Variation / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013927:Thrombosis / Q000235:genetics / Q000378:metabolism*; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D014841:von Willebrand Factor; D008666:Metalloendopeptidases