Extensive contacts between ADAMTS13 exosites and von Willebrand factor domain A2 contribute to substrate specificity.
The metalloprotease ADAMTS13 efficiently cleaves only the Tyr(1605)-Met(1606) bond in the central A2 domain of multimeric von Willebrand factor (VWF), even though VWF constitutes only 0.02% of plasma proteins. This remarkable specificity depends in part on binding of the noncatalytic ADAMTS13 spacer domain to the C-terminal alpha-helix of VWF domain A2. By kinetic analysis of recombinant ADAMTS13 constructs, we show that the first thrombospondin-1, Cys-rich, and spacer domains of ADAMTS13 interact with segments of VWF domain A2 between Gln(1624) and Arg(1668), and together these exosite interactions increase the rate of substrate cleavage by at least approximately 300-fold. Internal deletion of Gln(1624)-Arg(1641) minimally affected the rate of cleavage, indicating that ADAMTS13 does not require a specific distance between the scissile bond and auxiliary substrate binding sites. Smaller deletions of the P2-P9 or the P4'-P18' residues on either side of the Tyr(1605)-Met(1606) bond abolished cleavage, indicating that the metalloprotease domain interacts with additional residues flanking the cleavage site. Thus, specific recognition of VWF depends on cooperative, modular contacts between several ADAMTS13 domains and discrete segments of VWF domain A2.
Gao
Weiqiang
W
Department of Medicine, Howard Hughes Medical Institute, Washington University School of Medicine, St Louis, MO 63110, USA.
Anderson
Patricia J
PJ
Sadler
J Evan
JE
D051722:ADAM Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D014408:Biomarkers, Tumor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D014408:Biomarkers, Tumor; D002135:Calcium-Binding Proteins; D017931:DNA Primers; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C496188:VWA2 protein, human; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human