Extensive contacts between ADAMTS13 exosites and von Willebrand factor domain A2 contribute to substrate specificity

• Published in: Blood 2008-09, Vol.112 (5), p.1713-1719
• Main Authors: Gao, Weiqiang, Anderson, Patricia J., Sadler, J.Evan
• Format: Article
• Language: English
• Subjects: ADAM Proteins - chemistry; ADAM Proteins - genetics; ADAM Proteins - metabolism; ADAMTS13 Protein; Amino Acid Sequence; Base Sequence; Binding Sites; Biomarkers, Tumor - chemistry; Biomarkers, Tumor - genetics; Biomarkers, Tumor - metabolism; Calcium-Binding Proteins; DNA Primers - genetics; Hemostasis, Thrombosis, and Vascular Biology; Humans; In Vitro Techniques; Kinetics; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Interaction Domains and Motifs; Protein Structure, Tertiary; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Sequence Deletion; Substrate Specificity; Transcription Factors - chemistry; Transcription Factors - genetics; Transcription Factors - metabolism
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2008-04-148759

The metalloprotease ADAMTS13 efficiently cleaves only the Tyr1605-Met1606 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 α-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 Gln1624 and Arg1668, and together these exosite interactions increase the rate of substrate cleavage by at least approximately 300-fold. Internal deletion of Gln1624-Arg1641 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 Tyr1605-Met1606 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.