Characterization of a core binding site for ADAMTS-13 in the A2 domain of von Willebrand factor.
ADAMTS-13, a metalloprotease in plasma, specifically cleaves the Tyr-1605-Met-1606 bond in the A2 domain of von Willebrand factor (VWF) to regulate the polymer distribution of VWF in circulation, which is critical for primary hemostasis. A 73-aa peptide (VWF73) was previously identified as the minimal substrate cleavable by ADAMTS-13. In this study, VWF73 was enzymatically and chemically cleaved into shorter peptides, and the inhibition of cleavage of a VWF73-derived substrate by these purified peptides was measured in competition studies using a quantitative assay we recently reported. A 24-aa peptide encompassing Pro-1645-Lys-1668 (P'40-P'63) and situated 40 aa downstream from the cleavage site was the minimal peptide that could bind to and competitively inhibit ADAMTS-13 (K(i) = 12 microM). This peptide and longer peptides encompassing this core sequence also inhibited the cleavage of multimeric VWF by ADAMTS-13. These results suggest the presence of a complementary extended binding site, or exosite, on ADAMTS-13. Mutation of Asp-1653 and Asp-1663 to Ala in this region significantly reduced the rate of cleavage of the substrate peptide, whereas the Glu1655Ala mutation caused an enhanced rate of cleavage. These results suggest that ionic interactions of the Pro-1645-Lys-1668 region with the exosite on ADAMTS-13 play a significant role in mediating substrate recognition.
Wu
Jing-Jiang
JJ
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
Fujikawa
Kazuo
K
McMullen
Brad A
BA
Chung
Dominic W
DW
D051722:ADAM Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D001667:Binding, Competitive / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary / Q000235:genetics; D014841:von Willebrand Factor / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D010446:Peptide Fragments; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human