Identification of the Collagen-binding Site of the von Willebrand Factor A3-domain

Von Willebrand factor (vWF) is a multimeric glycoprotein that mediates platelet adhesion and thrombus formation at sites of vascular injury. vWF functions as a molecular bridge between collagen and platelet receptor glycoprotein Ib. The major collagen-binding site of vWF is contained within the A3 d...

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Published in:The Journal of biological chemistry 2001-03, Vol.276 (13), p.9985-9991
Main Authors: Romijn, Roland A.P., Bouma, Barend, Wuyster, Winnifred, Gros, Piet, Kroon, Jan, Sixma, Jan J., Huizinga, Eric G.
Format: Article
Language:English
Subjects:
Alanine - chemistry
Alanine - metabolism
Amino Acid Sequence
Antibodies, Monoclonal - metabolism
Arginine - chemistry
Binding Sites
Collagen - metabolism
Crystallography, X-Ray
Epitopes
Histidine - chemistry
Humans
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Mutation
Point Mutation
Protein Binding
Protein Conformation
Protein Structure, Tertiary
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
von Willebrand Factor - chemistry
von Willebrand Factor - metabolism
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Summary:Von Willebrand factor (vWF) is a multimeric glycoprotein that mediates platelet adhesion and thrombus formation at sites of vascular injury. vWF functions as a molecular bridge between collagen and platelet receptor glycoprotein Ib. The major collagen-binding site of vWF is contained within the A3 domain, but its precise location is unknown. To localize the collagen-binding site, we determined the crystal structure of A3 in complex with an Fab fragment of antibody RU5 that inhibits collagen binding. The structure shows that RU5 recognizes a nonlinear epitope consisting of residues 962–966, 981–997, and 1022–1026. Alanine mutants were constructed of residues Arg963, Glu987, His990, Arg1016, and His1023, located in or close to the epitope. Mutants were expressed as fully processed multimeric vWF. Mutation of His1023 abolished collagen binding, whereas mutation of Arg963 and Arg1016 reduced collagen binding by 25–35%. These residues are part of loops α3β4 and α1β2 and α-helix 3, respectively, and lie near the bottom face of the domain. His1023 and flanking residues display multiple conformations in available A3-crystal structures, suggesting that binding of A3 to collagen involves an induced-fit mechanism. The collagen-binding site of A3 is located distant from the top face of the domain where collagen-binding sites are found in homologous integrin I domains. AF2865871FE8
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M006548200