Mapping the Collagen-binding Site in the von Willebrand Factor-A3 Domain

• Published in: The Journal of biological chemistry 2003-04, Vol.278 (17), p.15035-15039
• Main Authors: Romijn, Roland A., Westein, Erik, Bouma, Barend, Schiphorst, Marion E., Sixma, Jan J., Lenting, Peter J., Huizinga, Eric G.
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Binding Sites - genetics; Collagen Type III - metabolism; Dimerization; Humans; Models, Molecular; Mutagenesis, Site-Directed; Peptide Mapping; Point Mutation; Protein Binding - genetics; Protein Structure, Tertiary; Transfection; von Willebrand Factor - chemistry; von Willebrand Factor - genetics; von Willebrand Factor - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M208977200

The multimeric glycoprotein von Willebrand factor (VWF) mediates platelet adhesion to collagen at sites of vascular damage. The binding site for collagen types I and III is located in the VWF-A3 domain. Recently, we showed that His1023, located near the edge between the “front” and “bottom” faces of A3, is critical for collagen binding (Romijn, R. A., Bouma, B., Wuyster, W., Gros, P., Kroon, J., Sixma, J. J., and Huizinga, E. G. (2001) J. Biol. Chem. 276, 9985–9991). To map the binding site in detail, we introduced 22 point mutations in the front and bottom faces of A3. The mutants were expressed as multimeric VWF, and binding to collagen type III was evaluated in a solid-state binding assay and by surface plasmon resonance. Mutation of residues Asp979, Ser1020, and His1023 nearly abolished collagen binding, whereas mutation of residues Ile975, Thr977, Val997, and Glu1001 reduced binding affinity about 10-fold. Together, these residues define a flat and rather hydrophobic collagen-binding site located at the front face of the A3 domain. The collagen-binding site of VWF-A3 is distinctly different from that of the homologous integrin α2 I domain, which has a hydrophilic binding site located at the top face of the domain. Based on the surface characteristics of the collagen-binding site of A3, we propose that it interacts with collagen sequences containing positively charged and hydrophobic residues. Docking of a collagen triple helix on the binding site suggests a range of possible engagements and predicts that at most eight consecutive residues in a collagen triple helix interact with A3.