title: Mapping the collagen-binding site in the von Willebrand factor-A3 domain.
abstract: 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 His(1023), 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 Asp(979), Ser(1020), and His(1023) nearly abolished collagen binding, whereas mutation of residues Ile(975), Thr(977), Val(997), and Glu(1001) 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 alpha(2) 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.
authors.affiliation: Thrombosis and Haemostasis Laboratory, Department of Haematology, University Medical Center and Institute of Biomembranes, HP G03.647, P. O. Box 85500, 3508 GA Utrecht, The Netherlands.
authors.forename: Roland A
authors.initials: RA
authors.lastname: Romijn
authors.forename: Erik
authors.initials: E
authors.lastname: Westein
authors.forename: Barend
authors.initials: B
authors.lastname: Bouma
authors.forename: Marion E
authors.initials: ME
authors.lastname: Schiphorst
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Sixma
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Lenting
authors.forename: Eric G
authors.initials: EG
authors.lastname: Huizinga
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D024061:Collagen Type III / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D014162:Transfection; D014841:von Willebrand Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D024061:Collagen Type III; D014841:von Willebrand Factor