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 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 Arg(963), Glu(987), His(990), Arg(1016), and His(1023), located in or close to the epitope. Mutants were expressed as fully processed multimeric vWF. Mutation of His(1023) abolished collagen binding, whereas mutation of Arg(963) and Arg(1016) reduced collagen binding by 25-35%. These residues are part of loops alpha3beta4 and alpha1beta2 and alpha-helix 3, respectively, and lie near the bottom face of the domain. His(1023) 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.
Romijn
R A
RA
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.
Bouma
B
B
Wuyster
W
W
Gros
P
P
Kroon
J
J
Sixma
J J
JJ
Huizinga
E G
EG
D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D003094:Collagen / Q000378:metabolism*; D018360:Crystallography, X-Ray; D000939:Epitopes; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014841:von Willebrand Factor / Q000737:chemistry* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D006639:Histidine; D003094:Collagen; D001120:Arginine; D000409:Alanine