Disulfide bond requirements for assembly of the platelet glycoprotein Ib-binding domain of von Willebrand factor.
von Willebrand factor (vWF) is a multimeric glycoprotein of plasma and the subendothelial matrix that interacts with specific platelet receptors to establish platelet adhesion at a site of vascular injury. The vWF domain containing the platelet receptor glycoprotein Ib-binding site can be expressed in heterologous cells as a recombinant homodimeric fragment that mediates platelet-platelet interaction, analogous to multimeric vWF. The recombinant domain, r116, contains 7 Cys residues within its 290-residue monomeric subunit paired in an unidentified intra- and intermolecular disulfide bond arrangement. In this report we define the disulfide bond-dependent framework of r116 that provides the domain with its essential structural features that support dimer formation and the generation of a disulfide bond-dependent epitope. The results demonstrate that a triplet of Cys residues at positions 459, 462, and 464 are essential for efficient dimer formation. An intramolecular Cys509/Cys695 disulfide loop is required for generating a functional dimeric molecule, and monomeric molecules containing a Cys509/Cys695 intramolecular disulfide bond are unable to support ristocetin-mediated platelet aggregation. The disulfide arrangement in r116 is similar, if not identical, to the proposed arrangement within the corresponding region of plasma vWF, and these studies document the inherent Cys-dependent maturation of an isolated vWF domain.
Azuma
H
H
Roon Research Laboratory for Arteriosclerosis and Thrombosis, Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
Hayashi
T
T
Dent
J A
JA
Ruggeri
Z M
ZM
Ware
J
J
D004220:Disulfides; D000939:Epitopes; D006801:Humans; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012310:Ristocetin / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014841:von Willebrand Factor / Q000378:metabolism*
D004220:Disulfides; D000939:Epitopes; D010980:Platelet Membrane Glycoproteins; D014841:von Willebrand Factor; D012310:Ristocetin