Abstract
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.
Highlights
Von Willebrand factor 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
Platelet adhesion to damaged vessel walls is the first step in the formation of an occluding platelet plug, which leads to the arrest of bleeding during normal hemostasis
In this process vWF serves as a molecular bridge that links collagen exposed by the damaged vessel wall to glycoprotein Ib located on the platelet surface
Summary
Purification of vWF-A3 and RU5—Recombinant selenomethionine (Se-Met) A3, comprising residues 920 –1111 of human vWF, was expressed and purified as described before [15]. Crystallization and Data Collection—Crystals of the (Se-Met) A31⁄7RU5 complex were grown by hanging-drop vapor diffusion at 4 °C using a protein concentration of 15 mg/ml and a precipitant solution consisting of 13% (v/v) isopropanol, 22% (v/v) 2-methyl-2,4-pentanediol, and 100 mM cacodylic acid, pH 5.3. A 518-base pair NheI-Csp45I fragment corresponding to amino acid residues 940 –1113 of mature vWF was subcloned into pBluescript SKII (Stratagene, La Jolla, CA). To this end a unique Csp45I restriction site was introduced at position 5628 of expression vector pNUT-vWFcas [8] in the following way. Ligation of this fragment into EcoRI-AccI-digested and Pwo-treated pBluescript SKII, produced mutagenesis plasmid pBSvWF-NC This plasmid retains the NheI and Csp45I restriction sites. A vWF concentration of 2.5 g/ml was used in the binding experiments
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