Analysis of structure-function relationships in the platelet membrane glycoprotein Ib-binding domain of von Willebrand's factor by expression of deletion mutants.
We have used a series of Escherichia coli-expressed deletion mutants of the glycoprotein (GP) Ib-binding domain of von Willebrand factor (vWF) to study the structural basis of its function. In addition to the prototypic molecule (rvWF441-733), we constructed 11 mutants; seven had deletions of sequence on the amino- and/or carboxyl-terminal side of the Cys509-Cys695 intrachain disulfide loop, and four had limited deletions inside the loop. Other cysteine residues in addition to 509 and 695, when present in the corresponding native sequence, were mutated to glycine; all molecules were purified in the oxidized as well as reduced and alkylated state. The smallest species retaining the ability to interact with GP Ib in the absence of modulators was the oxidized rvWF508-696; the latter, as well as rvWF441-696, became inactive after reduction and alkylation. In contrast, all the other fragments with deletions outside of the loop, but extending at least to residue 700, showed better binding to platelets after reduction and alkylation than when the Cys509-Cys695 disulfide bond was oxidized. Any limited deletion of sequence inside the loop caused complete loss of GP Ib-binding function both in the absence or in the presence of botrocetin, and this persisted even after reduction and alkylation. In contrast, all mutants with intact sequence between residues 509 and 695 bound to GP Ib in the presence of botrocetin, regardless of whether the 2 cysteine residues were oxidized or reduced and alkylated. Ristocetin, unlike botrocetin, appeared to have no effect in modulating the binding of any of the expressed fragments to platelets. Our findings suggest that the GP Ib-binding domain of vWF contains multiple interaction sites, but integrity of the sequence 509-695 is important for function.
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
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M
Sugimoto
J
J
Dent
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McClintock
J
J
Ware
Z M
ZM
Ruggeri
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003545:Cysteine; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014841:von Willebrand Factor / Q000378:metabolism*
D004220:Disulfides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D003545:Cysteine