Structural requirements of anticoagulant protein S for its binding to the complement regulator C4b-binding protein.
The vitamin K-dependent anticoagulant protein S binds with high affinity to C4b-binding protein (C4BP), a regulator of complement. Despite the physiological importance of the complex, we have only a patchy view of the C4BP-binding site in protein S. Based on phage display experiments, protein S residues 447-460 were suggested to form part of the binding site. Several experimental approaches were now used to further elucidate the structural requirements for protein S binding to C4BP. Peptides comprising residues 447-460, 451-460, or 453-460 of protein S were found to inhibit the protein S-C4BP interaction, whereas deletion of residues 459-460 from the peptide caused complete loss of inhibition. In recombinant protein S, each of residues 447-460 was mutated to Ala, and the protein S variants were tested for binding to C4BP. The Y456A mutation reduced binding to C4BP approximately 10-fold, and a peptide corresponding to residues 447-460 of this mutant was less inhibitory than the parent peptide. A further decrease in binding was observed using a recombinant variant in which a site for N-linked glycosylation was moved from position 458 to 456 (Y456N/N458T). A monoclonal antibody (HPSf) selective for free protein S reacted poorly with the Y456A variant but reacted efficiently with the other variants. A second antibody, HPS 34, which partially inhibited the protein S-C4BP interaction, reacted poorly with several of the Ala mutants, suggesting that its epitope was located in the 451-460 region. Phage display analysis of the HPS 34 antibody further identified this region as its epitope. Taken together, our results suggest that residues 453-460 of protein S form part of a more complex binding site for C4BP. A recently developed three-dimensional model of the sex hormone-binding globulin-like region of protein S was used to analyze available experimental data.
Giri
Tusar Kanti
TK
Department of Laboratory Medicine, Division of Clinical Chemistry, Lund University, University Hospital, S-20502 Malmö, Sweden.
Linse
Sara
S
García de Frutos
Pablo
P
Yamazaki
Tomio
T
Villoutreix
Bruno O
BO
Dahlbäck
Björn
B
D000595:Amino Acid Sequence; D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism*; D015935:Complement C4b / Q000378:metabolism*; D003169:Complement Inactivator Proteins; D018604:Epitope Mapping; D006023:Glycoproteins; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017293:Protein S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011951:Receptors, Complement / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012738:Sex Hormone-Binding Globulin / Q000378:metabolism; D020349:Surface Plasmon Resonance
D000925:Anticoagulants; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D017293:Protein S; D011951:Receptors, Complement; D011994:Recombinant Proteins; D012738:Sex Hormone-Binding Globulin; D015935:Complement C4b