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 ∼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.