A region of vitamin K-dependent protein S that binds to C4b binding protein (C4BP) identified using bacteriophage peptide display libraries.
Vitamin K-dependent protein S, a blood coagulation inhibitor, interacts with the C4b-binding protein (C4BP) in human plasma with high affinity (KD = 0.1 nM). Identification of a portion of protein S that binds to C4BP has been approached using random libraries of 6- and 15-mer peptides displayed on bacteriophage surfaces. Bacteriophage binding to the beta-chain of C4BP were selected in several rounds of affinity purification with intervening amplification in E. coli. Homology searches of the affinity purified peptide sequences against protein S led to the identification of four regions in protein S that were similar to several of the selected peptides. These regions were synthesized as linear peptides and tested in inhibition experiments. Only one distinct peak (around position 450) was observed when the homology scores versus human protein S sequence were averaged over all affinity purified peptides. A synthetic peptide comprising residues 439-460 in human protein S was found to inhibit protein S binding to C4BP. The same result was found with two overlapping peptides (residues 447-468 and 435-468, respectively) in a second set of synthetic peptides. Direct binding of the peptides to C4BP was inferred from titrations monitored by recording the near UV circular dichroism spectra or the polarization of tryptophan fluorescence. The results suggest that residues 447-460 constitute a portion of protein S that is important for the interaction with C4BP. These findings may have implications for patients suffering from thrombosis, due to the lack of free protein S, by directing the design of drugs that disrupt protein S binding to C4BP.
Linse
S
S
Department of Physical Chemistry 2, Lund University, S-221 00 Lund, Sweden. sara@bor.fkem2.lth.se
Härdig
Y
Y
Schultz
D A
DA
Dahlbäck
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D015935:Complement C4b / Q000378:metabolism; D003169:Complement Inactivator Proteins; D004247:DNA / Q000737:chemistry; D017931:DNA Primers; D006023:Glycoproteins; D000882:Haplorhini; D006801:Humans; D007256:Information Systems; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D011487:Protein Conformation; D017293:Protein S / Q000737:chemistry* / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011951:Receptors, Complement / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013552:Swine
D003169:Complement Inactivator Proteins; D017931:DNA Primers; D006023:Glycoproteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D017293:Protein S; D011951:Receptors, Complement; D011994:Recombinant Proteins; D015935:Complement C4b; D004247:DNA