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A protein S binding site on C4b-binding protein involves beta chain residues 31-45
1Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
Insights
C4b-binding protein (C4BP) binding to protein S involves specific residues on the C4BP beta chain. This interaction is crucial for regulating anticoagulant activity in the protein C pathway.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- C4b-binding protein (C4BP) regulates the anticoagulant cofactor activity of protein S.
- Only free protein S, not the protein S:C4BP complex, exhibits anticoagulant activity.
- Understanding the protein S binding site on C4BP is essential for elucidating this regulatory mechanism.
Purpose of the Study:
- To identify the specific beta chain residues of C4BP involved in binding protein S.
- To investigate the functional significance of the identified binding site in protein S:C4BP complex formation and anticoagulant activity.
Main Methods:
- Synthesis of overlapping pentadecapeptides covering the C4BP beta chain (residues 1-235).
- Inhibition assays using synthetic peptides to block C4BP:protein S complex formation.
- Coagulation assays to assess the effect of peptides on protein S cofactor activity.
- Binding studies using antipeptide antibodies and immobilized peptides to confirm direct interaction.
Main Results:
- Peptide beta(31-45) from the first short consensus repeat domain significantly inhibited C4BP:protein S binding.
- The sequence YxLVG within beta(31-45) was identified as crucial for binding.
- Peptide beta(31-45) specifically inhibited protein S cofactor activity in activated protein C (APC) assays.
- Protein S was shown to bind directly to immobilized peptide beta(31-45).
Conclusions:
- Residues 31-45 of the C4BP beta chain constitute a primary binding site for protein S.
- The C4BP beta chain residues 34-42 are spatially close to residues 420-434 of protein S within the complex.
- These findings provide critical insights into the molecular basis of protein S:C4BP interaction and its role in coagulation regulation.
Abstract:
C4b-binding protein (C4BP) down-regulates the anticoagulant cofactor activity of protein S in the protein C pathway since free protein S but not the protein S:C4BP complex is anticoagulantly active. To identify beta chain residues responsible for binding protein S, synthetic overlapping pentadecapeptides covering the entire 1-235 sequence were tested as inhibitors of complex formation. The peptide comprising residues 31-45 (VCIKGYHLVGKKTLF) from the first short consensus repeat domain inhibited the binding of C4BP to protein S with half-maximal inhibition at 20-45 microM, and studies suggested the sequence of YxLVG was crucial. Peptide beta(31-45) specifically inhibited the APC cofactor activity of purified protein S in Xa-1-stage coagulation assays with 50% inhibition at 15 microM peptide. Peptide beta(31-45) and related peptides such as beta(34-42) inhibited the binding of protein S to an antipeptide monoclonal antibody made against residues 420-434 of protein S (monoclonal antibody LJ-56). Polyclonal anti-beta(31-45) peptide antibodies inhibited complex formation. Dose-dependent binding studies showed that protein S bound directly to immobilized peptide beta(31-45). These results show that residues 31-45 of the C4BP beta chain provide a binding site for protein S, and they suggest that the C4BP beta chain residues 34-42 are located near residues 420-434 of protein S in the protein S:C4BP complex.