A protein S binding site on C4b-binding protein involves beta chain residues 31-45

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.