Intermolecular interactions between protein C inhibitor and coagulation proteases

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Protein C inhibitor (PCI) is a crucial plasma serine protease inhibitor.
• Understanding PCI's specificity is vital for elucidating serpin-protease interactions.
• Previous studies lacked detailed residue-specific insights into PCI's broad inhibitory activity.

Purpose of the Study:

• To identify specific residues in the PCI reactive site loop that dictate its inhibition specificity.
• To investigate the molecular interactions governing PCI's kinetics with thrombin, activated protein C (APC), and factor Xa.
• To correlate mutational effects with structural insights derived from molecular modeling.

Main Methods:

• Generated 19 mutations within the PCI reactive site loop (Thr352 to Arg357).
• Assayed inhibitory activity of mutant PCI against thrombin, APC, and factor Xa.
• Constructed a molecular model of PCI and performed docking simulations with target proteases.

Main Results:

• Mutations at the P2 position (Phe353) with Pro or Gly significantly enhanced thrombin inhibition (2- and 7-fold increases).
• Substitutions at the P3 position (Thr352) with Arg increased inhibition rates for APC (2-fold) and factor Xa (5-fold).
• Molecular modeling predicted specific interactions, such as salt bridges, explaining the observed kinetic changes.

Conclusions:

• Specific residues in the PCI reactive site loop, particularly at P2 and P3 positions, are critical for protease specificity.
• Mutations altering steric hindrance and forming new interactions (e.g., salt bridges) modulate inhibitory potency.
• PCI serves as a valuable model system for studying serpin-protease interactions through combined mutagenesis and molecular modeling.