Role of the A⁺ Helix in Heparin Binding to Protein C Inhibitor

Summary

Interactions between proteins and heparin(-like) structures involve electrostatic forces and structural features. Based on charge distributions in the linear sequence of protein C inhibitor (PCI), two positively charged regions of PCI were proposed as possible candidates for this interaction. The first region, the A⁺ helix, is located at the N-terminus (residues 1-11), whereas the second region, the H helix, is positioned between residues 264 and 280 of PCI. Competition experiments with synthetic peptides based on the sequence of these regions demonstrated that the H helix has the highest affinity for heparin. In contrast to previous observations we found that the A⁺ helix peptide competed for the interaction of PCI with heparin, but its affinity was much lower than that of the H helix peptide.

Recombinant PCI was also used to investigate the role of the A⁺ helix in heparin binding. Full-length (wild-type) rPCI as well as an A⁺ helix deletion mutant of PCI (rPCI-Δ2-l 1) were expressed in baby hamster kidney cells and both had normal inhibition activity with activated protein C and thrombin. The interaction of the recombinant PCIs with heparin was investigated and compared to plasma PCI. The A⁺ helix deletion mutant showed a decreased affinity for heparin in inhibition reactions with activated protein C and thrombin, but had similar association constants compared to wild-type rPCI. The synthetic A⁺ helix peptide competed with rPCI-Δ2-11 for binding to heparin. This indicated that the interaction between PCI and heparin is fairly non-specific and that the interaction is primarily based on electrostatic interactions.

In summary, our data suggest that the H helix of PCI is the main heparin binding region of PCI, but the A⁺ helix increases the overall affinity for the PCI-heparin interaction by contributing a second positively charged region to the surface of PCI.

• References

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