Role of the A+ helix in heparin binding to protein C inhibitor.
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-delta 2-11) 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-delta w-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.
Elisen
M G
MG
Department of Haematology, University Hospital, Utrecht, The Netherlands.
Maseland
M H
MH
Church
F C
FC
Bouma
B N
BN
Meijers
J C
JC
D000818:Animals; D006224:Cricetinae; D019077:Helix-Turn-Helix Motifs; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011486:Protein C / Q000378:metabolism; D018046:Protein C Inhibitor / Q000378:metabolism*
D011486:Protein C; D018046:Protein C Inhibitor; D006493:Heparin