title: Contribution of basic residues of the D and H helices in heparin binding to protein C inhibitor.
abstract: Protein C inhibitor (PCI) is a heparin-binding serine protease inhibitor (serpin) that regulates hemostatic proteases such as activated protein C (APC) and thrombin. The work described here provides further evidence that the PCI H helix, but not the D helix, has a major role in heparin-accelerated inhibition of APC and thrombin. We previously identified Arg-269 and Lys-270 of the H helix [R269A/K270A "H1" recombinant PCI (rPCI)] as important residues both for heparin-accelerated inhibition of thrombin and APC and for heparin-Sepharose binding (Shirk, R. A., Elisen, M. G. L. M., Meijers, J. C. M., and Church, F. C. (1994) J. Biol. Chem. 269, 28690-28695). H1 rPCI was used as a template for Ala-scanning mutagenesis of other H helix basic residues (H1-K266A, H1-K273A, and H1-K266A/K273A) and of the D helix basic residues (H1-K82A, H1-K86A, H1-R90A, and H1-K82A/K86A/R90A). Compared to wild-type rPCI/heparin (k2 = 2.2 x 10(7) M-1 min-1 for thrombin), heparin-accelerated thrombin inhibition was decreased 2.4-fold by H1 rPCI, 4.4-fold by H1-K266A rPCI, and 8-fold by H1-K273A rPCI. H1-K266A/K273A rPCI thrombin inhibition was essentially not accelerated by heparin. A similar trend was found for APC-heparin inhibition using these H helix rPCI mutants. In contrast, the D helix rPCI mutants did not have further reduced heparin-stimulated thrombin or APC inhibition compared to H1 rPCI. Interestingly, all of the H and D helix rPCI mutants had reduced heparin-Sepharose binding activity (ranging from 180 to 360 mM NaCl) compared to wild-type rPCI and H1 rPCI, which eluted at 650 and 430 mM NaCl, respectively. These data suggest that all four basic residues (Lys-266, Arg-269, Lys-270, Lys-273) in the H helix of PCI form a heparin binding site. Our results also imply that while the D helix basic residues (Lys-80, Lys-86, and Arg-90) contribute to overall heparin binding, they are not necessary for heparin-accelerated activity. We conclude that the primary heparin binding site of PCI is the H helix and not the D helix as found in other homologous heparin-binding serpins such as antithrombin III, heparin cofactor II, and protease nexin 1.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, North Carolina, 27599, USA.
authors.forename: L L
authors.initials: LL
authors.lastname: Neese
authors.forename: C A
authors.initials: CA
authors.lastname: Wolfe
authors.forename: F C
authors.initials: FC
authors.lastname: Church
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D006493:Heparin / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D018046:Protein C Inhibitor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D017931:DNA Primers; D018046:Protein C Inhibitor; D011994:Recombinant Proteins; D006493:Heparin