CONTRIBUTION OF BASIC RESIDUES OF THE D-HELICE AND H-HELICE IN HEPARIN-BINDING TO PROTEIN-C INHIBITOR

Protein C inhibitor (PCI) is a heparin-binding serine protease inhibit or (serpin) that regulates hemostatic proteases such as activated prot ein C (APC) and thrombin, The work described here provides further evi dence that the PCI H helix, but not the D helix, has a major role in h eparin-accelerated inhibition of APC and thrombin. We previously ident ified Arg-269 and Lys-270 of the H helix [R269A/K270A ''H1'' recombina nt PCI (rPCI)] as important residues both for heparin-accelerated inhi bition 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 (k(2) = 2.2 x 10(7) M-l min-l for thrombin), heparin-accelera ted 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 throm bin inhibition was essentially not accelerated by heparin. A similar t rend was found for APC-heparin inhibition using these H helix rPCI mut ants. In contrast, the D helix rPCI mutants did not have further reduc ed heparin-stimulated thrombin or APC inhibition compared to H1 rPCI, Interestingly, all of the H and D helix rPCI mutants had reduced hepar in-Sepharose binding activity (ranging from 180 to 360 mM NaCl) compar ed 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-26 6, Arg-269, Lys-270, Lys-273) in the H helix of PCI form a heparin bin ding site. Our results also imply that while the D helix basic residue s (Lys-80, Lys-86, and Arg-90) contribute to overall heparin binding, they are not necessary for heparin-accelerated activity. We conclude t hat 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. (C) 1998 Aeademic Press.