title: Characterization of the heparin-binding site of the protein z-dependent protease inhibitor.
abstract: High-molecular weight heparins promote the protein Z-dependent protease inhibitor (ZPI) inhibition of factors Xa (FXa) and XIa (FXIa) by a template mechanism. To map the heparin-binding site of ZPI, the role of basic residues of the D-helix (residues Lys-113, Lys-116, and Lys-125) in the interaction with heparin was evaluated by either substituting these residues with Ala (ZPI-3A) or replacing the D-helix with the corresponding loop of the non-heparin-binding serpin α(1)-proteinase inhibitor (ZPI-D-helix(α1-PI)). Furthermore, both the C-helix (contains two basic residues, Lys-104 and Arg-105) and the D-helix of ZPI were substituted with the corresponding loops of α(1)-proteinase inhibitor (ZPI-CD-helix(α1-PI)). All mutants exhibited near normal reactivity with FXa and FXIa in the absence of cofactors and in the presence of protein Z and membrane cofactors. By contrast, the mutants interacted with heparin with a lower affinity and the ~48-fold heparin-mediated enhancement in the rate of FXa inhibition by ZPI was reduced to ~30-fold for ZPI-3A, ~15-fold for ZPI-D-helix(α1-PI), and ~8-fold for ZPI-CD-helix(α1-PI). Consistent with a template mechanism for heparin cofactor action, ZPI-CD-helix(α1-PI) inhibition of a FXa mutant containing a mutation in the heparin-binding site (FXa-R240A) was minimally affected by heparin. A significant decrease (~2-5-fold) in the heparin template effect was also observed for the inhibition of FXIa by ZPI mutants. Interestingly, ZPI derivatives exhibited a markedly elevated stoichiometry of inhibition with FXIa in the absence of heparin. These results suggest that basic residues of both helices C and D of ZPI interact with heparin to modulate the inhibitory function of the serpin.
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO 63104, USA.
authors.forename: Likui
authors.initials: L
authors.lastname: Yang
authors.forename: Qiulan
authors.initials: Q
authors.lastname: Ding
authors.forename: Xin
authors.initials: X
authors.lastname: Huang
authors.forename: Steven T
authors.initials: ST
authors.lastname: Olson
authors.forename: Alireza R
authors.initials: AR
authors.lastname: Rezaie
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001798:Blood Proteins / Q000378:metabolism; D015945:Factor XIa / Q000037:antagonists & inhibitors; D065427:Factor Xa Inhibitors; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015843:Serpins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D001798:Blood Proteins; D065427:Factor Xa Inhibitors; D011994:Recombinant Proteins; C416370:SERPINA10 protein, human; D015843:Serpins; C026080:plasma protein Z; D006493:Heparin; D015945:Factor XIa