Protein Z-dependent Protease Inhibitor Binds to the C-terminal Domain of Protein Z

Alireza R Rezaie,Jong-Sup Bae,Chandrashekhara Manithody,Shabir H Qureshi,Likui Yang

doi:10.1074/jbc.m802639200

Abstract

Protein Z (PZ) is a multidomain vitamin K-dependent plasma protein that functions as a cofactor to promote the inactivation of factor Xa (fXa) by PZ-dependent protease inhibitor (ZPI) by three orders of magnitude. To understand the mechanism by which PZ improves the reactivity of fXa with ZPI, we expressed wild-type PZ, PZ lacking the gamma-carboxyglutamic acid domain (GD-PZ), and a chimeric PZ mutant in which both Gla and EGF-like domains of the molecule were substituted with identical domains of fXa. The ZPI binding and the cofactor function of the PZ derivatives were characterized in both binding and kinetic assays. The binding assay indicated that all PZ derivatives interact with ZPI with a similar dissociation constant (K(D)) of approximately 7 nm. However, the apparent K(D) for the chimeric PZ-mediated ZPI inhibition of fXa was elevated 6-fold on PC/PS vesicles and its capacity to function as a cofactor to accelerate the ZPI inhibition of fXa was also decreased 6-fold. The cofactor activity of GD-PZ was dramatically impaired; however, the deletion mutant exhibited a normal cofactor function in solution. A chimeric activated protein C mutant containing the Gla domain of fXa was susceptible to inhibition by ZPI in the presence of PZ. These results suggest that: (i) the ZPI interactive site of PZ is located within the C-terminal domain of the cofactor and (ii) a specific interaction between the Gla domains of PZ and fXa contributes approximately 6-fold to the acceleration of the ZPI inhibition of fXa on phospholipid membranes.

Highlights

Ulation proteases, two of the catalytic triad residues of the C-terminal domain have not been conserved in the homologous regions of Protein Z (PZ) [1, 2]
Expression and Purification of Recombinant Proteins—Recombinant PZ derivatives were expressed in HEK-293 or insect cells (GD-PZ) in fusion with the 12-residue epitope for the Ca2ϩ-dependent monoclonal HPC4 antibody to facilitate their purification by immunoaffinity chromatography as described under “Experimental Procedures.”
Interaction with ZPI—The PZ concentration dependence of the ZPI inhibition of factor Xa (fXa) on phosphatidylcholine and 20% phosphatidylserine (PC/PS) vesicles suggested that both pPZ and rPZ exhibit similar cofactor activity, promoting the ZPI inhibition of fXa with Kd(app) values of 1.1 nM and 1.4 nM, respectively, on PC/PS vesicles in the presence of calcium (Fig. 2A)

Summary

Introduction

Ulation proteases, two of the catalytic triad residues of the C-terminal domain have not been conserved in the homologous regions of PZ [1, 2]. An activated protein C chimera containing the Gla domain of fXa reacted with ZPI in the presence, but not in the absence of PZ on PC/PS vesicles in the presence of Ca2ϩ.

Results

Conclusion