Rezaie Alireza R, Bae Jong-Sup, Manithody Chandrashekhara, Qureshi Shabir H, Yang Likui
Edward A Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO 63104, USA.
J Biol Chem. 2008 Jul 18;283(29):19922-6. doi: 10.1074/jbc.M802639200. Epub 2008 May 23.
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.
蛋白Z（PZ）是一种多结构域的维生素K依赖性血浆蛋白，作为一种辅因子发挥作用，可使PZ依赖性蛋白酶抑制剂（ZPI）对因子Xa（fXa）的灭活作用增强三个数量级。为了了解PZ提高fXa与ZPI反应活性的机制，我们表达了野生型PZ、缺失γ-羧基谷氨酸结构域的PZ（GD-PZ）以及一种嵌合PZ突变体，该突变体分子的Gla和表皮生长因子样结构域均被fXa的相同结构域所取代。通过结合和动力学分析对PZ衍生物的ZPI结合及辅因子功能进行了表征。结合分析表明，所有PZ衍生物与ZPI相互作用的解离常数（K(D)）相似，约为7 nM。然而，在PC/PS囊泡上，嵌合PZ介导的ZPI对fXa的抑制作用的表观K(D)升高了6倍，其作为辅因子加速ZPI对fXa抑制作用的能力也降低了6倍。GD-PZ的辅因子活性显著受损；然而，该缺失突变体在溶液中表现出正常的辅因子功能。含有fXa的Gla结构域的嵌合活化蛋白C突变体在PZ存在的情况下易受ZPI的抑制。这些结果表明：（i）PZ的ZPI相互作用位点位于辅因子的C末端结构域内；（ii）PZ和fXa的Gla结构域之间的特异性相互作用使磷脂膜上ZPI对fXa的抑制作用加速约6倍。
