Huang Xin, Zhou Jian, Zhou Aiwu, Olson Steven T
From the Center for Molecular Biology of Oral Diseases and Department of Periodontics, University of Illinois at Chicago, Chicago, Illinois 60612 and
From the Center for Molecular Biology of Oral Diseases and Department of Periodontics, University of Illinois at Chicago, Chicago, Illinois 60612 and.
J Biol Chem. 2015 Apr 10;290(15):9906-18. doi: 10.1074/jbc.M114.633479. Epub 2015 Feb 20.
The anticoagulant serpin, protein Z-dependent protease inhibitor (ZPI), circulates in blood as a tight complex with its cofactor, protein Z (PZ), enabling it to function as a rapid inhibitor of membrane-associated factor Xa. Here, we show that N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-3-oxa-1,3-diazol-4-yl)ethylenediamine (NBD)-fluorophore-labeled K239C ZPI is a sensitive, moderately perturbing reporter of the ZPI-PZ interaction and utilize the labeled ZPI to characterize in-depth the thermodynamics and kinetics of wild-type and variant ZPI-PZ interactions. NBD-labeled K239C ZPI bound PZ with ∼3 nM KD and ∼400% fluorescence enhancement at physiologic pH and ionic strength. The NBD-ZPI-PZ interaction was markedly sensitive to ionic strength and pH but minimally affected by temperature, consistent with the importance of charged interactions. NBD-ZPI-PZ affinity was reduced ∼5-fold by physiologic calcium levels to resemble NBD-ZPI affinity for γ-carboxyglutamic acid/EGF1-domainless PZ. Competitive binding studies with ZPI variants revealed that in addition to previously identified Asp-293 and Tyr-240 hot spot residues, Met-71, Asp-74, and Asp-238 made significant contributions to PZ binding, whereas Lys-239 antagonized binding. Rapid kinetic studies indicated a multistep binding mechanism with diffusion-limited association and slow complex dissociation. ZPI complexation with factor Xa or cleavage decreased ZPI-PZ affinity 2-7-fold by increasing the rate of PZ dissociation. A catalytic role for PZ was supported by the correlation between a decreased rate of PZ dissociation from the K239A ZPI-PZ complex and an impaired ability of PZ to catalyze the K239A ZPI-factor Xa reaction. Together, these results reveal the energetic basis of the ZPI-PZ interaction and suggest an important role for ZPI Lys-239 in PZ catalytic action.
抗凝丝氨酸蛋白酶抑制剂、蛋白Z依赖性蛋白酶抑制剂（ZPI）作为与其辅因子蛋白Z（PZ）的紧密复合物在血液中循环，使其能够作为膜相关因子Xa的快速抑制剂发挥作用。在此，我们表明，N，N'-二甲基-N-（乙酰基）-N'-（7-硝基苯并-3-恶唑-1，3-二氮杂-4-基）乙二胺（NBD）荧光团标记的K239C ZPI是ZPI-PZ相互作用的灵敏、适度干扰的报告分子，并利用标记的ZPI深入表征野生型和变体ZPI-PZ相互作用的热力学和动力学。在生理pH和离子强度下，NBD标记的K239C ZPI以约3 nM的KD结合PZ，荧光增强约400%。NBD-ZPI-PZ相互作用对离子强度和pH非常敏感，但受温度影响最小，这与带电相互作用的重要性一致。生理钙水平使NBD-ZPI-PZ亲和力降低约5倍，类似于NBD-ZPI对γ-羧基谷氨酸/EGF1结构域缺失的PZ的亲和力。与ZPI变体的竞争性结合研究表明，除了先前确定的Asp-293和Tyr-240热点残基外，Met-71、Asp-74和Asp-238对PZ结合有显著贡献，而Lys-239则拮抗结合。快速动力学研究表明，其结合机制为多步骤，具有扩散限制的缔合和缓慢的复合物解离。ZPI与因子Xa的复合或裂解通过增加PZ解离速率使ZPI-PZ亲和力降低2-7倍。PZ从K239A ZPI-PZ复合物解离速率的降低与PZ催化K239A ZPI-因子Xa反应能力受损之间存在相关性，这支持了PZ的催化作用。总之，这些结果揭示了ZPI-PZ相互作用的能量基础，并表明ZPI Lys-239在PZ催化作用中起重要作用。
