title: Thermodynamic and kinetic characterization of the protein Z-dependent protease inhibitor (ZPI)-protein Z interaction reveals an unexpected role for ZPI Lys-239.
abstract: 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.
authors.affiliation: From the Center for Molecular Biology of Oral Diseases and Department of Periodontics, University of Illinois at Chicago, Chicago, Illinois 60612 and xinhuang@uic.edu.
authors.forename: Xin
authors.initials: X
authors.lastname: Huang
authors.affiliation: From the Center for Molecular Biology of Oral Diseases and Department of Periodontics, University of Illinois at Chicago, Chicago, Illinois 60612 and.
authors.forename: Jian
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Cell Differentiation and Apoptosis of Ministry of Education of China, Shanghai JiaoTong University School of Medicine, Shanghai 200025, China.
authors.forename: Aiwu
authors.initials: A
authors.lastname: Zhou
authors.affiliation: From the Center for Molecular Biology of Oral Diseases and Department of Periodontics, University of Illinois at Chicago, Chicago, Illinois 60612 and.
authors.forename: Steven T
authors.initials: ST
authors.lastname: Olson
mesh_terms: D000465:Algorithms; D001393:Azoles / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D001798:Blood Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D015951:Factor Xa / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009578:Nitrobenzenes / Q000737:chemistry; D009994:Osmolar Concentration; D011485:Protein Binding; D015843:Serpins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
chemical_list: C510797:7-nitrobenz-2-oxa-1,3-diazol-4-yl; D001393:Azoles; D001798:Blood Proteins; D005456:Fluorescent Dyes; D009578:Nitrobenzenes; C416370:SERPINA10 protein, human; D015843:Serpins; C026080:plasma protein Z; D015951:Factor Xa; D008239:Lysine; D002118:Calcium
keywords: Anticoagulant; Coagulation Factor; Factor Xa; Kinetics; Protease; Protease Inhibitor; Protein Z; Serpin; ZPI