Identification of factor Xa residues critical for interaction with protein Z-dependent protease inhibitor: both active site and exosite interactions are required for inhibition.
Protein Z-dependent protease inhibitor (ZPI) is a plasma serpin, which can rapidly inactivate factor Xa (fXa) in the presence of protein Z (PZ), negatively charged phospholipids, and Ca2+. To investigate the mechanism by which ZPI inactivates fXa, we expressed the serpin in mammalian cells and characterized its reactivity with both wild-type and selected mutants of fXa that 1) contained substitutions in the autolysis loop and the heparin binding exosite, 2) lacked the first EGF-like domain (fXa-des-EGF-1), or 3) contained the Gla domain of protein C (fXa/PC-Gla). Inhibition studies in both the presence and absence of PZ revealed that Arg-143, Lys-147, and Arg-154 of the autolysis loop and Lys-96, Lys-169, and Lys-236 of the heparin binding exosite are required for recognition of ZPI, with Arg-143 being essential for the interaction. Similar studies with fXa-des-EGF-1 and fXa/PC-Gla suggested that protein-protein interaction with either the Gla or the EGF-1 domain may not play a dominant role in the PZ-dependent recognition of fXa by the serpin on phospholipid vesicles. Further studies showed that an inactive Ser-195 to Ala mutant of fXa effectively competes with wild-type fXa for binding to the non-serpin inhibitors tissue factor pathway inhibitor and recombinant tick anticoagulant peptide, but does not compete for binding to ZPI. This suggests that the catalytic residue of fXa is required for interaction with ZPI.
Rezaie
Alireza R
AR
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri 63104, USA. rezaier@slu.edu
Manithody
Chandrashekhara
C
Yang
Likui
L
D000409:Alanine / Q000737:chemistry; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000737:chemistry; D015951:Factor Xa / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008074:Lipoproteins / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008954:Models, Biological; D016296:Mutagenesis; D009154:Mutation; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D015843:Serpins / Q000737:chemistry* / Q000378:metabolism
D008074:Lipoproteins; D010743:Phospholipids; D011994:Recombinant Proteins; C416370:SERPINA10 protein, human; D015843:Serpins; C051928:lipoprotein-associated coagulation inhibitor; D012694:Serine; D004815:Epidermal Growth Factor; D006493:Heparin; D001120:Arginine; D015951:Factor Xa; D008239:Lysine; D000409:Alanine