title: The high affinity calcium-binding site involved in protein C activation is outside the first epidermal growth factor homology domain.
abstract: Binding Ca2+ to a high affinity site in protein C and 4-carboxyglutamic acid (Gla)-domainless protein C results in a conformational change that is required for activation by the thrombin-thrombomodulin complex, the natural activator of protein C. It has been hypothesized that this high affinity Ca(2+)-binding site is located in the NH2-terminal epidermal growth factor (EGF) homology region of protein C. We have expressed in human 293 cells a deletion mutant of protein C (E2-PD) which lacks the entire Gla region as well as the NH2-terminal EGF homology region of protein C. Ca2+ inhibits activation of E2-PD or Gla-domainless protein C by thrombin with half-maximal inhibition occurring at Ca2+ concentrations of 103 +/- 11 and 70 +/- 7 microM, respectively, but is required for both E2-PD and Gla-domainless protein C activation by the thrombin-thrombomodulin complex with half-maximal acceleration occurring at Ca2+ concentrations of 87 +/- 8 and 89 +/- 8 microM, respectively. Both E2-PD and Gla-domainless protein C exhibit a reversible, Ca(2+)- but not Mg(2+)-dependent decrease (6 +/- 1%) in fluorescence emission intensity with Kd = 38 +/- 3 microM Ca2+. We conclude that the high affinity Ca(2+)-binding site important for the activation of protein C is located outside of the NH2-terminal EGF homology region and that the metal-binding site in the NH2-terminal EGF homology region may not be a high affinity site in intact protein C.
authors.affiliation: Howard Hughes Medical Institute, University of Oklahoma Health Sciences Center, Oklahoma City 73104.
authors.forename: A R
authors.initials: AR
authors.lastname: Rezaie
authors.forename: N L
authors.initials: NL
authors.lastname: Esmon
authors.forename: C T
authors.initials: CT
authors.lastname: Esmon
mesh_terms: D015055:1-Carboxyglutamic Acid / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011486:Protein C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D013050:Spectrometry, Fluorescence
chemical_list: D002413:Cations, Divalent; D011486:Protein C; D015055:1-Carboxyglutamic Acid; D004815:Epidermal Growth Factor; D002118:Calcium