title: Mechanism of the Ca2+-induced enhancement of the intrinsic factor VIIa activity.
abstract: The intrinsic activity of coagulation factor VIIa (FVIIa) is dependent on Ca(2+) binding to a loop (residues 210-220) in the protease domain. Structural analysis revealed that Ca(2+) may enhance the activity by attenuating electrostatic repulsion of Glu(296) and/or by facilitating interactions between the loop and Lys(161) in the N-terminal tail. In support of the first mechanism, the mutations E296V and D212N resulted in similar, about 2-fold, enhancements of the amidolytic activity. Moreover, mutation of the Lys(161)-interactive residue Asp(217) or Asp(219) to Ala reduced the amidolytic activity by 40-50%, whereas the K161A mutation resulted in 80% reduction. Hence one of these Asp residues in the Ca(2+)-binding loop appears to suffice for some residual interaction with Lys(161), whereas the more severe effect upon replacement of Lys(161) is due to abrogation of the interaction with the N-terminal tail. However, Ca(2+) attenuation of the repulsion between Asp(212) and Glu(296) keeps the activity above that of apoFVIIa. Altogether, our data suggest that repulsion involving Asp(212) in the Ca(2+)-binding loop suppresses FVIIa activity and that optimal activity requires a favorable interaction between the Ca(2+)-binding loop and the N-terminal tail. Crystal structures of tissue factor-bound FVIIa(D212N) and FVIIa(V158D/E296V/M298Q) revealed altered hydrogen bond networks, resembling those in factor Xa and thrombin, after introduction of the D212N and E296V mutations plausibly responsible for tethering the N-terminal tail to the activation domain. The charge repulsion between the Ca(2+)-binding loop and the activation domain appeared to be either relieved by charge removal and new hydrogen bonds (D212N) or abolished (E296V). We propose that Ca(2+) stimulates the intrinsic FVIIa activity by a combination of charge neutralization and loop stabilization.
authors.affiliation: Department of Protein Structure and Biophysics, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
authors.forename: Jais R
authors.initials: JR
authors.lastname: Bjelke
authors.forename: Ole H
authors.initials: OH
authors.lastname: Olsen
authors.forename: Michel
authors.initials: M
authors.lastname: Fodje
authors.forename: L Anders
authors.initials: LA
authors.lastname: Svensson
authors.forename: Susanne
authors.initials: S
authors.lastname: Bang
authors.forename: Gert
authors.initials: G
authors.lastname: Bolt
authors.forename: Birthe B
authors.initials: BB
authors.lastname: Kragelund
authors.forename: Egon
authors.initials: E
authors.lastname: Persson
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray / Q000379:methods; D015942:Factor VIIa / Q000737:chemistry*; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D006860:Hydrogen Bonding; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D055672:Static Electricity; D013925:Thromboplastin / Q000737:chemistry; D013997:Time Factors
chemical_list: D001224:Aspartic Acid; D018698:Glutamic Acid; D006859:Hydrogen; D013925:Thromboplastin; D015942:Factor VIIa; D002118:Calcium