title: Ca2+ in the first epidermal growth factor-like domain of activated factor VII.
abstract: Activated factor VII (FVIIa) needs to be bound to tissue factor (TF) to express biological activity, and biochemical and structural data show that the first epidermal growth factor (EGF)-like domain of FVIIa contributes essential contacts with TF. To investigate the role of Ca2+ binding to this domain in FVIIa we used site-directed mutagenesis to replace Asp46 and Asp63 by Asn, which abolished Ca2+ binding, and characterized the double mutant (D46,63N-FVIIa) with respect to its TF-binding properties and the functional status of its complex with TF. D46,63N-FVIIa had a lower amidolytic activity than FVIIa at optimal Ca2+ concentrations. A slightly lower amidolytic activity was also observed in complex with soluble TF, apparently due to a lower catalytic turnover rate of D46,63N-FVIIa. However, D46,63N-FVIIa and FVIIa bound to lipidated TF were equally efficient activators of factor X. The dissociation constant for the interaction between D46,63N-FVIIa and soluble TF, derived from amidolytic activity and direct binding measurements, was approximately 20-fold higher than that for the interaction between FVIIa and soluble TF. The same difference was observed in the affinity for lipidated TF. These findings suggest that a functional Ca2+-binding site in the first EGF-like domain adds approximately 7 kJ/mol to the total binding energy of the interaction with both lipidated and soluble TF.
authors.affiliation: Vessel Wall Biology, Health Care Discovery, Novo Nordisk A/S, Gentofte, Denmark. egpe@novo.dk
authors.forename: E
authors.initials: E
authors.lastname: Persson
authors.forename: L S
authors.initials: LS
authors.lastname: Nielsen
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D002118:Calcium / Q000737:chemistry*; D004815:Epidermal Growth Factor / Q000737:chemistry*; D015942:Factor VIIa / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D001224:Aspartic Acid; D004815:Epidermal Growth Factor; D001216:Asparagine; D015942:Factor VIIa; D002118:Calcium