Ca2+ in the first epidermal growth factor-like domain of activated factor VII.
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.
Persson
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Vessel Wall Biology, Health Care Discovery, Novo Nordisk A/S, Gentofte, Denmark. egpe@novo.dk
Nielsen
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LS
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
D001224:Aspartic Acid; D004815:Epidermal Growth Factor; D001216:Asparagine; D015942:Factor VIIa; D002118:Calcium