Site-directed fluorescence probing to dissect the calcium-dependent association between soluble tissue factor and factor VIIa domains.
We have used the site-directed labeling approach to study the Ca(2+)-dependent docking of factor VIIa (FVIIa) to soluble tissue factor (sTF). Nine Ca(2+) binding sites are located in FVIIa and even though their contribution to the overall binding between TF and FVIIa has been thoroughly studied, their importance for local protein-protein interactions within the complex has not been determined. Specifically we have monitored the association of the gamma-carboxyglutamic acid (Gla), the first EGF-like (EGF1), and the protease domains (PD) of FVIIa to sTF. Our results revealed that complex formation between sTF and FVIIa during Ca(2+) titration is initiated upon Ca(2+) binding to EGF1, the domain containing the site of highest Ca(2+) affinity. Besides we showed that a Ca(2+)-loaded Gla domain is required for an optimal association of all domains of FVIIa to sTF. Ca(2+) binding to the PD seems to be of some importance for the docking of this domain to sTF.
Carlsson
Karin
K
IFM-Department of Chemistry, Linköping University, Sweden.
Osterlund
Maria
M
Persson
Egon
E
Freskgård
Per-Ola
PO
Carlsson
Uno
U
Svensson
Magdalena
M
D002118:Calcium / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism; D015942:Factor VIIa / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes; D015335:Molecular Probes; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013925:Thromboplastin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
D005456:Fluorescent Dyes; D015335:Molecular Probes; D004815:Epidermal Growth Factor; D013925:Thromboplastin; D015942:Factor VIIa; D002118:Calcium