title: Spectroscopic probing of the influence of calcium and the gla domain on the interaction between the first EGF domain in factor VIIa and tissue factor.
abstract: The binding of factor VIIa (FVIIa) to tissue factor (TF) initiates blood coagulation. The binary complex is dependent on Ca2+ binding to several sites in FVIIa and is maintained by multiple contacts distributed throughout the various domains. Although the contributions from various residues and domains, including the Ca2+ coordination, to the global binding energy have been characterized, their importance for specific local interactions is virtually unknown. To address this aspect, we have attached four spectroscopic probes to an engineered Cys residue replacing Phe140 in soluble TF (sTF). This allows the monitoring of local changes in hydrophobicity and rigidity upon complex formation at the interface between the first epidermal growth factor-like (EGF1) domain of FVIIa and sTF. The fluorescent labels used sense a more hydrophobic environment and the spin labels are dramatically immobilized when FVIIa binds sTF. The results obtained with a 4-carboxyglutamic acid (Gla)-domainless derivative of FVIIa indicate that the Gla domain has no or minimal influence on the interaction between EGF1 and sTF. However, there is a difference in local Ca2+ dependence between Gla-domainless and full-length FVIIa.
authors.affiliation: IFM-Department of Chemistry, and IFM-Department of Chemical Physics, Linköping University, Sweden; Tissue Factor/Factor VII Research, Novo Nordisk A/S, Mâlov, Denmark.
authors.forename: M
authors.initials: M
authors.lastname: Osterlund
authors.forename: R
authors.initials: R
authors.lastname: Owenius
authors.forename: E
authors.initials: E
authors.lastname: Persson
authors.forename: M
authors.initials: M
authors.lastname: Lindgren
authors.forename: U
authors.initials: U
authors.lastname: Carlsson
authors.forename: P O
authors.initials: PO
authors.lastname: Freskgård
authors.forename: M
authors.initials: M
authors.lastname: Svensson
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D003198:Computer Simulation; D004578:Electron Spin Resonance Spectroscopy; D004815:Epidermal Growth Factor / Q000737:chemistry*; D015942:Factor VIIa / Q000737:chemistry* / Q000378:metabolism*; D014644:Genetic Variation; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D013925:Thromboplastin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D013925:Thromboplastin; D015942:Factor VIIa; D002118:Calcium