title: The length of the linker between the epidermal growth factor-like domains in factor VIIa is critical for a productive interaction with tissue factor.
abstract: Formation of the factor VIIa (FVIIa)-tissue factor (TF) complex triggers the blood coagulation cascade. Using a structure-based rationale, we investigated how the length of the linker region between the two epidermal growth factor (EGF)-like domains in FVIIa influences TF binding and the allosteric activity enhancement, as well as the interplay between the γ-carboxyglutamic acid (Gla)-containing and protease domains. Removal of two residues from the native linker was compatible with normal cofactor binding and accompanying stimulation of the enzymatic activity, as was extension by two (Gly-Ser) residues. In sharp contrast, truncation by three or four residues abolished the TF-mediated stabilization of the active conformation of FVIIa and abrogated TF-induced activity enhancement. In addition, FVIIa variants with short linkers associated 80-fold slower with soluble TF (sTF) as compared with wild-type FVIIa, resulting in a corresponding increase in the equilibrium dissociation constant. Molecular modeling suggested that the shortest FVIIa variants would have to be forced into a tense and energetically unfavorable conformation in order to be able to interact productively with TF, explaining our experimental observations. We also found a correlation between linker length and the residual intrinsic enzymatic activity of Ca(2+)-free FVIIa; stepwise truncation resulting in gradually higher activity with des(83-86)-FVIIa reaching the level of Gla-domainless FVIIa. The linker appears to determine the average distance between the negatively charged Gla domain and a structural element in the protease domain, presumably of opposite charge, and proximity has a negative impact on apo-FVIIa activity.
authors.affiliation: Haemophilia Biology, Novo Nordisk A/S, Novo Nordisk Park, DK-2760, Måløv, Denmark.
authors.forename: Egon
authors.initials: E
authors.lastname: Persson
authors.forename: Jesper J
authors.initials: JJ
authors.lastname: Madsen
authors.forename: Ole H
authors.initials: OH
authors.lastname: Olsen
mesh_terms: D018360:Crystallography, X-Ray; D066255:EGF Family of Proteins / Q000737:chemistry*; D015942:Factor VIIa / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D013925:Thromboplastin / Q000378:metabolism*
chemical_list: D066255:EGF Family of Proteins; D013925:Thromboplastin; D015942:Factor VIIa
keywords: allostery; factor VIIa; linker region; structural complementarity; tissue factor