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Probing inhibitor-induced conformational changes along the interface between tissue factor and factor VIIa
Novo Nordisk AS, Prot Biotechnol, DK-2880 Bagsvaerd, Denmark Linkoping Univ, IFM, Dept Chem, Linkoping, Sweden Linkoping Univ, IFM, Dept Phys Chem, Linkoping, Sweden Novo Nordisk AS, Vasc Biochem, Malov, Denmark.
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
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2001 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 40, no 31, 9324-9328 p.Article in journal (Refereed) Published
Abstract [en]

Upon injury of a blood vessel, activated factor VII (FVIIa) forms a high-affinity complex with its allosteric regulator, tissue factor (TF), and initiates blood clotting. Active site-inhibited factor VIIa (FVIIai) binds to TF with even higher affinity. We compared the interactions of FVIIai and FVIIa with soluble TF (sTF). Six residues in sTF were individually selected for mutagenesis and site-directed labeling. The residues are distributed along the extensive binding interface, and were chosen because they are known to interact with the different domains of FVIIa. Fluorescent and spin probes were attached to engineered Cys residues to monitor local changes in hydrophobicity, accessibility, and rigidity in the sTF-FVIIa complex upon occupation of the active site of FVIIa. The results show that inhibition of FVIIa caused the structures around the positions in sTF that interact with the protease domain of FVIIa to become more rigid and less accessible to solvent. Thus, the presence of an active site inhibitor renders the interface in this region less flexible and more compact, whereas the interface between sTF and the light chain of FVIIa is unaffected by active site occupancy.

Place, publisher, year, edition, pages
2001. Vol. 40, no 31, 9324-9328 p.
National Category
Engineering and Technology
URN: urn:nbn:se:liu:diva-49196DOI: 10.1021/bi010283nOAI: diva2:270092
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2012-02-17Bibliographically approved
In thesis
1. Tissue Factor in Complex: Studies of interactions between blood coagulation proteins
Open this publication in new window or tab >>Tissue Factor in Complex: Studies of interactions between blood coagulation proteins
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Many biological processes rely on specific protein-protein interactions, for example immune responses, cell signaling, transcription, and blood coagulation. Blood coagulation is initiated when a vessel wall is damaged, exposing tissue factor (TF) to the circulating factor VII/factor VIIa (FVII/FVIIa) which results in the formation of the TF:FVIIa complex and thereby the initiation of blood coagulation. One of the substrates for the TF:FVIIa complex is factor X (FX), which is activated to factor Xa (FXa), subsequently leading to a series of reactions resulting in clot formation. Tissue factor pathway inhibitor (TFPI) is the major physiological inhibitor of the sTF:FVIIa complex, involved in regulation of coagulation by forming the TF:FVIIa:FXa:TFPI complex. Occasionally, the blood coagulation mechanism malfunctions, resulting in conditions such as the inability to stop bleeding or thrombosis. The fact that TF is the main initiator of the coagulation makes this an interesting protein to study, in the hunt for means to interfere with players involved in the blood clotting process.

Throughout the studies included in this thesis the site-directed labeling technique is utilized to attach spectroscopic probes to cysteines, introduced at specific positions by mutagenesis, in the protein of interest. These fluorescent or spin-probes are sensitive for changes in their immediate environment and can thus, for example be used to monitor protein-protein complex formation and conformational changes.

No complete structure has been obtained as yet for the large complex involving sTF, FVIIa, FXa, and TFPI. Therefore, we introduced a fluorescent probe at specific positions in soluble tissue factor (sTF) and the changes in fluorescence emission were detected upon sTF:FVIIa:FXa:TFPI complex formation. From these measurements it was concluded that not only parts of the C-terminal domain of sTF (TF2), but also residues in the N-terminal domain (TF1) are involved in binding to FXa in the quaternary complex.

In order to investigate conformational changes occurring in the extended interface between sTF and FVIIa upon binding of different inhibitors spectroscopic probes were introduced in sTF, in the vicinity of the interaction region. From the obtained data it was concluded that the exosite-binding inhibitor E-76 induces equivalent structural changes at the interface of sTF and the protease domain (PD) of FVIIa, as do the active-site inhibitors FFR and TFPI, i.e. makes the region around the active-site more compact. Binding of these inhibitors shows similar effects despite their differences in size, binding site, and inhibitory mechanism.

In addition, the Ca2+ dependence of the formation of the sTF:FVIIa complex was studied. Association between sTF and FVIIa during Ca2+ titration begins by Ca2+ binding to the first EGF-like domain of FVIIa. However, Ca2+ saturation of the γ-carboxyglutamic acid-rich (Gla) domain of FVIIa is required for complete sTF:FVIIa complex formation, and we were also able to detect that a Gla domain with vacant Ca2+ sites hinders the docking to sTF.

Finally, we investigated the structural changes of free inhibited FVIIa upon sTF and Ca2+ binding by FRET and quenching measurements. From this it was concluded that inhibited FVIIa does not seem to undergo large global structural changes upon binding to sTF, when taking the dynamics of free FVIIa into account. However, Ca2+ binding induces minor local conformational changes in the active-site region of the PD of inhibited FVIIa and subsequent binding of sTF causesfurther structural rearrangements in this area.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2010. 75 p.
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1329
National Category
Natural Sciences
urn:nbn:se:liu:diva-63688 (URN)978-91-7393-355-1 (ISBN)
Public defence
2010-10-22, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 10:15 (English)
Available from: 2010-12-30 Created: 2010-12-30 Last updated: 2010-12-30Bibliographically approved

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