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Effects on the conformation of FVIIa by sTF and Ca(2+) binding: Studies of fluorescence resonance energy transfer and quenching
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
Haemostasis Biochemistry, Novo Nordisk A/S, DK-2760 Måløv, Denmark.
Department of Physics, Norwegian University of Science and Technology, Trondheim, Norway.
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
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2011 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 413, no 4, 545-549 p.Article in journal (Refereed) Published
Abstract [en]

The apparent length of FVIIa in buffer solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein linked to an inhibitor (FPR-chloromethyl ketone) and a rhodamine derivative (tetramethylrhodamine-5-maleimide), were covalently attached to FVIIa. The binding site of fluorescein was in the PD whereas rhodamine was positioned in the Gla domain, thus allowing a length measure over approximately the whole extension of the protein. From the FRET measurements the distances between the two probes were determined to 61.4 for free FVIIa and 65.5 Å for FVIIa bound to the soluble TF (sTF). Thus, the apparent distance from the FRET analysis was shown to increase with 4 Å upon formation of a complex with sTF in solution. However, by considering how protein dynamics, based on recently published molecular dynamics simulations of FVIIa and sTF:FVIIa (Ohkubo et al., 2010 J. Thromb. Haemost. 8, 1044-1053), can influence the apparent  fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF.

Moreover, it is known that Ca2+ binding leads to activation of FVIIa, and we have for the first time demonstrated conformational changes in the environment of the active site upon Ca2+ binding by direct measurements, previously suggested based on indirect measurements (Persson & Petersen, 1995 Eur. J. Biochem. 234, 293-300). Interestingly, this Ca2+-induced conformational change can be noted even in the presence of an inhibitor. By forming the sTF:FVIIa complex the conformational change of the active site is further developed, leading to a more inaccessible active-site located probe.

Place, publisher, year, edition, pages
Elsevier , 2011. Vol. 413, no 4, 545-549 p.
Keyword [en]
Factor VIIa, Fluorescence quenching, Fluorescence resonance energy transfer, Tissue factor, Fluorescein, Rhodamine, Conformational dynamics
National Category
Natural Sciences
URN: urn:nbn:se:liu:diva-63686DOI: 10.1016/j.bbrc.2011.08.135ISI: 000295912800010OAI: diva2:382172
Funding agencies|Swedish Research Council||Knut and Alice Wallenbergs Foundation||Available from: 2010-12-30 Created: 2010-12-30 Last updated: 2011-11-10Bibliographically 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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