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  • 1.
    Carlsson, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Freskgard, P.-O.
    Freskgård, P.-O., Protein Biotechnology, Novo Nordisk A/S, Novo Allé, Bagsværd, Denmark, Nuevolution A/S, Rønnegade 8, Copenhagen, Denmark.
    Persson, E.
    Haemostasis Biology, Novo Nordisk A/S, Novo Nordisk Park, Måløv, Denmark.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Svensson, Magdalena
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Probing the interface between factor Xa and tissue factor in the quaternary complex tissue factor-factor VIIa-factor Xa-tissue factor pathway inhibitor2003In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 270, no 12, p. 2576-2582Article in journal (Refereed)
    Abstract [en]

    Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). TF-FVIIa is inhibited by tissue factor pathway inhibitor (TFPI) in two steps: first TFPI is bound to the active site of factor Xa (FXa), and subsequently FXa-TFPI exerts feedback inhibition of TF-FVIIa. The FXa-dependent inhibition of TF-FVIIa activity by TFPI leads to formation of the quaternary complex TF-FVIIa-FXa-TFPI. We used site-directed fluorescence probing to map part of the region of soluble TF (sTF) that interacts with FXa in sTF-FVIIa-FXa-TFPI. We found that the C-terminal region of sTF, including positions 163, 166, 200 and 201, is involved in binding to FXa in the complex, and FXa, most likely via its Gla domain, is also in contact with the Gla domain of FVIIa in this part of the binding region. Furthermore, a region that includes the N-terminal part of the TF2 domain and the C-terminal part of the TF1 domain, i.e. the residues 104 and 197, participates in the interaction with FXa in the quaternary complex. Moreover, comparisons of the interaction areas between sTF and FX(a) in the quaternary complex sTF-FVIIa-FXa-TFPI and in the ternary complexes sTF-FVII-FXa or sTF-FVIIa-FX demonstrated large similarities.

  • 2.
    Carlsson, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Osterlund, Maria
    University Holding, Teknikringen 7, SE-581 83 Linköping, Sweden.
    Persson, Egon
    Vascular Biochemistry, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
    Freskgard, P.-O.
    Freskgård, P.-O., Protein Biotechnology, Novo Nordisk A/S, Novo Allé, DK-2880 Bagsværd, Denmark, Molecular Biology, Maxygen ApS, DK-2970, Hørsholm, Denmark.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Svensson, Magdalena
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Site-directed fluorescence probing to dissect the calcium-dependent association between soluble tissue factor and factor VIIa domains2003In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1648, no 1-2, p. 12-16Article in journal (Refereed)
    Abstract [en]

    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 Ca2+ 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 ?-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 Ca2+ titration is initiated upon Ca2+ binding to EGF1, the domain containing the site of highest Ca2+ affinity. Besides we showed that a Ca 2+-loaded Gla domain is required for an optimal association of all domains of FVIIa to sTF. Ca2+ binding to the PD seems to be of some importance for the docking of this domain to sTF. © 2003 Elsevier Science B.V. All rights reserved.

  • 3.
    Carlsson, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Persson, Egon
    Haemostasis Biochemistry, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Svensson, Magdalena
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Inhibitors of factor VIIa affect the interface between the protease domain and tissue factor2006In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 349, no 3, p. 1111-1116Article in journal (Refereed)
    Abstract [en]

    Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). The γ-carboxyglutamic acid-rich domain of FVIIa docks with the C-terminal domain of TF, the EGF1 domain of FVIIa contacts both domains of TF, and the EGF2 domain and protease domain (PD) form a continuous surface that sits on the N-terminal domain of TF. Our aim was to investigate the conformational changes that occur in the sTF·PD binding region when different types of inhibitors, i.e., one active-site inhibitor (FFR-chloromethyl ketone (FFR)), two different peptide exosite inhibitors (E-76 and A-183), and the natural inhibitor tissue factor pathway inhibitor (TFPI), were allowed to bind to FVIIa. For this purpose, we constructed two sTF mutants (Q37C and E91C). By the aid of site-directed labeling technique, a fluorescent label was attached to the free cysteine. The sTF·PD interface was affected in position 37 by the binding of FFR, TFPI, and E-76, i.e., a more compact structure was sensed by the probe, while for position 91 located in the same region no change in the surrounding structure was observed. Thus, the active site inhibitors FFR and TFPI, and the exosite inhibitor E-76 have similar effects on the probe in position 37 of sTF, despite their differences in size and inhibition mechanism. The allosteric changes at the active site caused by binding of the exosite inhibitor E-76 in turn induce similar conformational changes in the sTF·PD interface as does the binding of the active site inhibitors. A-183, on the other hand, did not affect position 37 in sTF, indicating that the A-183 inhibition mechanism is different from that of E-76.

  • 4.
    Carlsson, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Persson, Egon
    Haemostasis Biochemistry, Novo Nordisk A/S, DK-2760 Måløv, Denmark.
    Østergaard, Henrik
    Department of Physics, Norwegian University of Science and Technology, Trondheim, Norway.
    Lindgren, Mikael
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Svensson, Magdalena
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Effects on the conformation of FVIIa by sTF and Ca(2+) binding: Studies of fluorescence resonance energy transfer and quenching2011In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 413, no 4, p. 545-549Article in journal (Refereed)
    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.

  • 5.
    Carlsson, Uno
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Hammarström, Per
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Lindgren, M
    Persson, M
    Freskgård, Per-Ola
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Mårtensson, Lars-Göran
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Andersson, D
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Svensson, Magdalena
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Aggregation is site-specific in carbonic anhydrase and is prevented by GroEL: The interaction leads to a more flexible structure of both the protein substrate and the chaperonin.2000In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 78, no 1, p. 202Pos-Conference paper (Other academic)
  • 6.
    Carlsson, Uno
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Magnusson, Thomas
    Linköping University, Department of Management and Engineering, Project management, Innovations and Entrepreneurship . Linköping University, The Institute of Technology.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Svensson, Magdalena
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Tibell, Lena
    Linköping University, Department of Science and Technology. Linköping University, The Institute of Technology.
    To design a novel protein: A CDIO experience in Molecular Biotechnology at Linköping University2006Conference paper (Other academic)
  • 7.
    Jarl, Anngelica
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Andrésen, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Eriksson, Laila
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Svensson, Magdalena
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    AEDANS labeled MexR variants and their DNA interactionManuscript (preprint) (Other academic)
    Abstract [en]

    MexR is a transcriptional repressor of the multidrug efflux-pump operon in the bacterial pathogen Pseudomonas aeruginosa, and thus, malfunction in the DNA-binding of the MexR protein leads to multidrug resistance (Poole et al., 1996). lt has been shown that mutations in the MexR protein lead to lost DNA binding ability (Saito et al., 2003). X-ray studies of the MexR protein (Lim et al., 2002) show that the protein contains a winged helix-turn-helix motif and this type of motif is known to be involved in DNA-binding (Gajiwala & Burley, 2000), but no exact DNA-binding sequence in MexR has yet been determined.

    This study attempts to identify regions in the MexR protein involved in DNA-binding using a molecular probe as mapping tool. Three cysteines naturally occur in the MexR protein, located in positions 30, 62 and 138, respectively, and we have used two of them to map the DNA-binding site. Preliminary results show that the fluorescence of an TAEDANS probe attached to cys 62 is affected by DNA-binding, while a probe at cys 30 is not affected. Further studies are required to analyse the implications of these results.

  • 8. Lindgren, M.
    et al.
    Eaton, S.
    Eaton, G
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Hammarström, Per
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Svensson, Magdalena
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Electron Spin Echo Decay at a Probe of Aminoxyl Environment in Spin-labeled Mutants of Carbonic Anhydrase1997In: Journal of the Chemical Society. Perkin transactions II, ISSN 0300-9580, p. 2549-2554Article in journal (Refereed)
  • 9.
    Osterlund, Maria
    et al.
    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.
    Owenius, Rikard
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Carlsson, Karin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Persson, Egon
    Vascular Biochemistry, Novo Nordisk A/S, Denmark.
    Lindgren, Mikael
    Department of Laser Systems, Division of Sensor Technology, Swedish Defence Research Agency, P.O. Box 1165, SE- 581 11 Linko¨ping, Sweden..
    Freskgård, Per-Ola
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Svensson, Magdalena
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Probing inhibitor-induced conformational changes along the interface between tissue factor and factor VIIa2001In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 40, no 31, p. 9324-9328Article in journal (Refereed)
    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.

  • 10.
    Osterlund, Marie
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Owenius, Rikard
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Persson, E.
    Tissue Factor/Factor VII Research, Novo Nordisk A/S, Måløv, Denmark.
    Lindgren, M.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Freskgard, P.-O.
    Freskgård, P.-O., Tissue Factor/Factor VII Research, Novo Nordisk A/S, Måløv, Denmark.
    Svensson, Magdalena
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Spectroscopic probing of the influence of calcium and the Gla domain on the interaction between the first EGF domain in factor VIla and tissue factor2000In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 267, no 20, p. 6204-6211Article in journal (Refereed)
    Abstract [en]

    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-carboxy-glutamic 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.

  • 11.
    Osterlund, Marie
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Owenius, Rikard
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Svensson, Magdalena
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Lindgren, M
    Persson, E
    Freskgård, Per-Ola
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Mapping local interactions and resolving association kinetics for a receptor-ligand system2000Conference paper (Other academic)
  • 12.
    Owenius, Rikard
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Osterlund, Marie
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Svensson, Magdalena
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Lindgren, M
    Persson, E
    Freskgård, Per-Ola
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Spin and fluorescent probing of the binding interface between tissue factor and factor VIIa at multiple sites2001In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 81, no 4, p. 2357-2369Article in journal (Refereed)
    Abstract [en]

    The specific complex between the extracellular part of tissue factor (sTF) and factor Vlla (FVlla) was chosen as a model for studies of the binding interface between two interacting proteins. Six surface-exposed positions in sTF, residues known to contribute to the sTF-FVlla interaction, were selected for cysteine mutation and site-directed labeling with spin and fluorescent probes. The binding interface was characterized by spectral data from electron paramagnetic resonance (EPR) and steady-state and time-domain fluorescence spectroscopy. The labels reported on compact local environments at positions 158 and 207 in the interface region between sTF and the gamma -carboxyglutamic acid (Gla) domain of FVlla, and at positions 22 and 140 in the interface region between sTF and the first epidermal growth factor-like (EGF1) domain of FVlla. The tightness of the local interactions in these parts of the interface is similar to that seen in the interior of globular proteins. This was further emphasized by the reduced local polarity detected by the fluorescent label upon FVlla binding, especially in the sTF-Gla region. There were indications of structural rigidity also at positions 45 and 94 in the interface region between sTF and the protease domain (PD) of FVlla, despite the perturbed cofactor function of these sTF variants. The results of the present study indicate that the multi-probing approach enables comparison of the tightness and characteristics of interaction along the binding interface of a protein complex. This approach also increases the probability of acquiring reliable structural data that are descriptive of the wild-type proteins.

  • 13.
    Persson, Malin
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Hammarström, Per
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Lindgren, M.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Svensson, Magdalena
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    EPR Mapping of Interactions Between Spin-labeled Variants of Human Carbonic Anhydrase II and GroEL. Evidence for increased flexibility of the hydrophobic core by the interaction.1999In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 38, p. 432-441Article in journal (Refereed)
  • 14. Wirehn, J.
    et al.
    Carlsson, Karin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Herland, Anna
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics .
    Persson, E.
    Haemostasis Biochemistry, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Svensson, Magdalena
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Hammarström, Per
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Activity, folding, misfolding, and aggregation in vitro of the naturally occurring human tissue factor mutant R200W2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 18, p. 6755-6763Article in journal (Refereed)
    Abstract [en]

    Tissue factor (TF), a small transmembrane receptor, binds factor VIIa (FVIIa), and the formed complex initiates blood coagulation by proteolytic activation of substrate factors IX and X. A naturally occurring mutation in the human TF gene was recently reported, where a single-base substitution results in an R200W mutation in the TF extracellular domain [Zawadzki, C., Preudhomme, C., Gavériaux, V., Amouyel, P., and Jude, B. (2002) Thromb. Haemost. 87, 540-541]. This mutation appears to be associated with low monocyte TF expression and may protect against thrombosis but has not been associated with any pathological condition, and individuals who present the heterozygous trait appear healthy. Here, we report the activity, folding, and aggregation behavior of the R200W mutant of the 219-residue soluble extracellular domain of TF (sTFR200W) compared to that of the wild-type protein (sTF wt). No differences in stability or FVIIa cofactor activity but an impaired ability to promote FX activation at physiological conditions between the sTFR200W mutant and sTFwt were evident. Increased binding of 1-anilino-8-naphthalene-sulfonic acid (ANS) to sTFR200W indicated a population of partially folded intermediates during denaturation. sTFR200W showed a dramatically increased propensity for aggregate formation compared to sTFwt at mildly acidic pHs, with an increased rate of aggregation during conditions, promoting the intermediate state. The lowered pH resistance could explain the loss of sTFR200W in vivo because of aggregation of the mutant. The intrinsic structure of the sTF aggregates appears reminiscent of amyloid fibrils, as revealed by thioflavin T fluorescence, atomic force microscopy, and transmission electron microscopy. We conclude that the lowered activity for FX activation and the propensity of the mutant protein to misfold and aggregate will both contribute to decreased coagulation activity in TFR200W carriers, which could protect from thrombotic disease. © 2005 American Chemical Society.

  • 15.
    Österlund, Marie
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Persson, E
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Freskegård, P-O
    Svensson, Magdalena
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Sequential coagulation factor VIIa domain binding to tissue factor2005In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 337, no 4, p. 1276-1282Article in journal (Refereed)
    Abstract [en]

    Vessel wall tissue factor (TF) is exposed to blood upon vascular damage which enables association with factor VIIa (FVIIa). This leads to initiation of the blood coagulation cascade through localization and allosteric induction of FVIIa procoagulant activity. To examine the docking pathway of the FVIIa-TF complex, various residues in the extracellular part of TF (sTF) that are known to interact with FVIIa were replaced with cysteines labelled with a fluorescent probe. By using stopped-flow fluorescence kinetic measurements in combination with surface plasmon resonance analysis, we studied the association of the resulting sTF variants with FVIIa. We found the docking trajectory to be a sequence of events in which the protease domain of FVIIa initiates contact with sTF. Thereafter, the two proteins are tethered via the first epidermal growth factor-like and finally the γ-carboxyglutamic acid (Gla) domain. The two labelled sTF residues interacting with the protease domain of FVIIa bind or become eventually ordered at different rates, revealing kinetic details pertinent to the allosteric activation of FVIIa by sTF. Moreover, when the Gla domain of FVIIa is removed the difference in the rate of association for the remaining domains is much more pronounced. © 2005 Elsevier Inc. All rights reserved.

  • 16.
    Österlund, Marie
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Persson, E
    Svensson, Magdalena
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Freskegård, P-O
    Transition state analysis of the complex between coagulation factor VIIa and tissue factor: Suggesting a sequential domain-binding pathway2005In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 327, no 3, p. 789-793Article in journal (Refereed)
    Abstract [en]

    Injury of a blood vessel exposes membrane-bound tissue factor (TF) to blood, which allows binding of coagulation factor VIIa (FVIIa). This initiation of the coagulation cascade is dictated by a specific multi-domain interaction between FVIIa and TF. To examine the energies involved in the transition state of the FVIIa:TF complex, various residues in the extracellular part of TF (sTF) that are known to interact with FVIIa were replaced with a smaller cysteine residue. Determination of Φ values in each of the positions using surface plasmon resonance measurements enabled us to characterize the transition state complex between the resulting sTF variants and FVIIa. We found that the interactions in the transition state seemed to be most pronounced between the protease domain of FVIIa and sTF while detailed specific interactions between the Gla-domain and sTF were missing. Thus, the transition state energy data indicate a sequential binding event between these two macromolecules. © 2004 Elsevier Inc. All rights reserved.

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