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ABSTRACT: Fibroblast growth factor receptor (FGFR) 4 has been associated with progression of melanoma, breast, head and neck and hepatocellular carcinoma and is therefore an interesting target for therapeutic intervention (Ho et al. in J Hepatol 50:118-127, 2009). The extracellular D2 domain of the FGFR4 receptor contains a heparin binding site and the main interaction site with the fibroblast growth factor. We report the sequential backbone and side chain resonance assignment of the D2 domain of human FGFR4.
Biomolecular NMR Assignments 07/2012; · 0.72 Impact Factor
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Krishna Saxena,
Ulrich Schieborr,
Oliver Anderka,
Elke Duchardt-Ferner, Bettina Elshorst,
Santosh Lakshmi Gande,
Julia Janzon,
Denis Kudlinzki,
Sridhar Sreeramulu,
Matthias K Dreyer,
K Ulrich Wendt,
Corentin Herbert,
Philippe Duchaussoy,
Marc Bianciotto,
Pierre-Alexandre Driguez,
Gilbert Lassalle,
Pierre Savi,
Moosa Mohammadi,
Françoise Bono,
Harald Schwalbe
[show abstract]
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ABSTRACT: Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF.FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM(6)), octasaccharide (HM(8)), and decasaccharide (HM(10)), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM(8) and HM(10) are significantly more potent than HM(6) in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1.FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2.FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1.FGFR4 interaction site and a direct FGFR4.FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF.FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM(8) relative to HM(6) in stimulating FGF2.FGFR4 signaling correlates with the higher affinity of HM(8) to bind and dimerize FGF2. Notably FGF2.HM(8) exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF.FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.
Journal of Biological Chemistry 08/2010; 285(34):26628-40. · 4.77 Impact Factor
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Krishna Saxena,
Ulrich Schieborr,
Oliver Anderka,
Elke Duchardt-Ferner, Bettina Elshorst,
Santosh Lakshmi Gande,
Julia Janzon,
Denis Kudlinzki,
Sridhar Sreeramulu,
Matthias K. Dreyer,
K. Ulrich Wendt,
Corentin Herbert,
Philippe Duchaussoy,
Marc Bianciotto,
Pierre-Alexandre Driguez,
Gilbert Lassalle,
Pierre Savi,
Moosa Mohammadi,
Françoise Bono,
Harald Schwalbe
[show abstract]
[hide abstract]
ABSTRACT: Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated
in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF
signaling as they promote FGF·FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare
homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM6), octasaccharide (HM8), and decasaccharide (HM10), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM8 and HM10 are significantly more potent than HM6 in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1·FGFR4 signaling.
To understand the molecular basis for these differential activities in FGF1/2·FGFR4 signaling, we used NMR spectroscopy, isothermal
titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated
Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence
of both a secondary FGF1·FGFR4 interaction site and a direct FGFR4·FGFR4 interaction site thus supporting the formation of
the symmetric mode of FGF·FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM8 relative to HM6 in stimulating FGF2·FGFR4 signaling correlates with the higher affinity of HM8 to bind and dimerize FGF2. Notably FGF2·HM8 exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF·FGFR dimerization
model, which incorporates the differential ability of HM to dimerize FGFs.
Journal of Biological Chemistry 08/2010; 285(34):26628-26640. · 4.77 Impact Factor
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04/2008: pages 852 - 890; , ISBN: 9783527619375
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Journal of Biomolecular NMR 02/2006; 36 Suppl 1:52. · 3.61 Impact Factor
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Journal of Biomolecular NMR 11/2005; 33(2):136. · 3.61 Impact Factor
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ABSTRACT: Here we present an NMR-based approach to solving protein-ligand structures. The procedure is guided by biophysical, biochemical, or knowledge-based data. The structures are mainly derived from ligand-induced chemical-shift perturbations (CSP) induced in the resonances of the protein and ligand-detected saturated transfer difference signals between ligands and selectively labeled proteins (SOS-NMR). Accuracy, as judged by comparison with X-ray results, depends on the nature and completeness of the experimental data. An experimental protocol is proposed that starts with calculations that make use of readily available chemical-shift perturbations as experimental constraints. If necessary, more sophisticated experimental results have to be added to improve the accuracy of the protein-ligand complex structure. The criteria for evaluation and selection of meaningful complex structures are discussed. These are exemplified for three complexes, and we show that the approach bridges the gap between theoretical docking approaches and complex NMR schemes for determining protein-ligand complexes; especially for relatively weak binders that do not lead to intermolecular NOEs.
ChemBioChem 11/2005; 6(10):1891-8. · 3.94 Impact Factor
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Doris M Jacobs,
Susanne Grimme, Bettina Elshorst,
Barbara Pescatore,
Martin Vogtherr,
Marco Betz,
Ulrich Schieborr,
Thomas Langer,
Krishna Saxena,
Harald Schwalbe,
Klaus Fiebig
Journal of Biomolecular NMR 09/2005; 32(4):337. · 3.61 Impact Factor
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ABSTRACT: The catalytic subunit of cAMP-dependent protein kinase (PKA) can easily be expressed in Escherichia coli and is catalytically active. Four phosphorylation sites are known in PKA (S10, S139, T197 and S338), and the isolated recombinant protein is a mixture of different phosphorylated forms. Obtaining uniformly phosphorylated protein requires separation of the protein preparation leading to significant loss in protein yield. It is found that the mutant S10A/S139D/S338D has similar properties as the wild-type protein, whereas additional replacement of T197 with either E or D reduces protein expression yield as well as folding propensity of the protein. Due to its high sequence homology to Akt/PKB, which cannot easily be expressed in E. coli, PKA has been used as a surrogate kinase for drug design. Several mutations within the ATP binding site have been described to make PKA even more similar to Akt/PKB. Two proteins with Akt/PKB-like mutations in the ATP binding site were made (PKAB6 and PKAB8), and in addition S10, S139 and S338 phosphorylation sites have been removed. These proteins can be expressed in high yields but have reduced activity compared to the wild-type. Proper folding of all proteins was analyzed by 2D 1H, 15N-TROSY NMR experiments.
FEBS Letters 09/2005; 579(19):4049-54. · 3.54 Impact Factor
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ABSTRACT: A novel strategy is applied to obtain quantitative insights on factors influencing biological affinity in protein-ligand complexes. This approach is based on the detection of ligand binding by (15)N and (1)H amide chemical shift differences in two-dimensional (15)N-heteronuclear single-quantum correlation spectra. Essential structural features linked to affinity can be extracted using statistical analysis of (15)N and (1)H amide chemical shift differences in congeneric series relative to uncomplexed protein spectra, as demonstrated for 20 MMP-3 inhibitors in complex with human matrix metalloproteinase stromelysin (MMP-3). The statistical analysis using PLS led to a significant model, while its chemical interpretation, highlighting the importance of particular residues for affinity, are in agreement to an X-ray structure of one key compound in the homologue MMP-8 binding site.
Bioorganic & Medicinal Chemistry Letters 05/2005; 15(7):1779-83. · 2.55 Impact Factor
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ABSTRACT: Protein phosphorylation is one of the most important mechanisms used for intracellular regulation in eukaryotic cells. Currently, one of the best-characterized protein kinases is the catalytic subunit of cAMP-dependent protein kinase or protein kinase A (PKA). PKA has the typical bilobular structure of kinases, with the active site consisting of a cleft between the two structural lobes. For full kinase activity, the catalytic subunit has to be phosphorylated. The catalytic subunit of PKA has two main phosphorylation sites: Thr197 and Ser338. Binding of ATP or inhibitors to the ATP site induces large structural changes. Here we describe the partial backbone assignment of the PKA catalytic domain by NMR spectroscopy, which represents the first NMR assignment of any protein kinase catalytic domain. Backbone resonance assignment for the 42 kDa protein was accomplished by an approach employing 1) triply ((2)H,(13)C,(15)N) labeled protein and classical NMR assignment experiments, 2) back-calculation of chemical shifts from known X-ray structures, 3) use of paramagnetic adenosine derivatives as spin-labels, and 4) selective amino acid labeling. Interpretation of chemical-shift perturbations allowed mapping of the interaction surface with the protein kinase inhibitor H7. Furthermore, structural conformational changes were observed by comparison of backbone amide shifts obtained by 2D (1)H,(15)N TROSY of an inactive Thr197Ala mutant with the wild-type enzyme.
ChemBioChem 12/2004; 5(11):1508-16. · 3.94 Impact Factor
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Journal of Biomolecular NMR 11/2003; 27(2):191-2. · 3.61 Impact Factor
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ABSTRACT: Using NMR spectroscopy we show that the cellular prion protein constitutes a target for binding of various acridine and phenothiazine derivatives. We unambiguously map the quinacrine binding site of recombinant human prion protein to residues Tyr225, Tyr226, and Gln227 of helix alpha3, which is located near the "protein X" epitope. The millimolar dissociation constant of the complex suggests that in vivo inhibition of prion propagation occurs after 10000-fold concentration of quinacrine within endolysosomes.
Journal of Medicinal Chemistry 09/2003; 46(17):3563-4. · 5.25 Impact Factor
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Journal of Biomolecular NMR 07/2002; 23(2):163-4. · 3.61 Impact Factor
