Tavel, L., Jaquillard, L., Karsisiotis, A.I., Saab, F., Jouvensal, L., Brans, A., Delmas, A., Schoentgen, F., Cadene, M. and Damblon, C.
PLoS ONE 7 (4) e36187
par Frapart - publié le , mis à jour le
In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands : GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants KD for different ligands. Native mass spectrometry was used as an alternative method for measuring KD values. Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.