Interaction of the anticancer p28 peptide with p53-DBD as studied by fluorescence, FRET, docking and MD simulations

Investigation of a Direct Interaction between miR4749 and the Tumor Suppressor p53 by Fluorescence, FRET and Molecular Modeling

“…Figure 2A shows the fluorescence emission spectrum of DBD excited at 295 nm (black line), at which, the tryptophan residue (Trp146) of DBD still absorbed, while the other aromatic residues (Tyr and Phe) were substantially not excited. The spectrum was peaked at about 346 nm (see the arrow), indicating that Trp146 was almost fully exposed to the solvent, as also was the case in previous works [7,17]. Such a result finds a correspondence with the rather high SASA value (about 80 Å 2 ) for Trp146 from the DBD X-ray structure.…”

“…Additionally, no significant wavelength shift of the emission peak was observed; this being indicative that the addition of miR4749 did not affect the solvent exposition of Trp146, as also observed for the interaction of DBD with miR-21-3p [7]. It is interesting to note that a similar quenching behavior has been observed for the interaction of DBD with the anticancer peptide p28 [17]. Figure 2B shows the ratio, F0/F, between the fluorescence emission detected at 346 nm, of DBD alone and of DBD in the presence of progressively higher concentrations of the quencher Q (here miR4749).…”

“…The Zn ion is tetrahedrically coordinated to the side-chains of Cys176, His179, Cys238 and Cys242, forming a Zn-finger motif, which is connected to the L 2 and L 3 loops [13]. The interaction of the Zn ion with its ligands was treated through a bonded approach in which the Zn-N and Zn-S bonds and S-Zn-S angles, were set according to the parameters provided in references [14][15][16][17]. The binding of DBD to DNA occurs within L 1 and L 3 loops in a region, conventionally chosen to be the northern part of the molecule.…”

“…miR4749 was centered in a in a cubic box of edge 7.0 nm, while DBD and the DBD-miR4749 complex were centered in a cubic box of edge 9.0 nm 3 . Simulations were performed by following the procedures described in references [17,26]. Briefly, boxes were filled with water molecules, to reach a hydration level of 9 g water/g protein.…”

“…By taking into consideration that the FRET results indicated an experimental DDA distance within the 3.6-4.2 nm range and by assuming a contribution of 0.1 nm, as due to the dye attached to the 5′ end of miR4749, we preliminarily discarded those models whose DDA value was lower than 3.7 nm or higher than 4.3 nm. The remaining 16 models were then grouped by evaluating the RMSD between each couple of models by following the procedure as described in reference [17] In particular, models whose RMSD value differed less than 0.1 nm were grouped together, with the first ranked model taken as a representative of the corresponding group. After that, we obtained five models (labeled as Model 1-5), which are collectively shown in Figure 5B.…”