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

Abstract

The interactions between the DNA binding domain (DBD) of the tumor suppressor p53 and miR4749, characterized by a high sequence similarity with the DNA Response Element (RE) of p53, was investigated by fluorescence spectroscopy combined with computational modeling and docking. Fluorescence quenching experiments witnessed the formation of a specific complex between DBD and miR4749 with an affinity of about 10⁵ M. Förster Resonance Energy Transfer (FRET) allowed us to measure a distance of 3.9 ± 0.3 nm, between the lone tryptophan of DBD and an acceptor dye suitably bound to miR4749. Such information, combined with a computational modeling approach, allowed us to predict possible structures for the DBD-miR4749 complex. A successive docking refinement, complemented with binding free energy calculations, led us to single out a best model for the DBD-miR4749 complex. We found that the interaction of miR4749 involves the DBD L₃ loop and the H₁ helix, close to the Zn-finger motif; with this suggesting that miR4749 could directly inhibit the p53 interaction with DNA. These results might inspire new therapeutic strategies finalized to restore the p53 functional activity.

Keywords: FRET; computational docking; fluorescence quenching; miR4749; oncomiR; p53.

Figure 1

The structure of p53 DNA binding domain (DBD) derived from the chain B of PDB entry 1 TUP, with the addition of the 290–300 AA portion (colored in green). The Zn-finger and Trp146 are shown as sticks, while Zn as a yellow sphere.

Figure 2

(A) Fluorescence emission spectra of DBD (1 µM) alone (black line) and at progressively higher concentrations of miR4749 (0.5-2.5 µM; colored lines) at 298 °K, by exciting at 295 nm and corrected for the Raman scattering of the buffer. (B) Stern–Volmer plot of the fluorescence quenching of DBD (1 µM in PBS buffer) as a function of miR4749 concentration at 298 °K, shown as black squares. Continuous red line is the linear fit through Equation (1); the extracted Stern–Volmer constant, K_SV, being reported.

Figure 3

(A) Fluorescence emission spectra of DBD-miR4749 (black line) and of DBD-miR4749Atto390 (red dashed line), obtained at a concentration of 1 µM with a 1:1 molar ratio between DBD and miR4749 or miR4749Atto390. (B) Fluorescence emission spectra of DBD-miR4749Atto390 (black line) at a concentration of 1 µM and of miR4749Atto390 (red dashed line); both of them at a concentration of 1 µM, with a 1:1 molar ratio. All the spectra were excited at 295 nm and corrected for the Raman scattering of the buffer.

Figure 4

(A) Secondary structure of miR4749 together with the dot-bracket representation (bottom). (B) Five best models for the 3D structure of miR4749.

Figure 5

(A) Histogram of the D_DA distance in the 50 models for the DBD-miR4749 complex. The distance was measured between the 5′ end of miR4749 to which the Atto390 dye is bound and the center of the aromatic rings of the lateral chain of Trp146 of DBD. (B) Collective representation of the five best models for DBD-miR4749 complex. DBD is colored in magenta, while miR4749: Model 1 (red), Model 2 (blue), Model 3 (orange), Model 4 (yellow) and Model 5 (azur).

Figure 6

Temporal evolution of the all atom Root Mean Square Displacement (RMSD) for: (A) a representative run for each of the five DBD-miR4749 models and (B) the three replicate runs of Model 3.

Figure 7

(A) Best model for the complex between DBD and miR4749. The distance D_DA between Trp146 and the 5′ end of miR4749 and the center of the aromatic rings of the lateral chain of Trp146 of DBD (black dashed line) is reported. (B) X-ray structure of DBD (chain B) complexed with DNA (1 TUP PDB entry).