Biophysics and Nanoscience Centre, DEB, Università della Tuscia, 01100 Viterbo, Italy.
Biomolecules. 2020 Feb 22;10(2):346. doi: 10.3390/biom10020346.
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 10 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 L loop and the H 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.
研究了肿瘤抑制因子 p53 的 DNA 结合域（DBD）与 miR4749 之间的相互作用，miR4749 与 p53 的 DNA 反应元件（RE）具有高度的序列相似性，研究方法是结合计算建模和对接的荧光光谱法。荧光猝灭实验见证了 DBD 和 miR4749 之间形成特定复合物的过程，其亲和力约为 10M。Förster 共振能量转移（FRET）使我们能够测量 DBD 中单独的色氨酸与适当结合在 miR4749 上的受体染料之间的距离为 3.9±0.3nm。这些信息与计算建模方法相结合，使我们能够预测 DBD-miR4749 复合物的可能结构。随后进行对接细化，并结合结合自由能计算，我们确定了 DBD-miR4749 复合物的最佳模型。我们发现，miR4749 的相互作用涉及 DBD 的 L 环和 H 螺旋，靠近锌指基序；这表明 miR4749 可以直接抑制 p53 与 DNA 的相互作用。这些结果可能为恢复 p53 功能活性的新治疗策略提供启示。
