Mathivanan Subashini, Chunchagatta Lakshman Puneeth Kumar, Singh Manvi, Giridharan Saranya, Sathish Keerthana, Hurakadli Manjunath A, Bharatham Kavitha, Kamariah Neelagandan
Centre for Chemical Biology & Therapeutics, inStem & NCBS, Bellary Road, Bangalore 560065, India.
ACS Omega. 2022 Jul 5;7(28):24344-24352. doi: 10.1021/acsomega.2c01700. eCollection 2022 Jul 19.
The transcriptional activity of Forkhead Box O3 (FOXO3a) is inactivated by AKT-mediated phosphorylation on Serine 253 (S253), which enables FOXO3a binding to 14-3-3. Phosphorylated FOXO3a binding to 14-3-3 facilitates the nuclear exclusion of FOXO3a, causing cancer cell proliferation. The FOXO3a/14-3-3 interaction has, therefore, emerged as an important therapeutic target. Here, we report a comprehensive analysis using fluorescence polarization, isothermal titration calorimetry, small-angle X-ray scattering, X-ray crystallography, and molecular dynamics simulations to gain molecular-level insights into the interaction of FOXO3a phosphopeptide with 14-3-3ε. A high-resolution structure of the fluorophore-labeled FOXO3a:14-3-3ε complex revealed a distinct mode of interaction compared to other 14-3-3 phosphopeptide complexes. FOXO3a phosphopeptide showed significant structural difference in the positions of the -3 and -4 Arg residues relative to pSer, compared to that of a similar phosphopeptide, FOXO1 bound to 14-3-3σ. Moreover, molecular dynamics studies show that the significant structural changes and molecular interactions noticed in the crystal structure of FOXO3a:14-3-3ε are preserved over the course of the simulation. Thus, this study reveals structural differences between the binding to 14-3-3 isoforms of FOXO1 versus FOXO3a, providing a framework for the rational design of isoform-specific FOXO/14-3-3 protein-protein interaction inhibitors for therapy.
叉头框O3（FOXO3a）的转录活性通过丝氨酸253（S253）上AKT介导的磷酸化而失活，这使得FOXO3a能够与14-3-3结合。磷酸化的FOXO3a与14-3-3的结合促进了FOXO3a的核外排，导致癌细胞增殖。因此，FOXO3a/14-3-3相互作用已成为一个重要的治疗靶点。在此，我们报告了一项综合分析，使用荧光偏振、等温滴定量热法、小角X射线散射、X射线晶体学和分子动力学模拟，以深入了解FOXO3a磷酸肽与14-3-3ε相互作用的分子水平情况。与其他14-3-3磷酸肽复合物相比，荧光团标记的FOXO3a:14-3-3ε复合物的高分辨率结构揭示了一种独特的相互作用模式。与结合到14-3-3σ的类似磷酸肽FOXO1相比，FOXO3a磷酸肽在相对于pSer的-3和-4精氨酸残基位置上显示出显著的结构差异。此外，分子动力学研究表明，在FOXO3a:14-3-3ε晶体结构中观察到的显著结构变化和分子相互作用在模拟过程中得以保留。因此，本研究揭示了FOXO1与FOXO3a与14-3-3亚型结合之间的结构差异，为合理设计用于治疗的亚型特异性FOXO/14-3-3蛋白质-蛋白质相互作用抑制剂提供了框架。
