Biophysical investigation of the dual binding surfaces of human transcription factors FOXO4 and p53

Cellular senescence is protective against external oncogenic stress, but its accumulation causes aging-related diseases. Forkhead box O4 (FOXO4) and p53 are human transcription factors known to promote senescence by interacting each other and activating p21 transcription. Inhibition of the interaction is a strategy for inducing apoptosis of senescent cells, but the binding surfaces that mediate the FOXO4-p53 interaction remain elusive. Here, we investigated two binding sites involved in the interaction between FOXO4 and p53 by NMR spectroscopy. NMR chemical shift perturbation analysis showed that the binding between FOXO4’s forkhead domain (FHD) and p53’s transactivation domain (TAD), and between FOXO4’s C-terminal transactivation domain (CR3) and p53’s DNA binding domain (DBD), mediate the FOXO4-p53 interaction. Isothermal titration calorimetry data showed that both interactions have micromolar K d values, and FOXO4 FHD-p53 TAD interaction has a higher binding affinity. Also, we showed that the FOXO4 CR3-binding surface of FOXO4 FHD interacts with p53 TAD2, and FOXO4 CR3 interacts with the DNA/p53 TAD-binding surface of p53 DBD, suggesting a network of potentially competitive and/or coordinated interactions. Based on the results, we propose that the dual interaction contributes to two TF’s proper location on the p21 promoter site and consequently promotes p21 transcription and cell senescence. This work provides structural information at the molecular level that is key to understanding the interplay of two proteins responsible for cellular senescence. Conflicts of interest None