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

Abstract

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 in the promyelocytic leukemia bodies. Inhibiting their binding is a strategy for inducing apoptosis of senescent cells, but the binding surfaces that mediate the interaction of FOXO4 and p53 remain elusive. Here, we investigated two binding sites involved in the interaction between FOXO4 and p53 by using 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. Also, we showed that the CR3-binding surface of FOXO4 FHD interacts with p53 TAD2, and four residues of FOXO4 CR3 interact with the DNA-binding surface of p53 DBD. Further isothermal titration calorimetry experiments showed that the FOXO4 FHD-p53 TAD interaction takes precedence with high affinity and that the FOXO4 CR3-p53 DBD interaction follows. This work provides structural information at the molecular level that is key to understanding the interplay of two proteins responsible for cellular senescence.