LAUSR

Transcription factor p53 protects cells against tumorigenesis when subjected to various cellular stresses. Under these conditions, p53 interacts with transcription factor Forkhead box O (FOXO) 4, thereby inducing cellular senescence by upregulating the transcription of senescence‐associated protein p21. However, the structural details of this interaction remain unclear. Here, we characterize the interaction between p53 and FOXO4 by NMR, chemical cross‐linking, and analytical ultracentrifugation. Our results reveal that the interaction between p53 TAD and the FOXO4 Forkhead domain is essential for the overall stability of the p53:FOXO4 complex. Furthermore, contacts involving the N‐terminal segment of FOXO4, the C‐terminal negative regulatory domain of p53 and the DNA‐binding domains of both proteins stabilize the complex, whose formation blocks p53 binding to DNA but without affecting the DNA‐binding properties of FOXO4. Therefore, our structural findings may help to understand the intertwined functions of p53 and FOXO4 in cellular homeostasis, longevity, and stress response.