title: Biochemical and structural characterization of an intramolecular interaction in FOXO3a and its binding with p53.
abstract: FOXO3a, a forkhead transcription factor and member of the forkhead box class O (FOXO) subfamily, has been shown to promote the translocation of p53 to the cytoplasm, thereby inducing the mitochondria-associated apoptotic pathway. However, the binding sites that mediate this interaction between FOXO3a and p53 have not been identified. Here, we show that two regions within FOXO3a, the forkhead (FH) DNA binding domain and a conserved C-terminal transactivation domain (CR3), interact with the DNA binding domain of p53, with affinities in the low millimolar range and low micromolar range, respectively. Our data further suggest that within the FOXO3a molecule, the FH and CR3 domains engage in an intramolecular interaction with low micromolar affinity. Moreover, we used NMR to determine the solution structure of the FH domain. This enabled us to map the binding site for the CR3, which overlaps with the DNA binding site. We demonstrate that an intrinsically disordered linker between the FH and CR3 domains is required for full p53 binding activity. We also show that p53 disrupts the intramolecular interaction between FH and CR3. These results provide evidence for interplay of the FH and CR3 domains in association with p53.
authors.affiliation: Division of Signaling Biology, Ontario Cancer Institute, Toronto, Ontario, Canada M5G 2M9.
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Marshall
authors.forename: Kazuo
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Guang-Yao
authors.initials: GY
authors.lastname: Li
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Plevin
authors.forename: Han
authors.initials: H
authors.lastname: You
authors.forename: Tak W
authors.initials: TW
authors.lastname: Mak
authors.forename: Mitsuhiko
authors.initials: M
authors.lastname: Ikura
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis; D001665:Binding Sites; D002942:Circular Dichroism; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: C499517:FOXO3 protein, human; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53