Wang Feng, Marshall Christopher B, Yamamoto Kazuo, Li Guang-Yao, Plevin Michael J, You Han, Mak Tak W, Ikura Mitsuhiko
Division of Signaling Biology, Ontario Cancer Institute, Toronto, Ontario, Canada M5G 2M9.
J Mol Biol. 2008 Dec 19;384(3):590-603. doi: 10.1016/j.jmb.2008.09.025. Epub 2008 Sep 18.
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.
FOXO3a是一种叉头转录因子，属于叉头框O类（FOXO）亚家族成员，已被证明可促进p53转运至细胞质，从而诱导线粒体相关的凋亡途径。然而，介导FOXO3a与p53之间这种相互作用的结合位点尚未确定。在此，我们表明FOXO3a内的两个区域，即叉头（FH）DNA结合结构域和一个保守的C端反式激活结构域（CR3），分别以低毫摩尔范围和低微摩尔范围的亲和力与p53的DNA结合结构域相互作用。我们的数据进一步表明，在FOXO3a分子内，FH和CR3结构域以低微摩尔亲和力进行分子内相互作用。此外，我们利用核磁共振确定了FH结构域的溶液结构。这使我们能够绘制出CR3的结合位点，该位点与DNA结合位点重叠。我们证明FH和CR3结构域之间的一个内在无序连接子是完整p53结合活性所必需的。我们还表明p53破坏了FH和CR3之间的分子内相互作用。这些结果为FH和CR3结构域与p53相互作用提供了证据。
