Structures of KIX domain of CBP in complex with two FOXO3a transactivation domains reveal promiscuity and plasticity in coactivator recruitment Feng Wanga,b, Christopher B. Marshalla,b, Kazuo Yamamotob,c, Guang-Yao Lia,b, Geneviève M. C. Gasmi-Seabrooka,b, Hitoshi Okadab,c, Tak W. Makb,c, and Mitsuhiko Ikuraa,b,1 aOntario Cancer Institute, University Health Network (UHN), Toronto, ON, Canada M5G 1L7; bDepartment of Medical Biophysics, University of Toronto, Toronto, ON, Canada M5G 2M9; and cCampbell Family Institute for Breast Cancer Research, Ontario Cancer Institute, UHN, Toronto, ON, Canada M5G 2C1 Edited by Robert G. Roeder, The Rockefeller University, New York, NY, and approved March 5, 2012 (received for review November 20, 2011) Forkhead box class O 3a (FOXO3a) is a transcription factor and FOXOs recruit CBP/p300 to the FRE, which, in turn, recruits tumor suppressor linked to longevity that determines cell fate general transcriptional machinery and also remodels chromatin through activating transcription of cell differentiation, survival, and through histone acetyltransferase activity (10). However, CBP/ apoptotic genes. Recruitment of the coactivator CBP/p300 is a p300 also acetylates the FH domain, which impairs DNA binding crucial step in transcription, and we revealed that in addition to and attenuates transcription (11). The CR2, which consists of conserved region 3 (CR3) of FOXO3a, the C-terminal segment of CR2 three separate regions (A, B, and C) (Fig. 1A), may also play (CR2C) binds CBP/p300 and contributes to transcriptional activity. a role in the transactivation activity of FOXOs. Chromosomal CR2C and CR3 of FOXO3a interact with the KIX domain of CBP/p300 translocations create MLL-FOXO fusion proteins composed of at both “MLL” and “c-Myb” binding sites simultaneously. A FOXO3a the DNA binding region of MLL and the C terminus of either CR2C-CR3 peptide in complex with KIX exists in equilibrium be- FOXO3a (T228-G673) or FOXO4. In addition to CR3, im- tween two equally populated conformational states, one of which mortalization of myeloid progenitors by this fusion requires the has CR2C bound to the MLL site and CR3 bound to the c-Myb site, C-terminal segment of CR2 (CR2C) (5, 12), which contains whereas in the other, CR2C and CR3 bind the c-Myb and MLL sites, a ΦXXΦΦ motif (Fig. 1B). Whether CR2C is involved in BIOCHEMISTRY respectively. This promiscuous interaction between FOXO3a and transactivation by native FOXOs is unknown; however, it has CBP/p300 is further supported by additional binding sites on CBP/ been shown that CR2C binds Sirt1 (13). The histone deacetylase p300, namely, the TAZ1 and TAZ2 domains. In functional studies, activity of Sirt1 results in transcription silencing (14), but Sirt1 our structure-guided mutagenesis showed that both CR2C and CR3 also deacetylates the FH domain, restoring transcriptional ac- are involved in the activation of certain endogenous FOXO3a target tivity of FOXO3a; thus, the net effect of Sirt1 on transcription is genes. Further, phosphorylation of S626, a known AMP-dependent cell type- and gene-dependent (15). As such, the role of CR2C is protein kinase target in CR3, increased affinity for CBP/p300 and not fully understood. Moreover, the transactivation activity of the phosphomimetic mutation enhanced transactivation of lucifer- FOXO3a is regulated by AMP-dependent protein kinase ase. These findings underscore the significance of promiscuous mul- (AMPK) phosphorylation (16). Among six AMPK phosphory- tivalent interactions and posttranslational modification in the lation sites on FOXO3a, five are located in the C-terminal in- recruitment of transcriptional coactivators, which may allow tran- trinsically disordered region (IDR), including S626 in CR3; scription factors to adapt to various gene-specific genomic and however, the mechanism of phosphorylation-regulated tran- chromatin structures and respond to cell signals. scription is elusive. In this study, we found that in addition to CR3, CR2C interacts NMR solution structure | intrinsic disorder | promiscuous binding with the KIX domain and both regions bind KIX simultaneously. We determined the solution structures of KIX in complex with he forkhead box class O (FOXO) protein subfamily contains a fusion peptide of CR2C and CR3, which binds to the MLL and Tfour members: FOXO1, FOXO3a, FOXO4, and FOXO6 (1, c-Myb sites in equilibrium with two distinct orientations. The 2). FOXO3a is ubiquitously expressed in all tissues (2) and promiscuity of these TADs is further evidenced by our discovery activates the transcription of many genes that regulate differ- that they also bind the TAZ1 and TAZ2 domains of CBP/p300. entiation, cell cycle, DNA repair, stress resistance, metabolism, The transactivation function of CR2C and CR3 was characterized and apoptosis (1). FOXO3a is a 71-kDa intrinsically disordered by luciferase assays and examined in the context of full-length protein (IDP) that contains only one structured domain, the 100- FOXO3a by analysis of target gene expression. Our studies pro- residue forkhead (FH) domain (3), which binds a consensus vide insight into the mechanism by which CBP/p300 is recruited DNA sequence called forkhead response element (FRE). There by FOXO3a through multiple promiscuous and dynamic inter- are three additional conserved regions (CR1-CR3) across this actions between two TADs and KIX, TAZ1 and TAZ2, as well as subfamily (Fig. 1A), and it has been reported that CR3 is the how AMPK phosphorylation enhances this process. transactivation domain (TAD), mediating the association with the coactivator CBP/p300 through binding to its KIX domain (4, 5). We previously discovered an intramolecular interaction be- Author contributions: F.W., C.B.M., K.Y., H.O., and M.I. designed research; F.W., K.Y., G.-Y.L., and G.M.C.G.-S. performed research; H.O. and T.W.M. contributed new reagents/analytic tween the FH and CR3 domains (3), and demonstrated that FH tools; F.W., C.B.M., K.Y., and M.I. analyzed data; and F.W., C.B.M., K.Y., and M.I. wrote binding to FRE releases CR3, allowing it to bind KIX (6). The the paper. KIX domain has two distinct binding grooves that can simulta- The authors declare no conflict of interest. neously engage the TADs of c-Myb and MLL, thus designated This article is a PNAS Direct Submission. the c-Myb and MLL sites (7). MLL and c-Myb bind KIX in “Φ ΦΦ” Data deposition: The NMR structures, atomic coordinates, chemical shifts, and restraints a cooperative manner through their XX motifs, where have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 2LQH and “Φ” is a hydrophobic residue, and “X” is an arbitrary residue (7). 2LQI) and BioMagResBank, http://www.bmrb.wisc.edu/ (BMRB ID codes 18314 and 18315). A number of other transcription factors bearing this motif (e.g., 1To whom correspondence should be addressed. E-mail: [email protected]. CREB, c-Jun, p53) have also been found to interact with KIX This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (7–9) (Fig. 1B). 1073/pnas.1119073109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1119073109 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 Fig. 1. Characterization of interactions between FOXO3a and the CBP KIX domain. (A) Domain architecture of FOXO3a, indicating the FH domain and three conserved regions (CR1–3). Sequence alignments of CR2C and CR3 from FOXO proteins are shown with the predicted secondary structure. (B) Sequence alignment of KIX-interacting peptides. The hydrophobic residues of the “ΦXXΦΦ” motif are colored orange, and negatively charged residues are colored red. (C) NMR titration of 15N-labeled CR2C (0.2 mM) with KIX. (Left) HSQC of 15N-labeled CR2C (black) overlaid with spectra collected during titration with KIX: 0.1 (red), 0.35 (green), 0.85 (blue), 1.35 (yellow), 1.85 (pink), and 4.75-fold (cyan). Perturbed resonances are labeled. (Right) Normalized chemical shift change of each CR2C residue on addition of 4.75-fold excess KIX. The “LXXLL” motif is highlighted. (D) NMR titration of 15N-labeled CR2C-CR3 (0.16 mM) with KIX. (Left) HSQC of 15N-labeled CR2C-CR3 (black) overlaid with spectra collected during titration with KIX: 0.25 (red), 0.75 (green), 1.75 (blue), 3.25 (yellow), and 4.75-fold (pink). Perturbed resonances are labeled. Red circles indicate Gly and Ser residues in the engineered linker. (Right) Normalized chemical shift change of each CR2C-CR3 residue on addition of 4.75-fold excess KIX. CR2C and CR3 residues are plotted in black and red, respectively, and the most perturbed residues are labeled. (E) Probing CR2C-CR3 binding to KIX by PRE. (Left) Peak intensity ratio (Ipara /Idia) of each KIX residue in the presence of CR2C-CR3 peptide tagged with Mn2+ (para) vs. Ca2+ (dia). Cyan X’s indicate residues for which this ratio cannot be determined because of overlapped peaks. Residues exhibiting large broadening (ratio <0.2, red line) are labeled. (Right) Broadened residues mapped on the ternary KIX–c-Myb–MLL complex (PDB ID code 2AGH). The c-Myb and MLL sites are indicated, and broadened residues in each site are colored red and blue, respectively. All structure figures were prepared using PyMol (The PyMOL Molecular Graphics System, Schrödinger, LLC). Results CR2C-CR3 Interacts with KIX in Two Orientations. Titration with CR2C of FOXO3a Contributes to KIX Domain Binding. Characteriza- CR2C perturbed peaks from KIX residues in both the c-Myb and MLL sites (Fig. S2A), indicating that like CR3 (6), CR2C also tion of oncogenic MLL-FOXO fusion proteins has implicated 15 CR2C in transcription activation; however, little is known about binds both sites. We titrated N-labeled CR2C-CR3 with un- labeled KIX and observed that both CR2C and CR3 bound KIX the underlying mechanism or how CR2C and CR3 may cooperate simultaneously, whereas the linker was not affected by KIX.