Overview of the FOXO3a-DBD/DNA complex
We determined the crystal structure of FOXO3a-DBD (residues Gly158 to Ser253) bound to a 13-bp duplex DNA containing the 7-bp FOXO consensus binding sequence (GTAAACA) at 2.7 Å resolution (). In the asymmetric unit of the crystal, two FOXO3a-DBD molecules bound to two DNA duplexes with a pseudo 2-fold symmetry (A). The geometry of the DNA duplex was canonical B-form DNA with a few kinks. In the crystal, the DNA duplexes formed a pseudo-continuous DNA helix stabilized by base-stacking interactions with symmetry-related DNA. There were direct protein–protein interactions between the two FOXO3a-DBD molecules, ostensibly forming a homodimer; however, to date no biological function has been ascribed for such a dimer of FOXO3a-DBD.
Figure 2. Overall structure of the FOXO3a-DBD/DNA complex. (A) The dimeric structure of FOXO3a-DBD/DNA complex with a pseudo 2-fold symmetry within an asymmetric unit. Two FOXO3a-DBD molecules are shown as ribbon drawings with the DNA in orange. (B) Sequence of (more ...)
The two FOXO3a-DBD molecules bound to DNA in a similar manner. Briefly, the DNA-binding domain of FOXO3a was arranged in a compact winged-helix motif consisting of three α-helices, three β-strands, one wing (wing 1) and a C-terminal coil (C). The recognition helix (H3) docked into the major groove roughly perpendicular to the DNA axis and made numerous interactions with the FOXO consensus sequence. The protein–phosphate interactions were localized in two phosphate backbones forming the major groove of the core sequence. In the region between H2 and H3, the residues preceding the recognition helix formed a solvent-exposed loop structure. The typical wing 1 interacted with the phosphate group of the 3′ flanking region of the consensus sequence without making base-specific contacts. In the C-terminal region of FOXO3a-DBD, the residues following S3 formed a coil structure that aligned to the major groove and interacted with the DNA (C). To analyze structural deviations, two FOXO3a-DBD molecules related by pseudo 2-fold symmetry in the asymmetric unit were superimposed. The 0.64 Å root mean square deviation (RMSD) for Cα atoms of secondary structural elements showed no obvious structural disparity. D shows the electron density map of the interface between the recognition helix and major groove of DNA.
Major groove recognition within the consensus binding site
In most forkhead proteins, the recognition helices H3 within the DNA-binding domain share a highly conserved amino acid sequence (B). As expected, the recognition helix H3 in the FOXO3a-DBD structure docked perpendicular to the major groove and formed extensive contacts with the FOXO consensus binding sequence. The recognition helix H3 centered over the FOXO consensus sequence, with several residues within helix 3 forming direct hydrogen bonds as well as van der Waals contacts with bases of the major groove. Details of the interactions between recognition helix H3 and DNA are shown in A and . The conserved residues Asn208, Ser215, Arg211 and His212 within H3 were important for DNA recognition. Asn208 bound to the A4 base via two hydrogen bonds. Arg211 recognized T5′ and T7′ through van der Waals contacts and contributed to the specificity for G6′ with a direct hydrogen bond. The side chain of His212 protruded into the major groove and recognized bases T2 and T3′ through a van der Waals contact and a direct hydrogen bond, respectively. Ser215 recognized T4′ through a van der Waals contact. Among these interactions, we note that the methyl groups of thymine bases from the FOXO consensus binding sequence were crucial for FOXO3a-DBD promoter recognition. In addition to base recognition, Ser215 also interacted with the phosphate group of T8′ to further stabilize the complex.
Figure 3. Detailed contacts of the FOXO3a-DBD/DNA interface. (A) Stereo view of the recognition of the GTAAACA consensus DNA-binding sequence by recognition helix H3 of FOXO3a-DBD. Hydrogen bonding and van der Waals contacts participating in base recognition are (more ...)
Figure 4. Schematic diagram of interactions between FOXO3a-DBD and DNA. The phosphate groups of DNA are represented as red circles. Base pairs of the FOXO consensus DNA-binding sequence, GTAAACA, are in red. The residues of helix 3, wing 1 and the C-terminal coil (more ...)
The FOXO3a-DBD C terminus forms a coil to interact with the DNA major groove
In the FOXO3a-DBD/DNA complex, the C-terminal region adopted a coil structure and interacted with the major groove of DNA (C and B). Interestingly, the C-terminal region (242-KSGKAPRRRAVS-253) not only functions as a NLS, but also overlaps with the PKB phosphorylation site, 14-3-3 binding motif and CBP acetylation sites (A and B) (20
). The three basic residues Arg248-Arg249-Arg250, part of the PKB recognition motif, are highly conserved in other members of the FOXO family. In our structure, this region played an important role in DNA major groove binding, with each of these three residues forming an ion pair with a phosphate group in the major groove of the DNA backbone without base-specific contacts (B). Ser253, a PKB phosphorylation site, immediately following the Arg248–250 motif also made direct contact with a DNA phosphate group (B), implying that Ser253 (when non-phosphorylated) participates in DNA binding. PKB-mediated phosphorylation at this residue may disrupt this contact and thereby inhibit DNA binding by introduction of a negative charge. In addition, previous work has shown that two positively charged residues, Lys242 and Lys245 of FOXO1 (corresponding to Lys242 and Lys245 in FOXO3a) located in the C-terminal region of the DNA-binding domain, are prone to acetylation by CBP, leading to reduced DNA binding and increased sensitivity to PKB-induced phosphorylation at Ser253 (34
). As shown in C, Lys245 interacted directly with the DNA phosphate group, but Lys242 was too far away from the DNA to participate directly in binding. This is consistent with the results from our mutagenesis study (see below) where a K245A mutation resulted in a greater apparent loss of DNA binding than a K242A mutation. The protein–DNA interactions of Lys245 could be explained if the acetylation of Lys residues led to the reduction of the DNA-binding affinity. Interestingly, no apparent intramolecular interaction was found between the C-terminal coil and FOXO3a-DBD, suggesting that the C-terminal region is highly mobile in the absence of DNA. Hence, in the presence of DNA the C-terminal coil may be stabilized by interactions with the major groove. The C-terminal region of FOXO3a thus plays several important roles in DNA binding, and charge variations induced by phosphorylation or acetylation may attenuate the DNA-binding affinity of FOXO proteins.
Interaction between wing 1 and DNA
The FOXO3a-DBD/DNA complex structure revealed that wing 1 of FOXO3a also contributes to DNA binding by interacting with the phosphate backbone in the 3′-flanking region of the consensus sequence (D). The terminal amine of Lys230 formed an ion pair with the phosphate group of T7′. In addition, the amide group of Ser232 made a hydrogen bond with the phosphate group of G6′. Arg222 and Trp234, located at the stem of wing 1, also made contact with the phosphate groups of G6′ (D). Analysis of the amino acid composition of wing 1 of FOX proteins showed that FOXO3a is two residues shorter in the wing 1 region (B). The lack of these two residues may change the shape and size of the wing 1 loop, such that wing 1 of FOXO3a is not long enough to push Lys230 far enough into the minor groove for DNA recognition. Thus, although wing 1 of FOXO3a may help to stabilize DNA binding, it does not play a direct role in protein–DNA recognition.
Structural comparison with other FOX proteins
A superimposition of the FOXO3a-DBD/DNA complex with the previously reported FOXA3, FOXK1a and FOXP2 DNA complexes showed a high degree of structural similarity in the core region (H1 ~ H3 and S1 ~ S3) with RMSDs of 0.64, 0.66 and 0.56 Å for Cα positions, respectively (). However, there are several major structural deviations located in the H2–H3 turn, wing 1 and C-terminal regions, as the amino acid compositions and lengths of these regions are not well conserved. In the FOXA3 and FOXP2 complexes, both H2–H3 turns formed short α-helices. In contrast, the corresponding regions in FOXO3a and FOXK1a are coil structures. In particular, the H2–H3 turn region of FOXO3a has an insertion of five additional residues (198-GDSNS-202) that are solvent-exposed. The function of these extra residues is unknown, because there was no protein–DNA interaction in this region within the FOXO3a-DBD/DNA complex structure.
Figure 5. Structural comparison of FOXO3a-DBD with other FOX/DNA complexes. Stereo diagram of superposition of the FOXO3a, FOXA2, FOXK1a and FOXP2/DNA complexes showing the Cα trace. For clarity, only the DNA in FOXO3a-DBD is shown, in light blue. FOXO3a, (more ...)
The wing 1 region of these FOX proteins also varies in sequence and length, which may lead to various conformations of the wing 1 structure (). In the FOXK1a/DNA complex, wing 1 utilizes Lys73 (corresponding to Lys230 in FOXO3a) to insert into the minor groove to recognize the base (35
). In contrast to FOXK1a, no direct base interaction was found for residue Lys230 in the FOXO3a-DBD/DNA complex. However, in FOXP2, the five amino acids preceding the corresponding Lys residue were truncated, resulting in a simple turn that did not make any contact with DNA (41
). Interestingly, in the FOXP3/NFAT/DNA ternary complex structure, the same Lys residue mediates a protein–protein interaction with NFAT (41
). Due to the sequence variation in the wing 1 region, we speculate that the differences in length and amino acid composition of wing 1 allow it to play various roles in the mediation of DNA recognition, the stabilization of protein–DNA complex or the protein–protein interactions in the various FOX proteins.
The most structurally divergent region of these FOX/DNA complex structures is the C-terminus of the DNA-binding domains, which contains many positively charged residues and functions in DNA binding or recognition specificity. The C-terminal regions of FOXK1a and FOXP2 form an α-helix. In contrast, the corresponding regions of FOXO3a and FOXA3 adopt coiled structures. Although the C-terminal regions of FOXA3, FOXK1a and FOXP2 have different structures, they all position the protein in the same binding orientation, whereas the corresponding region in FOXO3a was in the opposite orientation (). Due to the different arrangement of these coiled structures, FOXO3a interacted with DNA in the major groove whereas FOXA3 interacts with DNA in the minor groove.
DNA conformation in the FOXO3a-DBD/DNA complex
In the FOXO3a-DBD/DNA complex, the DNA exhibited a bend of 20° toward FOXO3a-DBD, mainly in the major groove, which was bound by the recognition helix. The base steps 2/3 and 6/7 were heavily kinked with roll angles 12.2° and 16.3°, respectively. The major groove was slightly wider (~3–4 Å wider than the canonical B-DNA) at positions where the recognition helix of FOXO3a-DBD was inserted. The minor groove was also slightly wider in the core sequence region. The helical twist per base pair varied from 24.0° to 40.5°. The DNA was 3.1% shorter than canonical B-DNA with the same number of nucleotides.
Mutational analyses of FOXO3a-DBD
To further characterize the roles of the residues involved in protein–DNA interactions in the FOXO3a-DBD/DNA structure, we studied the effect of various mutations on the ability of FOXO3a-DBD to bind to a DNA duplex containing either the FOXO consensus binding sequence 5′-GTAAACA-3′ or insulin response sequence (IRS) 5′-CAAAATA-3′ (50
). Gel shift studies (A and B) revealed that the FOXO3a-DBD158–240
lacking the C-terminal region almost lost the ability to bind DNA, indicating that the C-terminal region of FOXO3a-DBD contributes significantly to the formation of a stable protein–DNA complex. Substitutions of the three basic residues (Arg248 ~ 250) with alanine also substantially diminished DNA binding. Because Lys242 and Lys245 are located in the C-terminus of FOXO3a-DBD, and Lys245 interacted with the phosphate group, we mutated these Lys residues to alanine and examined their effect on DNA binding. The K245A mutation reduced the DNA-binding ability more than the K242A mutation, consistent with the observed direct interaction between Lys245 and the DNA in the crystal structure. Although Lys242 did not contact the DNA, the K242A mutant showed a slight decrease in DNA affinity. We hypothesize that the K242A mutant impaired DNA binding by destabilizing the C-terminal region. We also examined the effect on DNA binding with the double mutant, K242A/K245A. Interestingly, DNA binding in this double mutant was dramatically reduced (A and B), suggesting that K242 and K245 might have a synergistic effect on DNA binding.
Figure 6. Electrophoretic mobility shift assay (EMSA). (A) EMSA was performed with wild-type, mutated and FOXO3a-DBD158–240 and a 32P-labeled oligonucleotide probe containing the FOXO consensus DNA-binding sequence, GTAAACA. In the control lane, only 32 (more ...)
Recently, CDK2 was shown to interact with the DNA-binding domain of FOXO1 by phosphorylating the C-terminus at Ser246 (corresponding to Ala246 in FOXO3a), which resulted in translocation from the nucleus to the cytoplasm (19
). In the FOXO3a-DBD/DNA complex, Ala246 was close to the DNA major groove (C). We constructed an aspartic acid substitution mutant, A246D, to mimic the Ser246-phosphorylated state of FOXO1 and to investigate its effect on DNA-binding ability. Indeed, the A246D mutant exhibited substantially decreased DNA binding, indicating that introduction of a negative charge at A246 reduced the DNA-binding ability of FOXO3a by decreasing the stability of the interaction between the C-terminus and DNA. In addition, we also created two mutants, K230A (wing 1 region) and R211A (recognition helix H3), to examine the importance of these residues in DNA binding. Both the R211A and K230A mutants showed an apparent loss of DNA-binding ability, consistent with the crystallographic data showing that both wing 1 and the C-terminal region of FOXO3a-DBD participated in DNA binding. Moreover, the FOXO consensus sequence had higher affinity for DNA than the IRS sequence, consistent with a previous report (50
To compare the DNA-binding affinity of the FOXO3a-DBD proteins, we performed fluorescence anisotropy assay to measure the binding ability of these mutant proteins using a fluorescently labeled DNA containing the FOXO consensus binding sequence. As shown in , we observed that the K24A and K245A mutants are defective in binding DNA compared to wild-type FOXO3a-DBD. The wild-type FOXO3a-DBD bound to DNA with an apparent Kd
of 295 ± 26 nM (). However, the apparent Kd
value for K242A and K245A mutants is 399 ± 25 nM and 511 ± 63 nM, respectively. These data support the results of gel shift studies that substitutions of K242 and K245 for alanine reduced the DNA-binding affinity of FOXO3a-DBD. In the structure, residue Ser253 interacted with phosphate group of DNA. To further study the role of PKB-induced phosphorylation at Ser253, we created a S253D mutant to mimic the phosphorylated state. The result showed that S253D mutant caused a decrease on its DNA-binding ability (Kd
= 511 ± 63 nM), indicating that phosphorylation at Ser253 influences the stability of protein–DNA interaction. This effect is also consistent with the result that S256D of FOXO1 (Ser253 in FOXO3a) led a decrease in DNA-binding affinity (52
). In addition, substitutions of the three basic residues (Arg248 ~ 250) with alanine also significantly impaired DNA-binding affinity with an apparent Kd
of 1178 ± 168 nM. Furthermore, the truncation of C-terminus at the FOXO3a-DBD also caused a 5-fold decrease for DNA-binding (Kd
= 1492 ± 197 nM), indicating that the C-terminus of FOXO3a-DBD is important for DNA binding.
Figure 7. DNA-binding properties of FOXO3a-DBD proteins examined by fluorescence anisotropy assay. Wild-type FOXO3a-DBD158–253, K242A, K245A, S253D, RRR/AAA and FOXO3a-DBD158–240 were diluted and incubated with a fluorescein-labeled DNA at 25°C (more ...)
DNA-binding properties of FOXO3a-DBD proteinsa
FOXO protein recognizes an AT-rich consensus sequence
In the FOXO3a-DBD/DNA structure, methyl groups of thymine bases within the 5′-GTAAACA-3′ consensus sequence formed numerous van der Waals contacts with the recognition helix. This phenomenon suggested that these methyl groups are also important for FOXO protein recognition of the AT-rich consensus DNA. To test this hypothesis and analyze the binding contribution from each methyl group, we substituted individual T nucleotides with U and measured the affinity of wild-type FOXO3a-DBD for the new oligonucleotides. As shown in C, the substitution U2 decreased protein–DNA complex formation to 30%, whereas base substitution at positions U4, U5 and U7 reduced binding to 30, 15 and 20%, respectively. Base substitution at position U3 did not apparently decrease in DNA binding, which was consistent with the structure result where no interaction was found between the methyl group of T3′ and the recognition helix (A). In the FOXO3a-DBD/DNA complex structure, the methyl groups of DNA bases T5′ and T7′ interacted with residue Arg211 through van der Waals contacts (A and ), which explained why the R211A mutant in FOXO3a-DBD had substantially less affinity for DNA. Based on the structure and on these DNA substitution experiments, we suggest that van der Waals contacts between the methyl groups of thymine bases and side chains of the recognition helix are important for FOXO3a recognition of the FOXO consensus sequence.