14-3-3ζ interacts with DNA-binding domain of FOXO3a and competitively dissociates DNA by dual-motif tethering

Domain: neuroscience
Published: 2026-02-16
Editorial outcome: major_revision

Abstract
In cancer cells, Ras protein mutations activate a signaling cascade that phosphorylates kinases, transcription, and translation factors, driving cancer cell proliferation. One such factor, FOXO3a, promotes apoptosis-related gene transcription. However, in many cancer cells, FOXO3a is phosphorylated and is bound to 14-3-3ζ at phosphorylation sites. The 14-3-3ζ binding displaces phosphorylated FOXO3a from DNA, suppressing apoptosis. Since the phosphorylation sites are far from the DNA-binding domain (DBD) of FOXO3a, the mechanism of displacement remains unclear. Using isothermal titration calorimetry and fluorescence-detection size-exclusion chromatography, we find that 14-3-3ζ strongly displaces DNA from di-phosphorylated FOXO3a (dpFOXO3a), despite similar dissociation constants for dpFOXO3a–14-3-3ζ and dpFOXO3a–DNA. Nuclear magnetic resonance data identify weak, but direct binding of 14-3-3ζ to the DBD, suggesting direct competition. These findings suggest that 14-3-3ζ enhances its competitive ability by dual tethering to the DBD of FOXO3a via phosphorylation sites, effectively displacing DNA.

Introduction
In cancer cells, Ras proteins are activated by mutations independently of upstream regulators, initiating a signaling cascade. This cascade consists of the sequential phosphorylation of three or more kinases, ultimately leading to the phosphorylation of effector proteins involved in transcription and translation1. Phosphorylation up- or down-regulates effector protein function, causing uncontrolled cell proliferation, apoptosis evasion, invasion, and metastasis1. One such effector protein is FOXO3a, which is a transcriptional activator of genes encoding a number of apoptosis-related proteins such as Bim and FasL that inhibit the cell proliferation2–4. However, in tumor cells transcriptional activity of FOXO3a is inhibited upon its phosphorylation and subsequent binding of an adaptor protein, 14-3-3ζ, resulting in cancer progression via cancer cell apoptosis suppression2.

FOXO3a (a 71 kDa protein consisting of 673 residues) is a member of the forkhead box (FOX) protein family. FOXO3a comprises a DNA-binding domain (DBD), also called the forkhead domain (residues 157–250), and intrinsically disordered regions in the N- and C-terminals (Fig. 1)5,6. The DBD binds to specific DNA sequences (referred to as FOXO-responsive elements, FREs), triggering apoptosis-related gene transcription. In tumor cells, FOXO3a is phosphorylated at three residues (T32, S253, and S315) in the intrinsically disordered regions by an upstream kinase, AKT. Subsequently, its transcriptional activity is inhibited by the binding of an adaptor protein, 14-3-3ζ2, which is highly expressed in cancer cells7.

14-3-3ζ, a member of the 14-3-3 protein family, is a 245-residue acidic protein, consisting of nine α-helices. 14-3-3ζ exists and functions as an axisymmetric dimer8. 14-3-3ζ binds to the Ser/Thr-phosphorylated motifs in its target proteins and prevents their dephosphorylation, prolonging the functional regulation by phosphorylation9. 14-3-3ζ recognizes two phosphorylated motifs in the N-terminal region of FOXO3a (P1: RPRSC-pT-WPLQR, P2: RRRAV-pS-MDNSN)2,10,11. Upon binding to the phosphorylated FOXO3a, 14-3-3ζ dissociates FOXO3a from FREs, thereby inhibiting its transcriptional activity2,12,13. Furthermore, 14-3-3ζ masks the nuclear localization signal (RRR) of FOXO3a located within the P2 site, facilitating the export of FOXO3a from the nucleus to the cytoplasm14,15. In the cytoplasm, FOXO3a is ubiquitinated and subject to degradation16,17. Consequently, FOXO3a-mediated apoptosis is suppressed, leading to malignant cancer transformation. Reportedly, cancer cell apoptosis is enhanced by inhibiting the interaction between phosphorylated FOXO3a and 14-3-3ζ18. Therefore, inhibiting the interaction is a viable target for anticancer drugs. Although understanding the mechanism of action is important, it remains unknown how the 14-3-3ζ binding dissociates FOXO3a from its target DNA sequence, as the 14-3-3ζ binding sites, P1 and P2, located remotely from the DBD.

This dissociation mechanism has been mainly studied using FOXO4, which has a similar structure to a member of the FOXO family19. FOXO4 has high sequence identity with FOXO3a at P1 (100%), P2 (86%), and the DBD (83%), supporting the assumption that their interaction modes with DNA and 14-3-3ζ are highly similar, and thus that Kd values of FOXO4 provide a reliable reference for FOXO3a. A previous study12 showed that 14-3-3ζ dissociates di-phosphorylated FOXO4 (dpFOXO4) from the DNA with the human insulin response element (hIRE). This result was attributed to the difference in dissociation constants (Kds), which were reported to be 30 nM for dpFOXO4–14-3-3ζ and 500 nM for dpFOXO4–hIRE. However, as the consensus DNA sequence recognized by the FOXO family (e.g., daf-16 binding element, DBE)20 has higher affinity for FOXO3a than for hIRE21, the relative binding affinity of phosphorylated FOXO3a for DBE and 14-3-3ζ should be analyzed quantitatively under the same conditions to conclude that the dissociation is caused by the difference in their Kd values.

In another study, the binding mode of the DBD of FOXO4 and the 14-3-3ζ dimer was deduced based on the distance constraints obtained from fluorescence resonance energy transfer (FRET) experiments using 14-3-3ζ ‘monomeric mutants’ and a FOXO4 fragment, which contained the DBD and was mono-phosphorylated at the P2 site22. However, the direct interaction between the 14-3-3ζ dimer and the DBD within dpFOXO4 has not yet been experimentally observed.

Here, we quantitatively analyze the binding affinity and competition of di-phosphorylated FOXO3a (dpFOXO3a), DNA, and 14-3-3ζ using isothermal titration calorimetry (ITC) and fluorescence-detection size-exclusion chromatography (FSEC). In addition, we execute NMR analyses on the interaction between dpFOXO3a and 14-3-3ζ. Based on these results, we propose a mechanism of the FOXO3a dissociation from DNA by the 14-3-3ζ binding.

Results
14-3-3ζ binds to dpFOXO3a and completely dissociates DNA from the dpFOXO3a-DNA complex

We expressed and purified FOXO3a residues 1–284 (hereafter referred to as FOXO3a) containing the DBD and two 14-3-3ζ-binding motifs, each of which possesses a phosphorylation site, T32 in P1 and S253 in P2, respectively, as previously reported (Fig. 1 and Supplementary Fig. 1a)23. Di-phosphorylated FOXO3a (dpFOXO3a) was prepared by phosphorylating FOXO3a using AKT (Supplementary Fig. 2a). The truncated construct FOXO3a (residues 1–284) used in this study is expected to have essentially the same binding affinities to DNA and to 14-3-3ζ as the full-length protein. For DNA binding, the interaction is dominated by the DBD5, and while the transactivation domain (TAD) can compete with DNA for the DBD6,13, the DNA–DBD interaction is strong enough to displace the TAD13. For 14-3-3ζ binding, the recognition of the two phosphorylated motifs (P1 and P2) of FOXO3a is known to be dominant. Isothermal titration calorimetry (ITC) analysis indicated the Kd value of 130 nM for the dpFOXO3a – DNA (DBE sequence, see “Methods” section) interaction (Table 1 and Supplementary Fig. 4a). 14-3-3ζ was also expressed and purified to homogeneity (Supplementary Fig. 1f). Size-exclusion chromatography – multi-angle light scattering (SEC-MALS) analysis indicated that the 14-3-3ζ dimer forms a 1:1 complex with dpFOXO3a (Supplementary Fig. 3). The Kd value for the dpFOXO3a interaction with 14-3-3ζ was 60 nM (Table 1 and Supplementary Fig. 4b), which is ~half that of the interaction with DNA (130 nM). These Kd values, together with binding stoichiometry, are consistent with previous reports for dpFOXO42,12, which has the similar binding mode, indicating that the samples we prepared possessed appropriate binding activities.

First, we quantitatively analyzed the competition between 14-3-3ζ and DNA for binding to dpFOXO3a by fluorescence-detection size-exclusion chromatography (FSEC). The fluorescein-labeling of the DNA duplex containing the DBE sequence enabled selective detection of the DNA based on the fluorescence from the fluorescein moiety, while the proteins (dpFOXO3a and/or 14-3-3ζ) were detected by their tryptophan fluorescence (Fig. 2). When a sample containing 10 μM dpFOXO3a and 11 μM DNA was analyzed by FSEC, the elution profiles showed a peak of the dpFOXO3a–DNA complex at the elution volume of 13.1 mL (Black in Figs. 2a, b). As the increasing amounts of 14-3-3ζ were added to the 1:1 mixture of dpFOXO3a and DNA, the peak height of the dpFOXO3a-bound DNA and free DNA decreased and increased, respectively (Fig. 2a), whereas the peak of DNA-bound dpFOXO3a gradually shifted toward the peak of the dpFOXO3a–14-3-3ζ dimer complex (Fig. 2b). These changes were saturated when one equivalent of the 14-3-3ζ dimer was added. These results indicate that the equimolar amount of 14-3-3ζ completely dissociated dpFOXO3a from the DNA.

We then simulated the competitiveness of 14-3-3ζ against the DNA on dpFOXO3a. Here, ‘competitiveness’ refers to the relative ability of 14-3-3ζ or DNA to bind to dpFOXO3a under the same concentration conditions. In quantitative terms, this can be expressed as the ratio of concentration to the dissociation constant (concentration/Kd), as described in the supplementary discussion. The simulation results (Figs. 2c–e) showed that the experimental FSEC results (solid lines) were not explained by the factor of two (dashed black lines), which is based on the reciprocal Kd ratio calculated using the Kd values obtained by ITC (Kd (dpFOXO3a – DNA) / Kd (dpFOXO3a – 14-3-3ζ) = 130/60) (Supplementary Fig. 4). However, the FSEC results are well explained by assuming the factor of 100 (thick dashed lines), demonstrating that 14-3-3ζ possesses ~100-fold competitiveness over DNA for dpFOXO3a. As 100-fold higher competitiveness is notably higher than the two-fold, which is expected from the experimental Kd values, an unknown mechanism seems to underlie the high competitiveness. On the other hand, NMR analysis of [2H, 15N]-dpFOXO3a bound to DNA upon addition of 14-3-3ζ showed that most signals did not change; however, the signals corresponding to P1 and P2 disappeared or shifted in a manner identical to that observed when 14-3-3ζ bound to dpFOXO3a in the absence of DNA (Supplementary Fig. 5a, b, and 6a). This indicates that 14-3-3ζ can bind to dpFOXO3a at the P1 and P2 sites even while dpFOXO3a remains associated with DNA. SEC of the same sample showed that this ternary complex was unstable, with ~74% of DNA free at 126 μM protein concentration and ~91% free at 16 μM (Supplementary Fig. 5c, d, 6b, and c), indicating strong concentration dependence.

14-3-3ζ binding to pS253 of dpFOXO3a mainly contributes to the DNA dissociation, whereas pT32 binding is necessary for complete DNA dissociation

To investigate the functional role of the phosphorylation sites, P1 and P2, of dpFOXO3a in the 14-3-3ζ binding and the high competitiveness against the DNA, we prepared the mono-phosphorylated mutants, pT32-FOXO3a(S253A) and pS253-FOXO3a(T32A), and analyzed their interactions with 14-3-3ζ and DNA. ITC analyses indicated that the binding affinities of these mutants for the DNA were similar to the wild type (Table 1 and Supplementary Fig. 7a, b), while those for 14-3-3ζ were weaker than the wild type protein but sufficiently high for these FSEC analyses at the concentrations of 10 μM (Table 1 and Supplementary Fig. 8b, c). When the equimolar mixture of pT32-FOXO3a(S253A), DNA, and 14-3-3ζ dimer was applied to FSEC, the elution volume of the peak of the protein-bound DNA slightly shifted from 13.1 to 13.0 mL and the peak shape was broadened, while the peak position and height of the unbound DNA did not change (Fig. 3a). In the meantime, the elution volume of the peak observed by the tryptophan fluorescence corresponded to the protein-bound DNA at 12.9 mL (Fig. 3b). These results indicate that 14-3-3ζ binds to the pT32-FOXO3a(S253A)–DNA complex, forming a ternary complex.

In contrast, when the equimolar mixture of the pS253-FOXO3a(T32A), DNA, and 14-3-3ζ dimer was applied, the peak height of the protein-bound DNA decreased by 42%, whereas that of unbound DNA increased by more than two-fold, indicating that the DNA partially dissociated from pS253-FOXO3a(T32A). Simultaneously, the peak observed by the tryptophan fluorescence shifted forward (Figs. 3c, d), demonstrating that 14-3-3ζ binds to pS253-FOXO3a(T32A). The full FSEC-based competition analysis revealed that at one equivalent of 14-3-3ζ dimer, only ~50% of the DNA complex was dissociated, compared with ~95% for dpFOXO3a, and even with four equivalents dissociation reached only ~83% (Supplementary Fig. 9).

Notably, the P1 site containing pT32 has higher binding affinity for 14-3-3ζ (Kd = 1.2 μM) than the P2 site containing pS253 (Kd = 21 μM), as confirmed by the ITC experiments using phosphorylated peptides, P1-peptide (RPRSC-pT-WPLQR), and P2-peptide (RRRAV-pS-MDNSN) (Table 1 and Supplementary Fig. 8).

We also performed this competition analysis using unphosphorylated FOXO3a as a control. Figure 3e shows that there were no changes on the elution profile of the DNA, while the peak for 14-3-3ζ appeared on the DNA-bound FOXO3a when one equivalent of 14-3-3ζ dimer was added (Fig. 3f), indicating that the phosphorylation at both two motifs of FOXO3a is necessary for the 14-3-3ζ binding and DNA dissociation.

Altogether, these FSEC results using di-/mono-phosphorylated FOXO3a, DNA, and 14-3-3ζ suggest that the binding of the P2 site to 14-3-3ζ mainly contributes to the DNA dissociation from dpFOXO3a. However, P1 site binding, which has a higher affinity than the P2 site, is needed for the full competitiveness against DNA. It remains unclear how 14-3-3ζ binds to both phosphorylation sites, which are located 126- and 11-residue apart from the structural region of the DBD, and facilitates DNA dissociation from the DBD. To elucidate the mechanism, we investigated the dpFOXO3a–14-3-3ζ interaction using nuclear magnetic resonance (NMR) spectroscopy.

14-3-3ζ directly interacts with dpFOXO3a at the DNA-binding domain (DBD) and the two phosphorylation motifs

First, we analyzed the NMR spectral changes of dpFOXO3a after adding 14-3-3ζ. The assignments of the backbone resonances of dpFOXO3a in the free state (BMRB: 26350) were described in a previous study23. A series of the 1H-15N transverse relaxation-optimized spectroscopy (TROSY) spectra of uniformly 2H, 15N-labeled dpFOXO3a were observed upon sequential additions of unlabeled 14-3-3ζ (Fig. 4). As the added amount of 14-3-3ζ increased, 66 signals disappeared, and 54 new signals appeared, suggesting that the spectral changes occurred apparently in a slow exchange manner. The changes almost saturated when one equivalent of the 14-3-3ζ dimer was added.

Next, we analyzed the chemical shift perturbation to identify the regions in dpFOXO3a affected by the 14-3-3ζ binding (Fig. 4c). The backbone assignments of the DBD of dpFOXO3a bound to 14-3-3ζ were transferred from those of C-terminal domain (CTD) construct of FOXO3a (residues 141-284, Fig. 1), which contains the DBD together with the C-terminal unstructured tail, rather than the DBD alone (Supplementary Fig. 10). Consequently, the dpFOXO3a residues that showed chemical shift changes above 0.03 ppm were not only around the P1 and P2 sites, but also around the helix 3 of the DBD (Fig. 4c). Mapping of the affected residues onto the DBD structure revealed that these residues were localized to one side of the DBD that includes helix 3 (Figs. 4c, d). These results suggest that in addition to the P1 and P2 sites, the region around the helix 3 on the DBD is the 14-3-3ζ-binding interface, which overlap well with the DNA-binding interface in the crystal structure of the DBD–DNA complex (Fig. 4e)24. NMR analysis monitoring the spectral changes of the CTD upon additions of 14-3-3ζ confirmed the 14-3-3ζ-binding region on the DBD of dpFOXO3a (Supplementary Fig. 10c–e). Notably, the curve fitting of the chemical shift changes of [2H, 15N]-CTD was established by the 14-3-3ζ titration that resulted in a Kd value of 5–30 μM for the DBD–14-3-3ζ interaction (Supplementary Fig. 10b).

Grooves of the 14-3-3ζ dimer accommodate the two phosphorylation motifs of dpFOXO3a and the upper regions of the grooves interact with the DBD

We analyzed the NMR spectral changes of uniformly 2H, 15N-labeled 14-3-3ζ, upon the sequential additions of unlabeled dpFOXO3a. Backbone resonances have been assigned for full length 14-3-3ζ for 211 of the total 244 observable residues (86%, BMRB: 26348) by a series of triple resonance experiments with reference to the previously reported assignments for the residues 1–23325 (Supplementary Fig. 11). In the free state of 14-3-3ζ, each residue provides only one peak, indicating that the 14-3-3ζ dimer possesses a symmetric structure in the NMR timescale.

Through sequential additions of dpFOXO3a, 116 signals of 14-3-3ζ disappeared from 1H-15N TROSY spectra, and 133 new signals appeared in a slow exchange manner. The signal changes almost saturated when one equivalent of dpFOXO3a was added to the 14-3-3ζ dimer (Fig. 5a). The increase in the number of signals is mainly due to splitting of the signals as shown in Fig. 5b. The splitting reflects that the 14-3-3ζ dimer is asymmetric in the complex with dpFOXO3a (Fig. 5b), where each protomer of the 14-3-3ζ dimer binds to the P1 or P2 sites of dpFOXO3a, respectively, which possess different amino acid sequences. Establishing the backbone assignments of 14-3-3ζ in the dpFOXO3a-bound state by triple resonance experiments was difficult, due to the line broadening and low sensitivity arising from high molecular mass of the complex (ca. 90 kDa) as well as the signal splitting caused by the asymmetry. Therefore, we evaluated whether each signal of 14-3-3ζ in the free state is affected or not by the addition of dpFOXO3a. The signals whose intensities decreased to 15% or less with the addition of 1.5 equivalents of dpFOXO3a were judged as the affected residues. Mapping of the affected residues indicates that these residues lie not only in the grooves to which the P1 and P2 sites bind, but also in the upper regions of the grooves formed by the helices 8–9 (Figs. 5c, d), suggesting that these upper regions of 14-3-3ζ interact with the dpFOXO3a regions other than the phosphorylated sites, P1 and P2. In addition, the spectral overlay of 14-3-3ζ upon binding to dpFOXO3a with those upon binding to the N-terminal unstructured region upstream of the DBD that is phosphorylated at T32 (referred to as pNTD containing the P1 site) and with those upon binding to the P2-peptide, showed that many signals with pNTD or the P2-peptide overlapped those with dpFOXO3a but 18 and 44 signals in the pNTD-bound state and P2-peptide-bound state, respectively, did not overlap with those in the dpFOXO3a-bound state (Supplementary Fig. 12a). These residues clustered on the 14-3-3ζ surface formed by its helices 8–9 (Supplementary Fig. 12b and c). These spectral differences were likely caused by the DBD that exists only in dpFOXO3a, neither in pNTD nor in the P2-peptide.

Based on these results, we hypothesized that this upper region of 14-3-3ζ interacts with the DBD of dpFOXO3a. To confirm the hypothesis, we analyzed the changes in 1H-15N TROSY spectra of uniformly 2H, 15N-labeled 14-3-3ζ, upon the sequential additions of the unlabeled CTD, which consists of the DBD and the C-terminal tail (Fig. 6). The chemical shift changes were observed in a fast exchange manner, enabling the assignments of the CTD-bound state by tracking the chemical shift changes (Fig. 6b). The residues whose chemical shifts changed more than 0.045 ppm with the addition of 2.5 equivalent of the CTD were clustered on the upper sites of the grooves of 14-3-3ζ (Figs. 6c, d). Given that the DBD in dpFOXO3a interacts with 14-3-3ζ (Fig. 4), we conclude that these upper sites of the grooves (helices 8–9) are the DBD-binding regions. From the observed chemical shift changes, the Kd value for the 14-3-3ζ binding to the CTD was calculated to be ca. 10 μM (Fig. 6b).

However, CSP identifies residues whose local chemical environment changes upon binding, which can result from either direct contact or conformational changes. To obtain direct evidence for DBD–14-3-3ζ interaction beyond CSP mapping, we performed NMR cross-saturation experiments, which selectively detect residues in close spatial proximity (~5 Å) to dpFOXO3a. This analysis identified specific contact residues on 14-3-3ζ, several of which are located outside the canonical phospho-motif binding groove and positioned to interact with the DBD (Fig. 7).

Quadruple-alanine mutants targeting these sites (D197A/E198A/E202A/D204A or T215A/L216A/Q219A/R222A) showed no reduction in DNA displacement ability in FSEC assays (Supplementary Fig. 13), consistent with a broad interaction interface in which partial disruption does not abolish binding. In contrast, NMR spectral changes of [2H, 15N]-CTD titrated with the T215A/L216A/Q219A/R222A 14-3-3ζ mutant demonstrated that signals, mainly in helix 3, shifted in a different manner or decreased in intensity compared with the titration using wild type 14-3-3ζ. The titration with the D197A/E198A/E202A/D204A mutant was not possible because the sample precipitated and could not be concentrated sufficiently for NMR analysis. Together, these NMR data strongly suggest that the helix 3 of DBD directly interacts with the residues around T215/L216/Q219/R222 of 14-3-3ζ (Supplementary Fig. 14).

DBD undergoes exchange between protomer 1- and 2-bound states in the dpFOXO3a and 14-3-3ζ dimer complex

Some signals of the residues at the DBD interaction site (helices 8–9) of 14-3-3ζ split into two signals (hereafter referred to as signals A and B) when dpFOXO3a was added, reflecting the asymmetry of the 14-3-3ζ dimer, where two protomers bind to P1 and P2, respectively. Then, to assign the split signals whether it is from the P1-bound protomer or P2-bound protomer, we prepared three types of symmetric dimers, in which both protomers are bound to pNTD (N-terminal unstructured region containing P1), pCTD (DBD and C-terminal unstructured region containing P2), or the P2-peptide. Notably, neither signal (A) nor (B) for E202 in the dpFOXO3a-bound state overlapped with the corresponding signals in the symmetric dimers (pNTD-, pCTD-, nor the P2-peptide-bound states) (Fig. 8a). The signal (A) is located in the middle of the signal of pCTD-bound and the P2-peptide-bound states.

It is tempting to speculate that the signal (A) is observed as a result of the fast exchange between the DBD-bound and -unbound states of the P2-bound protomer, where the population of the DBD-bound and -unbound state is almost 1:1. Under this assumption, the signal (B) is regarded as the signal from the P1-bound protomer, which seems due to the fast exchange between the DBD-bound and -unbound states of the P1-bound protomer.

The other five residues in the DBD binding site (D204, T205, E208, S214, and the signal overlapped by E209 and W228) provided split signals, where each signal was broad and distorted (Supplementary Fig. 15a–e), suggesting the multiple states are averaged in a fast-intermediate exchange regime. For the 14-3-3ζ residues in the region not interacting with the DBD, the signal that split into two upon addition of dpFOXO3a overlapped with those upon addition of pNTD, pCTD or the P2-peptide, respectively (e.g., H164) (Supplementary Fig. 15f).

In addition, high-field (1 GHz) NMR experiments provided direct evidences supporting the dynamic exchange of the DBD between the two protomers of the 14-3-3ζ dimer. At 1 GHz, the E202 ‘A’ signal observed in the fast-exchange regime at 800 MHz split into two peaks, consistent with distinct P2-peptide-bound and [P2+DBD]-bound states (Supplementary Fig. 16b, c). The E202 ‘B’ signal remained single at 1 GHz but split into two peaks when the temperature was lowered from 303 K to 293 K, with the ‘A’ peaks merging, indicating that ‘B’ contains both pNTD-bound and [pNTD+DBD]-bound states (Supplementary Fig. 16d, e). These field- and temperature-dependent changes indicate that the DBD dynamically exchanges between the two protomers of the 14-3-3ζ dimer.

Altogether, we propose that in the dpFOXO3a in complex with a 14-3-3ζ dimer, the DBD binds to upper region (helices 8–9) of either protomer of the 14-3-3ζ dimer at one time, where the DBD rapidly exchanges between the two protomers of the 14-3-3ζ dimer (Fig. 8b). The DNA binding interface of the DBD is covered by the upper region of either protomer of 14-3-3ζ most of the time, which competitively inhibits the DNA binding.

Methods
Protein expression and purification
As previously reported23, we constructed human FOXO3a (residues 1-284), the NTD (residues 1-146) and the CTD (residues 141-284) and purified them. In addition, the mutants FOXO3a (T32A) and FOXO3a (S253A) were generated according to the QuikChange® mutagenesis protocol (Stratagene, La Jolla, CA, USA) using the pGEX-6P-1-FOXO3a plasmid as the template and were purified in the same way as the FOXO3a purification. FOXO3a, FOXO3a (T32A), NTD, and CTD were phosphorylated by AKT23 and FOXO3a (S253A) required the addition of three times the amount of AKT for the phosphorylation. The phosphorylation reaction proceeded completely, confirmed by MALDI-TOF mass spectrometry, using MALDI-8020 spectrometer (SHIMADZU). In the MALDI-TOF MS, α-cyano-4-hydroxycinnamic acid was used as the matrix for FOXO3a, FOXO3a (T32A), FOXO3a (S253A), and the NTD, and sinapinic acid was used for the measurement with CTD. These experiments were performed in a buffer containing 50% acetonitrile and 0.1% TFA. pNTD was additionally purified by reversed-phase chromatography.

The DNA sequence encoding full-length human 14-3-3ζ (residues 1-245), D197A/E198A/E202A/D204A mutant, and T215A/L216A/Q219A/R222A mutant were cloned into the pGEX-6P-1 (Cytiva). The glutathione-S-transferase-fusion proteins were expressed in Escherichia coli C41 (DE3) cells and induced by addition of isopropyl β-D-thiogalactopyranoside at a final concentration of 1 mM for 4 h at 37 °C. The cells were collected by centrifugation and disrupted by sonication in a buffer containing phosphate-buffered saline (PBS) (pH 7.4), 1 M NaCl, 10% glycerol, 1 mM phenylmethanesulfonyl fluoride, 1 mM DTT, and 0.1% polyethylenimine. Cell debris was removed by centrifugation, and the supernatant was purified using a Glutathione-SepharoseTM 4B column (Cytiva) in a buffer containing PBS (pH 7.4), 500 mM NaCl, 1 mM DTT and 10 mM reduced glutathione. Followed by digestion with PreScission Protease (Cytiva), the cleaved glutathione-S-transferase and noncleaved fusion proteins were removed using a Glutathione-SepharoseTM 4B column. The impurities were removed using an anion exchange column (CaptoTM HiRes Q 5/50 GL; Cytiva) in a linear gradient of 0–1 M NaCl in 20 mM Tris-HCl (pH 7.5) and 1 mM DTT. 14-3-3ζ mutants were additionally purified by gel filtration using SuperdexTM 200 increase 10/300 GL (Cytiva) in a buffer containing 20 mM NaPi (pH 7.4), 150 mM NaCl and 1 mM DTT.

Uniformly 15N or 13C, 15N-labeled proteins were expressed by growing E. coli host cells in M9 minimal medium containing 1 g/L 15NH4Cl or 1 g/L 15NH4Cl and 2 g/L 13C-glucose. 2H, 15N-labeled proteins were expressed by growing E. coli host cells in M9 minimal medium in 2H2O containing 1 g/L 15NH4Cl, 2 g/L 2H-glucose, and 0.2 g/L 2H, 15N-celtone. 2H, 13C, 15N-labeled proteins were expressed by growing E. coli host cells in M9 minimal medium in 2H2O containing 1 g/L 15NH4Cl, 2 g/L 2H, 13C-glucose and 0.2 g/L 2H, 13C, 15N-celtone. These isotopically labeled proteins were purified using a similar procedure as used for non-labeled proteins.

Preparation of FOXO3a-phosphorylated peptides
We purchased the following FOXO3a-phosphorylated peptides (Shanghai Royo Biotech Co., LTD), and dissolved in Milli-Q water for NMR experiments or a buffer containing 20 mM NaPi (pH 7.4), 150 mM NaCl, and 1 mM DTT for ITC experiments.

P1-peptide: RPRSC-pT-WPLQR (FOXO3a residues 27-37, MW = 1478.64)
P2-peptide: RRRAV-pS-MDNSN (FOXO3a residues 248-258, MW = 1384.43)

Preparation of DNA and fluorescein-labeled DNA
We purchased the following oligonucleotides containing the consensus FOXO binding sequence (DBE sequence) (Eurofins and Thermo Fisher) and prepared dsDNA:
sense: 5′-CTATGTAAACAAC-3′,
antisense: 5′-GTTGTTTACATAG-3′ (DBE),
and fluorescein-antisense: 5′-fluorescein-GTTGTTTACATAG-3′.
The molar concentrations of oligonucleotides were evaluated by measuring the UV absorption at 260 nm based on Lambert–Beer’s law:
c = Abs260 / εl
where c is the molar concentration of the oligonucleotide, Abs260 and ε is the UV absorption and the molar extinction coefficient value at a wavelength of 260 nm, respectively, and l is the optical path length of 1 cm. ε was calculated using the nearest neighbor method34,35. εsense was 133140 and εantisense was 132180. εfluorescein-antisense was calculated as 153080 as the sum of εantisense 132180 and εfluorescein 2090036. Sense and anti-sense were mixed in 1 equivalent each and annealed by decreasing the temperature from 100 °C to 25 °C at 1 °C/min in a buffer containing 20 mM NaPi (pH 6.4) and 150 mM NaCl for ITC experiments, 20 mM NaPi (pH 6.4) and 100 mM NaCl for NMR experiments, or 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 1 mM EDTA for FSEC experiments.

SEC-MALS analysis
20 μL of the complex of dpFOXO3a–14-3-3ζ dimer (12.3 mg/mL) was loaded onto WTC-030S5 column (Wyatt) pre-equilibrated with 20 mM Tris (pH 7.5), 300 mM NaCl, 1 mM DTT at 0.4 mL/min at room temperature. DAWN HELEOS II detector (Wyatt) was used to measure MALS, while the RH-101 detector (Shodex) was used to detect refractive index. Data analysis was performed using ASTRA 6.1.7 software (Wyatt) with a dn/dc value of 0.185 mL/g. The samples of dpFOXO3a (8.5 mg/mL) and 14-3-3ζ (8.3 mg/mL) were also analyzed in the same way.

ITC experiments
The binding of 14-3-3ζ or DNA to each FOXO3a was measured by ITC37 using MicroCal PEAQ-ITC (Malvern Instruments). All experiments were performed at 30 °C.

In a buffer containing 20 mM NaPi (pH 7.4), 150 mM NaCl and 1 mM DTT, dpFOXO3a (49 μM and 114 μM) was titrated with the DNA (4.8 μM) or 14-3-3ζ dimer (9.4 μM). pT32-FOXO3a (S253A) (46 μM), pS253-FOXO3a (T32A) (49 μM), and the CTD (48 μM) were titrated with the DNA (4.8 μM, 4.3 μM, 4.5 μM), respectively.

The significant heat exchanges of the 14-3-3ζ binding to pT32-FOXO3a (S253A) and pS253-FOXO3a (T32A) were not observed in the phosphate buffer, although their bindings were observed in FSEC experiments. Therefore, we performed the ITC experiments in a buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl and 1 mM DTT. In the HEPES buffer, dpFOXO3a (138 μM), P1-peptide (988 μM), and P2-peptide (989 μM) were titrated with 14-3-3ζ dimer (12 μM, 48 μM, and 49 μM), respectively. 14-3-3ζ dimer (150 μM and 88 μM) was titrated with pT32-FOXO3a (S253A) (30 μM) and pS253-FOXO3a (T32A) (18 μM), respectively.

The heats of the injectant dilution were determined by titration of the injectant into the buffer, followed by subtraction from the row titration data before analysis using MicroCal PEAQ-ITC Analysis Software (Malvern), except for the P1-peptide–14-3-3ζ experiment in the HEPES buffer. A ‘One Set of Sites’ model was applied for curve fitting. Thermodynamic parameters with error values were calculated from the fitted data.

FSEC analysis
All experiments were conducted at 4 °C using EnrichTM SEC 650 10×300 Column (BIO-RAD) with LC-20AD, DGU-20A5, SIL-20A, CBM-20A, and RF-20A XS (SHIMADZU). The fluorescein-labeled DNA was detected by the fluorescence of fluorescein (λex = 465 nm, λem = 540 nm) and the proteins were detected by that of tryptophan (λex = 280 nm, λem = 350 nm). The running buffer contains 20 mM NaPi (pH 7.4), 150 mM NaCl, and 1 mM DTT, and the flow rate is 0.5 mL/min. Before analyzing the mixture of dpFOXO3a, the DNA, and 14-3-3ζ, 1% BSA was applied to suppress the sample adsorption. Then, the buffer was run to remove excess BSA.

In FSEC experiments for the competition of dpFOXO3a, the DNA, and 14-3-3ζ, the sample concentrations were as follows: dpFOXO3a (10 μM), DNA (11 μM), and 14-3-3ζ dimer (0, 2.5, 5, 7.5, 10, 12.5 μM). Each experiment was repeated independently 3–4 times on the same sample (technical replicates). In FSEC experiments with pT32-FOXO3a (S253A) and pS253-FOXO3a (T32A), the sample concentrations were as follows: pT32-FOXO3a (S253A) (10 μM), pS253-FOXO3a (T32A) (10 μM), DNA (15 μM), and 14-3-3ζ dimer (10 μM). FOXO3a (10 μM) was mixed with the DNA (11 μM) and 14-3-3ζ dimer (10 μM). The full FSEC-based competition analysis for pS253-FOXO3a (T32A) (10 μM) and DNA (15 μM) titrated 0, 2.5, 5, 7.5, 10, 12.5, 20, 40 μM 14-3-3ζ dimer. Measurements were performed in triplicate on the same sample (technical replicates). Competition analyses using 14-3-3ζ mutants were conducted with dpFOXO3a (10 μM), DNA (11 μM), and 14-3-3ζ mutants dimer (10 μM).

The fluorescence intensity was normalized using the following equations:
Fnorm = (Fn - F0eq) / (F1.25eq - F0eq)
where Fn is the fluorescence intensity observed with the addition of n equivalent amount of 14-3-3ζ dimer. The competition between 14-3-3ζ and the DNA for dpFOXO3a were simulated using the following equation:
Kd = [A][B]/[A-B]
where [A] is the concentration of 14-3-3ζ or the DNA, [B] is that of dpFOXO3a, and [A–B] is that of the 14-3-3ζ–dpFOXO3a complex or DNA–dpFOXO3a complex. The concentration of the DNA (11 μM), that of dpFOXO3a (10 μM), and the Kd value for DNA–dpFOXO3a binding were considered as constants. The concentration of 14-3-3ζ dimer and the Kd value for 14-3-3ζ–dpFOXO3a binding were as variables. The simulations were most consistent with the FSEC results when the 14-3-3ζ stoichiometry was set to 0.8.

NMR analysis
Data were collected on Bruker Avance 600 and 800, or Ascend Evo 1.0 GHz spectrometers (Billerica) equipped with TXI or TCI probes. All spectra were processed using Bruker TopSpin 4.2.0 software, and data were analyzed using Sparky (T.D. Goddard and D.G. Kneller, Sparky 3.190, University of California). All experiments were performed at 303 K in the buffer containing 20 mM NaPi (pH 6.4), 100 mM NaCl, 1 mM DTT, and 8–10% 2H2O.

The backbone NMR resonances for dpFOXO3a and CTD were assigned based on our previous report (the accession number of the BMRB: 26350 and 26346)23. The assignments of the backbone NMR resonances for 2H, 13C, 15N-labeled 14-3-3ζ were archived using HNCO, HNCA, HN(CO)CA, HN(CA)CO, HNCACB, 1H-15N NOESY-TROSY experiments, 1H-15N HSQC with Arg, Phe, Leu, or Met-selective 15N-labeled 14-3-3ζ, and the assignment data of 14-3-3ζ ΔC12 (residues 1-233) (BMRB: 25231)25. We deposited the assignments of 14-3-3ζ to the BMRB (accession number: 26348).

2H, 15N-labeled dpFOXO3a (169 μM) and 2H, 15N-labeled CTD (150 μM) were titrated with unlabeled 14-3-3ζ dimer and 2H, 15N-labeled CTD (150 μM) was also titrated with T215A/L216A/Q219A/R222A 14-3-3ζ mutant. 2H, 15N-labeled 14-3-3ζ dimer (82 μM, 100 μM, 128 μM, 92 μM, and 123 μM) was titrated with unlabeled dpFOXO3a, pNTD, pCTD, P2-peptide, and CTD, respectively. The experiments were monitored using a 1H-15N TROSY spectrum. Chemical shift changes were calculated using the following equation:
Δδ = sqrt(Δδ1H^2 + (Δδ15N / 6.5)^2)
where Δδ1H and Δδ15N are the chemical shift changes in the 1H and 15N dimensions, respectively.

When unlabeled 14-3-3ζ was titrated to 2H, 15N-labeled CTD, and unlabeled CTD to 2H, 15N-labeled 14-3-3ζ, the chemical shift changes of the signals were observed as the fast exchange regime. Therefore, the dissociation constant and the stoichiometry of the CTD and 14-3-3ζ were calculated from the chemical shift changes using the following equation:
Δδ = Δδmax * (x + [P]tot + Kd - sqrt((x + [P]tot + Kd)^2 - 4 * x * [P]tot)) / (2 * [P]tot)
where Δδmax is maximum of the chemical shift changes, [P]tot is the concentration of the host protein, x is that of the ligand protein, and n is the stoichiometry. Curve fitting was performed using Origin 2015. NMR cross-saturation experiments were performed with 222 μM 2H, 15N-labeled 14-3-3ζ in complex with 1.25 equivalent of dpFOXO3a, containing 20% 2H2O/80% 2H2O and selectively saturated dpFOXO3a by irradiating the aliphatic proton resonances, using the mlevsp180.p30 decoupling program. The saturation frequency was set at 1.0 ppm, and the saturation time was set to 1.0 s.

Competition analysis of 126 μM 2H, 15N-labeled dpFOXO3a with 1.1 equivalents of DNA and 1.5 equivalents of 14-3-3ζ dimer were monitored using 1H-15N TROSY spectrum at 600 and 800 MHz. The diluted sample (16 μM dpFOXO3a with 18 μM DNA plus 24 μM 14-3-3ζ) was also monitored using 1H-15N TROSY spectrum at 600 MHz. NMR samples were analyzed by gel filtration using SuperdexTM 200 increase 10/300 GL (Cytiva).

In the magnetic field- and temperature-dependent analyses, we prepared 209 μM 2H, 15N-labeled 14-3-3ζ with 1.25 equivalent of dpFOXO3a and measured 1H-15N TROSY spectra at 1 GHz at different temperatures: 303 K, 298 K, and 293 K.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript \"14-3-3ζ interacts with DNA binding domain of FOXO3a and competitively dissociates DNA by dual motif tethering\" by Enomoto et al. studies the FOXO3a/14-3-3 ζ /DNA ternary system by ITC, FSEC and NMR. This is another example of a protein fully displacing DNA in spite of similar affinities. At first glance, this is counter-intuitive, but it adds to a growing number of systems where such behavior has been identified.\n\nIn principle I think the manuscript is scientifically sound and of broad interest to warrant publication in Nature Communications. However, I would request additional experiments and edits from the authors as follows:\n1) Can the authors confirm their key finding (full displacement of DNA by an equimolar amount of 14-3-3 to dpFOXO3a) by a method different from FSEC? FSEC is not an equilibrium method, and on/off kinetics may play a role and confound the analysis. Could the authors demonstrate this key finding e.g. by NMR?\n2) The affinity of 14-3-3 to FOXO3a is 2-fold higher than the affinity of DNA to FOXO3a, hence displacement of DNA by 14-3-3 is only quantitatively, but not qualitatively, surprising. The authors also have the monophosphorylated system pS253-FOXO3a (T32A) which binds much weaker to 14-3-3 but still partially displaces DNA. Could the authors please perform a titration and present the full competition analysis with pS253-FOXO3a (T32A), as they did for dpFOXO3a ?\n3) Please define the term “competitiveness” briefly and verbally in the main text, without the equation used in the Supplemental Information\n4) Please move Supplementary Table 2 to the main text, as it is important to understand the argument.\n5) The Kd value of 5–30 μM for the FOXO3a –14-3-3ζ interaction is quite confusing. Which FOXO3a construct was used for this experiment? I assume it is only the DBD, but this is not clearly stated, and why does the paragraph then often refer to the protein as dpFOXO3a ?\n6) The Figure numbering is off for several Figures\nPoints 1 and 2 will need additional experiments, but I feel only these additional experiments will make the paper really valuable for publication in Nature Communications.", "reviewer_2": "Foxo3a is a well-established tumor-suppressive transcription factor that activates the expression of various anti-proliferative and pro-apoptotic genes. The 14-3-3 protein binds to diphosphorylated Foxo3a at threonine 32 (T32) and serine 253 (S253), inhibiting its DNA-binding activity. However, the precise mechanism of this inhibition remains unclear.\nThis manuscript investigates the potential mechanism by which 14-3-3 binding displaces Foxo3a from DNA, despite the phosphorylation sites of Foxo3a being distant from the DNA-binding domain (DBD). Using a combination of biophysical approaches—FSEC, ITC, and NMR—the authors propose that 14-3-3 interacts not only with the phosphorylated sites but also directly with the DBD of Foxo3a. They further suggest a two-step mechanism: initial binding of 14-3-3 to site P1 of Foxo3a forms a ternary complex, followed by the binding of a second 14-3-3 molecule that facilitates dissociation of Foxo3a from DNA.\nMajor Points for Consideration:\n1. Novelty of the Findings:\nThe novelty of the findings needs to be clearly demonstrated. A similar study was published in ACS Omega (Vol. 7, Issue 28, July 5, 2022), titled “Structure of a 14-3-3ε:FOXO3a-pS253 Phosphopeptide Complex Reveals 14-3-3 Isoform-Specific Binding of Forkhead Box Class O Transcription Factor (FOXO) Phosphoproteins.” That paper reported that 14-3-3ε binding induces conformational changes that reposition critical arginine residues in Foxo3a, reducing its DNA-binding affinity.\nWhile the current manuscript presents NMR-based data identifying additional critical residues upon protein interaction, the novelty and functional significance of these residues in regulating Foxo3a's DNA-binding activity have not been fully validated. For instance, site-directed mutagenesis of 14-3-3 residues in contact with the Foxo3a DBD could provide direct evidence of their role in Foxo3a displacement. Crosslinking experiments using chemical or UV crosslinkers, followed by identification of the crosslinked amino acids, would also significantly strengthen the conclusions.\nAdditionally, the NMR data indicate several residues involved in direct contact between the proteins. However, it remains unclear to an NMR nonspecialist whether these residues reflect direct interactions or result from allosteric conformational changes. This distinction should be clarified.\nThe weak binding of DBD to 14-3-3ζ was indicated only by NMR chemical shift and affinity measurements (Page 8, The Kd value for the DBD – 14-3-3ζ interaction was calculated to be 5–30 μM). However, without high-resolution structural data (such as crystal structures or cryo-EM) it is hard to support the mechanistic conclusions of this manuscript. Furthermore, the dynamic exchange model was primarily based on NMR signal splitting phenomena (e.g., splitting of the E202 signal into A and B, Figure 9a), but it was not validated through blocking exchange with mutants or real-time kinetic experiments, nor supported by structural data. Moreover, the hypothesis that DBD rapidly exchanges between 14-3-3ζ dimers was not substantiated by resolving the complex structure with individual monomers or through direct functional experiments. These gaps may lead to conclusions that exceed the scope of data support.\n2. Figure Mislabeling:\nThe manuscript contains mislabeled or missing figure numbers (e.g., no Figure 6), leading to confusion and hindering the review process. This oversight suggests a lack of thorough proofreading prior to submission and should be corrected to maintain clarity and professionalism.\nMinor Points:\n1. Kd Determination Using Truncated Protein:\nThe dissociation constant (Kd) was determined using a truncated version of Foxo3a (residues 1–284). It is important to address whether this truncated construct behaves similarly to the full-length protein in Kd values. Is there any evidence that the full-length and truncated forms have comparable Kd values?\n2. Incorrect Literature Citation:\nOn page 5, line 96, the cited papers focus on Foxo4 rather than Foxo3a. Please ensure that the references are relevant and specific to the subject of the manuscript.\n3. Limited Evidence for Direct DBD Interaction:\nThe proposed direct binding of 14-3-3 to the DBD of Foxo3a and the suggested hybrid binding model are currently supported solely by NMR data, which were obtained using the truncated protein and peptides. This weakens the overall conclusion. Crosslinking experiments and site-specific mutagenesis would provide more robust support for these claims.\n4. Inadequate Comparison with Existing Studies:\nThe authors only mentioned the similar mechanism of FOXO4 but did not conduct a detailed comparison of the phosphorylation site specificity, DBD structural differences, or functional regulatory similarities and differences between FOXO3a and FOXO4, leading to limited innovation in this study. It is recommended to include a structural and functional comparison between FOXO3a and FOXO4 in the Discussion section and to discuss other subtypes of the 14-3-3 family.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-by-point response to REVIEWER COMMENTS\nIn accordance with the reviewers’ comments, we have thoroughly revised our manuscript and\nprepared a detailed point-by-point response. We are grateful for constructive feedback, which has\nhelped us to improve the quality and clarity of our work. In the point-by-point response, reviewers’\ncomments and our responses are shown in oblique and normal text, respectively. The revisions in\nmanuscript are highlighted by yellow marker. We hope that these revisions sufficiently address all\nconcerns from reviewers.\nReviewer #1 (Remarks to the Author):\nThe manuscript \"14-3-3ζ interacts with DNA binding domain of FOXO3a and competitively\ndissociates DNA by dual motif tethering\" by Enomoto et al. studies the FOXO3a/14-3-3 ζ /DNA\nternary system by ITC, FSEC and NMR. This is another example of a protein fully displacing\nDNA in spite of similar affinities. At first glance, this is counter-intuitive, but it adds to a growing\nnumber of systems where such behavior has been identified.\nIn principle I think the manuscript is scientifically sound and of broad interest to warrant\npublication in Nature Communications. However, I would request additional experiments and\nedits from the authors as follows:\nWe greatly appreciate Reviewer #1’s time and effort. We thank you for your positive evaluation\nof our study and providing valuable suggestions for additional experiments and edits. We have\nrevised the manuscript accordingly, as shown below.\n1\n1) Can the authors confirm their key finding (full displacement of DNA by an equimolar amount\nof 14-3-3 to dpFOXO3a) by a method different from FSEC? FSEC is not an equilibrium method,\nand on/off kinetics may play a role and confound the analysis. Could the authors demonstrate this\nkey finding e.g. by NMR?\nThank you for your suggestion. We performed NMR experiments to test whether the key\nfinding from FSEC—full displacement of DNA from dpFOXO3a by an equimolar amount of 14-3-\n3ζ—could be reproduced. In these experiments, full displacement was not confirmed; instead, the\nspectra indicated that the dpFOXO3a–DNA complex can bind 14-3-3ζ at the P1 and P2 sites without\nreleasing the DNA, forming a ternary complex (Supplementary Fig. 5a, 5b, and 6a).\nSEC analysis of the same NMR sample revealed that this ternary complex is unstable: ~74% of\nthe DNA was free at 126 μM protein concentration and ~91% was free at 16 μM (Supplementary Fig.\n5c, 5d, 6b, and 6c), indicating strong concentration dependence.\nWe have revised the Results section (page 6, lines 127–134) to describe these NMR findings. In\naddition, we added the interpretation—comparison with the NF-κB–IκBα–DNA system and the\nconclusion that FSEC and NMR detect different aspects of the competition—to the Discussion\nsection for clarity (page 14, lines 294–303).\nOn page 6, lines 127–134\n“On the other hand, NMR analysis of [2H, 15N]-dpFOXO3a bound to DNA upon addition of 14-3-3ζ\nshowed that most signals did not change; however, the signals corresponding to P1 and P2\ndisappeared or shifted in a manner identical to that observed when 14-3-3ζ bound to dpFOXO3a in\nthe absence of DNA (Supplementary Fig. 5a, 5b, and 6a). This indicates that 14-3-3ζ can bind to\ndpFOXO3a at the P1 and P2 sites even while dpFOXO3a remains associated with DNA. SEC of the\nsame sample showed that this ternary complex was unstable, with ~74% of DNA free at 126 μM\nprotein concentration and ~91% free at 16 μM (Supplementary Fig. 5c, 5d, 6b, and 6c), indicating\nstrong concentration dependence.”\nOn page 14, lines 294–303\n“In this study, we quantitatively evaluated the competitiveness of 14-3-3ζ against DNA on\ndpFOXO3a by FSEC and NMR, as well as the binding affinities of dpFOXO3a for 14-3-3ζ and\nDNA by ITC. Whereas FSEC revealed efficient displacement of DNA by 14-3-3ζ, NMR further\ndemonstrated the formation of a transient dpFOXO3a–DNA–14-3-3ζ ternary complex whose\nstability is strongly concentration-dependent. These findings suggest that DNA can still associate\nwith the dpFOXO3a–14-3-3ζ complex, but with reduced accessibility compared to free dpFOXO3a,\nleading to an unstable ternary assembly. This interpretation is consistent with previous observations\n2\nin the NF-κB–IκBα–DNA system (PNAS 108, 4, 2011; Cell 61, 2, 1990), in which NMR detected a\ntransient ternary complex despite DNA displacement being evident in other assays. Thus, while\nFSEC detects efficient displacement under assay conditions, NMR captures the presence of a short-\nlived ternary complex whose stability is strongly concentration-dependent.”\n3\nfor dpFOXO3a at high concentration.\na Overlay of the 1H-15N TROSY spectra of [2H, 15N]-dpFOXO3a in the free state (gray)\nsuperimposed on the spectrum in the presence of 1.1 equivalents of the DNA (cyan; 126 μM\ndpFOXO3a with 139 μM DNA). Black squares highlight the cyan signals that underwent chemical\nshift changes upon DNA binding. b Overlay of the 1H-15N TROSY spectra of [2H, 15N]-dpFOXO3a\nbound to DNA in the absence of 14-3-3ζ (cyan; 126 μM dpFOXO3a with 139 μM DNA) and\npresence of 1.5 equivalents of the 14-3-3ζ dimer (magenta; 126 μM dpFOXO3a with 139 μM DNA\nplus 189 μM 14-3-3ζ dimer). The black squares mark the same cyan signals as in panel a, allowing\ndirect comparison before and after the addition of 14-3-3ζ. Signals that disappeared or shifted upon\naddition of 14-3-3ζ were labeled with residue names when assignments were available, and indicated\nby red arrows when unavailable. c, d SEC profiles of NMR sample containing 126 μM dpFOXO3a,\n139 μM DNA, and 189 μM 14-3-3ζ dimer (c) and 139 μM DNA alone prepared at the same\nconcentration as in the NMR experiments (d). Black profiles represent the absorbance at 280 nm and\nmagenta profiles represent the absorbance at 254 nm. The fractions indicated by cyan bar in (c) were\nanalyzed by SDS-PAGE, and the magenta-colored peak areas (14.5—22 mL) were integrated to\nquantify free DNA.\nfor dpFOXO3a at low concentration.\na Overlay of the 1H-15N TROSY spectra of [2H, 15N]-dpFOXO3a in the presence of 1.1 equivalents\nof DNA and 1.5 equivalents of 14-3-3ζ dimer at high concentration (magenta; 126 μM dpFOXO3a\nwith 139 μM DNA plus 189 μM 14-3-3ζ dimer) and at low concentration (black; 16 μM dpFOXO3a\nwith 18 μM DNA plus 24 μM 14-3-3ζ). Black squares highlight the same signals that shifted upon\nDNA binding as in Supplementary Fig. 5a. b, c SEC profiles of the NMR sample containing 10 μM\ndpFOXO3a, 11 μM DNA and 15 μM 14-3-3ζ dimer (b) and 11 μM DNA at the same concentration\nas used in the FSEC experiments (c).\n6\n2) The affinity of 14-3-3 to FOXO3a is 2-fold higher than the affinity of DNA to FOXO3a, hence\ndisplacement of DNA by 14-3-3 is only quantitatively, but not qualitatively, surprising. The\nauthors also have the monophosphorylated system pS253-FOXO3a (T32A) which binds much\nweaker to 14-3-3 but still partially displaces DNA. Could the authors please perform a titration\nand present the full competition analysis with pS253-FOXO3a (T32A), as they did for\ndpFOXO3a?\nThank you for your comment. We performed a full FSEC-based competition analysis of pS253-\nFOXO3a (T32A) with titrated 14-3-3ζ, as requested. As the amount of 14-3-3ζ increased, the peak\nheight of DNA-bound pS253-FOXO3a (T32A) decreased and the free DNA peak increased\n(Supplementary Fig. 9a and 9b). At 1 equivalent of 14-3-3ζ dimer, only ~50% of the DNA complex\nwas dissociated, compared with ~95% for dpFOXO3a, and even with 4 equivalents, dissociation\nreached ~83% (Supplementary Fig. 9c). These results indicate that strong DNA displacement\nrequires phosphorylation at both T32 (P1) and S253 (P2), and that the absence of P1 phosphorylation\nin pS253-FOXO3a (T32A) markedly reduces the competitive ability of 14-3-3ζ against DNA.\nWe have revised the Results section (page 7, lines 154–157) to include these new FSEC\ntitration data for pS253-FOXO3a (T32A) alongside the dpFOXO3a results, highlighting that the\nreduced competitiveness arises from the absence of P1 phosphorylation, as follows:\n“The full FSEC-based competition analysis revealed that at one equivalent of 14-3-3ζ dimer, only\n~50% of the DNA complex was dissociated, compared with ~95% for dpFOXO3a, and even with\nfour equivalents dissociation reached only ~83% (Supplementary Fig. 9).”\nThese results directly address the reviewer’s request and clarify the functional importance of dual\nphosphorylation for efficient DNA displacement.\n7\nFOXO3a (T32A).\na, b FSEC profiles of pS253-FOXO3a (T32A), DNA, and 14-3-3ζ mixtures at different molar ratios\nof 14-3-3ζ dimer (0 – 4 equivalents). a DNA profiles detected by fluorescein fluorescence. b Protein\nprofiles of pS253-FOXO3a (T32A) and 14-3-3ζ detected by tryptophan fluorescence. c Dissociation\ncurves showing the fraction of DNA-bound pS253-FOXO3a (T32A) (dashed line) compared with\ndpFOXO3a (solid line). Data points represent mean ± standard deviation (n = 3).\n3) Please define the term “competitiveness” briefly and verbally in the main text, without the\nequation used in the Supplemental Information\nThank you for your comment. In the revised manuscript, we have added a brief verbal\ndefinition of “competitiveness” in the main text to improve clarity. Specifically, we now explain that\n“competitiveness” represents the relative ability of 14-3-3ζ or DNA to bind dpFOXO3a under the\nsame concentration conditions, and can be expressed as the ratio of concentration to the dissociation\nconstant (concentration/K ). This description has been inserted in the Results section (page 6, lines\nd\n117–120) where the FSEC competition data are introduced, as follows:\n“Here, ‘competitiveness’ refers to the relative ability of 14-3-3ζ or DNA to bind to dpFOXO3a under\nthe same concentration conditions. In quantitative terms, this can be expressed as the ratio of\nconcentration to the dissociation constant (concentration/K ), as described in the supplementary\nd\ndiscussion.”\n4) Please move Supplementary Table 2 to the main text, as it is important to understand the\nargument.\nThank you for your comment. In the revised manuscript, we have merged Supplementary Table\n1 and Supplementary Table 2, which summarize the thermodynamic parameters of the interaction of\nFOXO3a with DNA and with 14-3-3ζ, into a single Table 1 and moved it from the Supplementary\nInformation to the main text to facilitate understanding of our argument. The new Table 1 now\nappears in the Results section (page 5, line 97), where it is first discussed. To comply with the\njournal’s limit of 10 display items, we relocated the figure showing the raw ITC data (“ITC results\nfor the binding activity of dpFOXO3a and DNA (a) or dpFOXO3a and 14-3-3ζ (b).”), to\nTable 1 Thermodynamic parameters obtained via ITC analysis of the interaction of each\nFOXO3a with DNA and with 14-3-3ζ in the phosphate or HEPES buffer.\nIn the phosphate buffer\nK ΔH ΔG - TΔS\nFOXO3a DNA/14-3-3ζ N[a] d\n(μM) (kJ/mol) (kJ/mol) (kJ/mol)\ndpFOXO3a 14-3-3ζ dimer 1.02 ± 0.01 0.06 ± 0.02 - 27 ± 1 - 42 -15\ndpFOXO3a DNA 0.70 ± 0.02 0.13 ± 0.03 - 68 ± 3 - 40 28\npT32-FOXO3a\nDNA 0.63 ± 0.02 0.09 ± 0.03 - 54 ± 3 - 41 13\n(S253A)\npS253-FOXO3a\nDNA 0.61 ± 0.02 0.11 ± 0.04 - 61 ± 4 - 40 21\n(T32A)\nCTD DNA 0.83 ± 0.01 0.03 ± 0.01 - 66 ± 2 - 44 22\nIn the HEPES buffer\nK ΔH ΔG - TΔS\nFOXO3a 14-3-3ζ N[a] d\n(μM) (kJ/mol) (kJ/mol) (kJ/mol)\ndpFOXO3a 14-3-3ζ dimer 1.23 ± 0.01 0.04 ± 0.01 - 34 ± 1 - 43 -9.0\npT32-FOXO3a\n14-3-3ζ dimer 2.38 ± 0.03 0.8 ± 0.1 - 11.3 ± 0.3 - 35 - 24\n(S253A)\npS253-FOXO3a\n14-3-3ζ dimer 2.12 ± 0.09 5 ± 1 - 36 ± 4 - 31 5.4\n(T32A)\nP1-peptide 14-3-3ζ dimer 2.37 ± 0.04 1.2 ± 0.4 - 16 ± 1 - 34 - 18\nP2-peptide 14-3-3ζ dimer 1.84 ± 0.07 21 ± 5 - 32 ± 3 - 27 4.9\n[a] N is the molar ratio of each FOXO3a to DNA or 14-3-3ζ dimer.\n5) The Kd value of 5–30 μM for the FOXO3a –14-3-3ζ interaction is quite confusing. Which\nFOXO3a construct was used for this experiment? I assume it is only the DBD, but this is not\nclearly stated, and why does the paragraph then often refer to the protein as dpFOXO3a ?\nThank you for your comment. We understand the potential confusion arising from our\noccasional ambiguous reference to the DBD as if it were a separate protein construct. In this study,\nwe did not use a construct expressing only the DBD. Instead, we used the CTD, which contains the\nDBD together with the C-terminal tail, for the experiments that yielded the K value of 5–30 μM.\nd\nThis construct (CTD, residues 141–284) was consistently used for the affinity measurement that\nyielded the 5–30 μM value, and we have revised the terminology in the manuscript to explicitly state\n10\n“CTD” instead of ambiguously referring to “DBD.” We have revised the manuscript to clarify this\npoint and to avoid any ambiguity in terminology (page 9, line 185–188), as follows:\n“The backbone assignments of the DBD of dpFOXO3a bound to 14-3-3ζ were transferred from\nthose of C-terminal domain (CTD) construct of FOXO3a (residues 141-284, Fig. 1), which contains\nthe DBD together with the C-terminal unstructured tail, rather than the DBD alone (Supplementary\nFig. 10).”\n11\n6) The Figure numbering is off for several Figures\nThank you for pointing out the errors in figure numbering. We apologize for these mistakes —\nthe issue appears to have arisen during file conversion to PDF on the Nature Communications\nwebsite, but we acknowledge that it was our responsibility to detect and correct it before submission.\nThe numbering has now been corrected in the revised manuscript.\nPoints 1 and 2 will need additional experiments, but I feel only these additional experiments will\nmake the paper really valuable for publication in Nature Communications.\nWe appreciate Reviewer #1’s time and effort again. Your valuable suggestions and comments\nhave helped us to substantially improve the manuscript.\nFoxo3a is a well-established tumor-suppressive transcription factor that activates the expression\nof various anti-proliferative and pro-apoptotic genes. The 14-3-3 protein binds to\ndiphosphorylated Foxo3a at threonine 32 (T32) and serine 253 (S253), inhibiting its DNA-binding\nactivity. However, the precise mechanism of this inhibition remains unclear.\nThis manuscript investigates the potential mechanism by which 14-3-3 binding displaces Foxo3a\nfrom DNA, despite the phosphorylation sites of Foxo3a being distant from the DNA-binding\ndomain (DBD). Using a combination of biophysical approaches—FSEC, ITC, and NMR—the\nauthors propose that 14-3-3 interacts not only with the phosphorylated sites but also directly with\nthe DBD of Foxo3a. They further suggest a two-step mechanism: initial binding of 14-3-3 to site\nP1 of Foxo3a forms a ternary complex, followed by the binding of a second 14-3-3 molecule that\nfacilitates dissociation of Foxo3a from DNA.\nThank you for your thorough review and clear summary of our manuscript. We appreciate your\nvaluable suggestions for additional experiments and edits. We agree with Reviewer #2’s comments\nand have revised the manuscript accordingly, as detailed below.\n1. Novelty of the Findings:\n1-1. The novelty of the findings needs to be clearly demonstrated. A similar study was published in\nACS Omega (Vol. 7, Issue 28, July 5, 2022), titled “Structure of a 14-3-3ε:FOXO3a-pS253\nPhosphopeptide Complex Reveals 14-3-3 Isoform-Specific Binding of Forkhead Box Class O\nTranscription Factor (FOXO) Phosphoproteins.” That paper reported that 14-3-3ε binding\ninduces conformational changes that reposition critical arginine residues in Foxo3a, reducing its\nDNA-binding affinity.\nThank you for your comment. The novelty of our study lies in demonstrating that 14-3-3ζ\ninhibits the dpFOXO3a–DNA binding primarily through direct interaction with the DBD, rather than\nsolely via the phosphorylated P2 motif, and in revealing a two-step displacement mechanism. While\nwe are aware of the ACS Omega study reporting 14-3-3ε binding to the P2 phosphopeptide\n( RRRAV-pS -MD ) and the resulting reduction in DNA-binding affinity, our work shows that,\n248 253 255\nin the dpFOXO3a–DNA interaction, the contribution of helix 3 in the DBD to DNA binding is\nsubstantially greater than that of the P2 motif. This conclusion is supported by a previous Nucleic\nAcids Research study (Vol. 35, No. 20, 2007) reporting a K of 295 nM (ΔG = 37 kJ mol⁻¹) for wild-\nd\ntype FOXO3a (158–253) binding to DNA and 1178 nM (ΔG = 34 kJ mol⁻¹) for the\nR248A/R249A/R250A mutant. The ΔG difference of 3 kJ mol⁻¹ corresponds to only ~8% of the total\nbinding free energy, indicating that residues R248–R250 make a minor contribution compared to the\nDBD.\nImportantly, our NMR competition experiments demonstrated that a ternary dpFOXO3a–DNA–\n14-3-3ζ complex can form, indicating that binding of 14-3-3ζ to the P2 site alone does not induce\nDNA dissociation (Supplementary Fig. 5 and 6). This finding argues against a P2-centric model (as\nsuggested in ACS Omega, 2022) and supports our conclusion that DNA displacement requires the\nadditional engagement of 14-3-3ζ with the DBD.\nTherefore, inhibition via the 14-3-3ζ–DBD interaction is expected to be more effective than\nthrough the 14-3-3ζ–P2 interaction. We have revised the manuscript to clarify this point in the\nDiscussion section describing the novelty of our findings (pages 14–15, line 312–319) as follows:\n“The novelty of our study lies in two points: (i) demonstrating that 14-3-3ζ inhibits dpFOXO3a–\nDNA binding primarily through direct interaction with the DBD rather than solely via the P2 motif,\nand (ii) revealing a two-step displacement mechanism. Although the P2 also interacts with the DNA\n(Nucleic Acids Research, Vol. 35, No. 20, 2007) and behaves as the competitive site for DNA and\n14-3-3ζ (ACS Omega, Vol. 7, No. 28, 2022), its contribution to DNA binding is substantially minor\nto that of helix 3 in the DBD (Nucleic Acids Research, Vol. 35, No. 20, 2007). Moreover, our NMR\n14\ncompetition data revealed that P2 binding alone is insufficient for DNA displacement, further\nhighlighting the critical role of the DBD in mediating effective competition with DNA. Therefore,\nthe DBD is a more crucial site than P2 for the competition between DNA and 14-3-3ζ.”\n15\n1-2. While the current manuscript presents NMR-based data identifying additional critical\nresidues upon protein interaction, the novelty and functional significance of these residues in\nregulating Foxo3a's DNA-binding activity have not been fully validated. For instance, site-\ndirected mutagenesis of 14-3-3 residues in contact with the Foxo3a DBD could provide direct\nevidence of their role in Foxo3a displacement. Crosslinking experiments using chemical or UV\ncrosslinkers, followed by identification of the crosslinked amino acids, would also significantly\nstrengthen the conclusions.\nThank you for this important suggestion. On the 14-3-3ζ side, CSP mapping was already\npresented in the original manuscript; however, because CSP can reflect not only direct\nintermolecular contacts but also conformational changes upon binding, in this revision we applied\nNMR cross-saturation experiments, which selectively detect residues in close spatial proximity (~5\nÅ) to dpFOXO3a, making them particularly suitable for directly identifying the interaction surface.\nAmong the residues identified on 14-3-3ζ, several were located away from the canonical phospho-\nmotif binding groove and positioned such that they are likely to interact with the DBD of FOXO3a.\nFrom these, we selected two residue clusters (D197/E198/E202/D204 and\nT215/L216/Q219/R222) as representative contact sites for mutagenesis. We generated quadruple-\nalanine mutants targeting these residues and tested them in FSEC-based competition assays;\nalthough the mutants did not show a major reduction in DNA displacement ability (Supplementary\nFig. 13), this outcome is likely due to the broad protein–protein interaction surface, where disruption\nof only part of the interface is insufficient to abolish binding.\nIn contrast, NMR spectral changes of [2H, 15N]-CTD titrated with the\nT215A/L216A/Q219A/R222A mutant of 14-3-3ζ revealed that signals mainly in helix 3 shifted in\ndifferent direction or decreased in intensity compared with titration using wildtype 14-3-3ζ.\nThis supports a direct contribution of these residues to the DBD – 14-3-3ζ interface. In the case of the\nD197A/E198A/E202A/D204A mutant, precipitation prevented collection of sufficient NMR data.\nThese NMR observations therefore strongly suggest that the helix 3 of the DBD directly interacts\nwith residues around T215/L216/Q219/R222 of 14-3-3ζ (Supplementary Fig. 14).\nFor completeness, we note that on the FOXO3a side, CSP analysis identified residues around\nhelix 3 of the DBD as part of the interface; however, because this region overlaps with the DBD–\nDNA binding site, mutagenesis was not feasible without perturbing DNA binding directly.\nTaken together, these results provide direct experimental evidence for the physical interaction\nbetween dpFOXO3a and 14-3-3ζ, and clarify why even multiple-site mutations on 14-3-3ζ were\ninsufficient to eliminate the interaction. We have clarified these points in the Results sections (page\n11, lines 240–255) and added new figures highlighting the identified residues on both proteins, as\nfollows:\n16\n“However, CSP identifies residues whose local chemical environment changes upon binding, which\ncan result from either direct contact or conformational changes. To obtain direct evidence for DBD –\n14-3-3ζ interaction beyond CSP mapping, we performed NMR cross-saturation experiments, which\nselectively detect residues in close spatial proximity (~5 Å) to dpFOXO3a. This analysis identified\nspecific contact residues on 14-3-3ζ, several of which are located outside the canonical phospho-\nmotif binding groove and positioned to interact with the DBD (Fig. 7).\nQuadruple-alanine mutants targeting these sites (D197A/E198A/E202A/D204A or\nT215A/L216A/Q219A/R222A) showed no reduction in DNA displacement ability in FSEC assays\n(Supplementary Fig.13), consistent with a broad interaction interface in which partial disruption does\nnot abolish binding. In contrast, NMR spectral changes of [2H, 15N]-CTD titrated with the\nT215A/L216A/Q219A/R222A 14-3-3ζ mutant demonstrated that signals, mainly in helix 3, shifted\nin a different manner or decreased in intensity compared with the titration using wildtype 14-3-3ζ.\nThe titration with the D197A/E198A/E202A/D204A mutant was not possible because the sample\nprecipitated and could not be concentrated sufficiently for NMR analysis. Together, these NMR data\nstrongly suggest that the helix 3 of DBD directly interacts with the residues around\nT215/L216/Q219/R222 of 14-3-3ζ (Supplementary Fig. 14).”\n18\na Spectra of 222 μM [2H, 15N]-14-3-3ζ with 1.25 equivalent of dpFOXO3a without radiofrequency\n(RF) irradiation (black) and with RF irradiation (red). b Enlarged spectra and 1H-1D slices of\nS37_NC, D197_C, and V46_C. “N” indicates the signal from P1-bound protomer and “C” indicates\nthe signal from P2-bound protomer. “X” represents unassigned signals from 14-3-3ζ residues. c\nRatio of the intensity with RF irradiation (irr (+)) to that without RF irradiation (irr (-)). The error\nrange was calculated as √(1⁄𝑆𝑁 )2+(1⁄𝑆𝑁 )2, where SN is the signal-to-noise ratio.\n𝑖𝑟𝑟(+) 𝑖𝑟𝑟(−)\nResidues with an intensity ratio below 0.75 are colored red and unanalyzable residues are shown in\nlight gray. Residues whose signals disappeared upon RF irradiation are indicated with red arrows.\nd Affected residues in c mapped onto the 14-3-3ζ dimer (PDB: 5JM4). Prolines are shown in gray and\nunassigned and unanalyzable residues in black.\nSupplementary Fig. 13 FSEC analysis of the competition between DNA and 14-3-3ζ mutants\nfor dpFOXO3a.\na, b FSEC profiles of dpFOXO3a, DNA, and wildtype 14-3-3ζ mixture solutions. c, d Profiles of\ndpFOXO3a, DNA, and D197A/E198A/E202A/D204A 14-3-3ζ mixture solutions. e, f Profiles of\ndpFOXO3a, DNA, and T215A/L216A/Q219A/R222A 14-3-3ζ mixture solutions. Upper panels a, c,\nand e show the profiles of DNA detected by fluorescein fluorescence. Lower panels b, d, and f show\nthe profiles of FOXO3a and various 14-3-3ζ detected by tryptophan fluorescence. Black lines\nrepresent profiles in the absence of 14-3-3ζ (or mutants), and colored lines represent profiles in the\npresence of one equivalent of 14-3-3ζ (or mutants).\n19\nT215A/L216A/Q219A/R222A 14-3-3ζ.\na Overlay of the 1H-15N TROSY spectrum of the CTD (151 μM) in the absence (black) and presence\nof one equivalent of the 14-3-3ζ dimer (magenta) or the T215A/L216A/Q219A/R222A 14-3-3ζ\ndimer (cyan). Square boxes indicate the signals which changed to different direction (red labels) and\nweakened (blue labels) upon binding of T215A/L216A/Q219A/R222A 14-3-3ζ, relative to wildtype\n14-3-3ζ. b Zommed spectra for the signals indicated by square boxes in (a). Arrows show the\ndirections of signal changes. c The chemical shift changes of the CTD upon addition of one\nequivalent of the T215A/L216A/Q219A/R222A 14-3-3ζ dimer (black and cyan) or the 14-3-3ζ\ndimer (gray and magenta). The residues corresponding to CSP ≥ 0.03 ppm (cyan and magenta) were\nregarded as the residues affected by the 14-3-3ζ binding. The signals of K207, L216, and R219\n(circled) were also regarded as the residues affected because their CSP is more than 0.03 ppm upon\naddition of 0.25 equivalent of the 14-3-3ζ dimer, although they disappeared or overlapped with other\nsignals upon addition of one equivalent of the 14-3-3ζ dimer. The red and blue labels are\ncorresponded to (a). d Mapping of the residues affected by T215A/L216A/Q219A/R222A 14-3-3ζ\nbiding on the DBD structure as a ribbon (PDB: 2UZK). The residues whose signals differently\nchanged upon binding of T215A/L216A/Q219A/R222A 14-3-3ζ or wildtype 14-3-3ζ are shown as\nspheres. Black indicates unassigned and unanalyzable residues, and gray indicates proline.\n1-3. Additionally, the NMR data indicate several residues involved in direct contact between the\nproteins. However, it remains unclear to an NMR nonspecialist whether these residues reflect\ndirect interactions or result from allosteric conformational changes. This distinction should be\nclarified.\nThank you for this valuable comment. We agree that NMR chemical shift perturbation (CSP)\ncan arise from both direct intermolecular contacts and allosteric conformational changes, and that\nthis distinction is not always obvious to a nonspecialist. To address this, in the revised manuscript we\nhave explicitly stated in the Results section that CSP identifies residues whose local chemical\nenvironment changes upon binding, which may be due to direct contact or to structural\nrearrangements. We also clarify that, to unambiguously identify direct contact residues, we\nperformed NMR cross-saturation experiments, which selectively detect nuclei in close spatial\nproximity (<5 Å) to the partner protein. This approach allowed us to distinguish residues directly\ncontacting dpFOXO3a from those affected indirectly by conformational change.\nOn the FOXO3a side, only CSP analysis indicated residues around helix 3 of the DBD as part\nof the interface. However, as described in our response to Major Point Review 1-2 from Reviewer #2,\n21\nthe NMR analyses using [2H, 15N]-CTD and T215A/L216A/Q219A/R222A 14-3-3ζ strongly suggest\nthe residues around helix 3 of DBD for the direct interaction with 14-3-3ζ. These clarifications have\nbeen added to the Results (page 11, lines 240–255).\n“However, CSP identifies residues whose local chemical environment changes upon binding, which\ncan result from either direct contact or conformational changes. To obtain direct evidence for DBD–\n14-3-3ζ interaction beyond CSP mapping, we performed NMR cross-saturation experiments, which\nselectively detect residues in close spatial proximity (~5 Å) to dpFOXO3a. This analysis identified\nspecific contact residues on 14-3-3ζ, several of which are located outside the canonical phospho-\nmotif binding groove and positioned to interact with the DBD (Fig. 7).\nQuadruple-alanine mutants targeting these sites (D197A/E198A/E202A/D204A or\nT215A/L216A/Q219A/R222A) showed no reduction in DNA displacement ability in FSEC assays\n(Supplementary Fig.13), consistent with a broad interaction interface in which partial disruption does\nnot abolish binding. In contrast, NMR spectral changes of [2H, 15N]-CTD titrated with the\nT215A/L216A/Q219A/R222A 14-3-3ζ mutant demonstrated that signals, mainly in helix 3, shifted\nin a different manner or decreased in intensity compared with the titration using wildtype 14-3-3ζ.\nThe titration with the D197A/E198A/E202A/D204A mutant was not possible because the sample\nprecipitated and could not be concentrated sufficiently for NMR analysis. Together, these NMR data\nstrongly suggest that the helix 3 of DBD directly interacts with the residues around\nT215/L216/Q219/R222 of 14-3-3ζ (Supplementary Fig. 14).”\n1-4. The weak binding of DBD to 14-3-3ζ was indicated only by NMR chemical shift and affinity\nmeasurements (Page 8, The Kd value for the DBD – 14-3-3ζ interaction was calculated to be 5–30\nμM). However, without high-resolution structural data (such as crystal structures or cryo-EM) it\nis hard to support the mechanistic conclusions of this manuscript. Furthermore, the dynamic\nexchange model was primarily based on NMR signal splitting phenomena (e.g., splitting of the\nE202 signal into A and B, Figure 9a), but it was not validated through blocking exchange with\nmutants or real-time kinetic experiments, nor supported by structural data. Moreover, the\nhypothesis that DBD rapidly exchanges between 14-3-3ζ dimers was not substantiated by\nresolving the complex structure with individual monomers or through direct functional\nexperiments. These gaps may lead to conclusions that exceed the scope of data support.\nThank you for this important suggestion. We agree that further validation of the proposed\ndynamic exchange of the DBD between 14-3-3ζ protomers is important. While high-resolution\n22\nstructural approaches such as X-ray crystallography or cryo-EM would have been ideal, they were\nnot feasible for this system. Extensive crystallization trials (423 buffer conditions) failed, likely due\nto the intrinsic flexibility of the DBD and NTD of dpFOXO3a. Cryo-EM was also deemed\nunsuitable for the relatively small 72 kDa DBD–14-3-3ζ dimer complex, as confirmed in\nconsultation with our co-authors and structural biology experts, Dr. Mikio Tanabe and Prof. Toshiya\nSenda (specialists in X-ray crystallography and cryo-EM).\nTo directly test the exchange model, we performed high-field NMR experiments at 1 GHz,\nwhich improve the temporal resolution of exchange processes. If the DBD rapidly exchanges\nbetween 14-3-3ζ protomers, resonances such as E202—observed in the fast-exchange regime at 800\nMHz—should shift to the intermediate/slow-exchange regime at 1 GHz. Indeed, at 1 GHz the E202\n“A” signal split into two peaks, consistent with separate populations corresponding to the P2-\npeptide-bound state and the [P2 + DBD]-bound state. Conversely, the E202 “B” signal remained\nsingle at 1 GHz but split into two peaks when the temperature was lowered from 303 K to 293 K,\nwith the “A” signals merging. This temperature dependence indicates that “B” also contains two\npopulations: the pNTD-bound state and the [pNTD + DBD]-bound state.\nThese field- and temperature-dependent changes in the E202 signals provide direct kinetic\nevidence that the DBD of dpFOXO3a dynamically exchanges between the two protomers of the 14-\n3-3ζ dimer. We have incorporated these results into the revised Results and Discussion sections\n(pages 12–13, lines 279–286; pages 17–18, lines 391–393), as follows:\nOn pages 12–13, lines 279–286\n“In addition, high-field (1 GHz) NMR experiments provided direct evidence supporting the dynamic\nexchange of the DBD between the two protomers of the 14-3-3ζ dimer. At 1 GHz, the E202 ‘A’\nsignal observed in the fast-exchange regime at 800 MHz split into two peaks, consistent with distinct\nP2-peptide-bound and [P2 + DBD]-bound states (Supplementary Fig. 16b and 16c). The E202 ‘B’\nsignal remained single at 1 GHz but split into two peaks when the temperature was lowered from\n[pNTD + DBD]-bound states (Supplementary Fig. 16d and 16e). These field- and temperature-\ndependent changes indicate that the DBD dynamically exchanges between the two protomers of the\n14-3-3ζ dimer.”\nOn pages 17–18, lines 391–393\n“Although these NMR data do not provide high-resolution structural information, they serve as\ndirect evidence supporting the proposed dynamic exchange model between the two protomers of the\n14-3-3ζ dimer.”\nfield strength and temperature.\na Same as Fig. 8a. Spectral overlays of the E202 signals of [2H, 15N]-labeled 14-3-3ζ upon binding\nto dpFOXO3a (red), pNTD (blue), P2-peptide (green), and pCTD (orange) at 800 MHz and 303 K. b\nSpectrum of the E202 signal of 14-3-3ζ bound to dpFOXO3a at 800 MHz and 303 K. c–e Spectra of\nthe E202 signal of 14-3-3ζ bound to dpFOXO3a at 1 GHz at different temperatures: 303 K (c), 298\nK (d), and 293 K (e). The split signals are denoted as (A-1) / (A-2) for dpFOXO3a (A) and (B-1) /\n(B-2) for dpFOXO3a (B), corresponding to the ‘A’ and ‘B’ states observed in Fig. 8a. Dashed lines\nindicate linear spectral changes dependent on temperature.\n2. Figure Mislabeling:\nThe manuscript contains mislabeled or missing figure numbers (e.g., no Figure 6), leading to\nconfusion and hindering the review process. This oversight suggests a lack of thorough\nproofreading prior to submission and should be corrected to maintain clarity and professionalism.\nThank you for pointing out the errors in figure numbering. We apologize for this oversight and\nhave corrected all mislabeled or missing figure numbers in the revised manuscript. We have also\nthoroughly re-checked the entire text, figure legends, and cross-references to ensure consistency and\nto prevent such errors in the future.\n1. Kd Determination Using Truncated Protein:\nThe dissociation constant (Kd) was determined using a truncated version of Foxo3a (residues 1–\n284). It is important to address whether this truncated construct behaves similarly to the full-\nlength protein in Kd values. Is there any evidence that the full-length and truncated forms have\ncomparable Kd values?\nThank you for your comment. We agree that it is important to clarify whether FOXO3a\n(residues 1–284) behaves similarly to the full-length protein in binding affinity measurements. Based\non previous studies, the DNA-binding activity of full-length FOXO3a is dominated by the DBD\n(Cell Death Dis. 14, 702, 2023). While the transactivation domain (TAD) can interact with the DBD\nand compete with DNA (J. Mol. Biol. 384, 3, 2008; ACS Chem. Biol. 4, 12, 2009), the DNA–DBD\ninteraction is strong enough to displace the TAD (ACS Chem. Biol. 4, 12, 2009), suggesting minimal\ninfluence on DNA binding.\nFor 14-3-3ζ binding, it has been generally assumed that only the two phosphorylated sites, P1\nand P2, interact with 14-3-3ζ. In our study, we additionally identified a direct interaction between the\nDBD and 14-3-3ζ. While the TAD of the full-length protein could, in principle, compete with 14-3-\n3ζ for the DBD, its competitiveness is expected to be lower than that of 14-3-3ζ, as it is also lower\nthan that of DNA.\nTaken together, these considerations indicate that phosphorylated full-length FOXO3a is\nexpected to have essentially the same binding affinity to DNA and to 14-3-3ζ as dpFOXO3a\n(residues 1–284). We have clarified this point in the Results section (page 5, lines 91–96), as\nfollows:\n“The truncated construct FOXO3a (residues 1–284) used in this study is expected to have essentially\nthe same binding affinities to DNA and to 14-3-3ζ as the full-length protein. For DNA binding, the\ninteraction is dominated by the DBD (Cell Death Dis. 14, 702, 2023), and while the transactivation\ndomain (TAD) can compete with DNA for the DBD (J. Mol. Biol. 384, 3, 2008; ACS Chem. Biol. 4,\n12, 2009), the DNA–DBD interaction is strong enough to displace the TAD (ACS Chem. Biol. 4, 12,\n2009). For 14-3-3ζ binding, the recognition of the two phosphorylated motifs (P1 and P2) of\nFOXO3a is known to be dominant.”\n25\n2. Incorrect Literature Citation:\nOn page 5, line 96, the cited papers focus on Foxo4 rather than Foxo3a. Please ensure that the\nreferences are relevant and specific to the subject of the manuscript.\nThank you for your comment. We have confirmed that the cited references are relevant to the\nsubject of our manuscript. Although the specific studies cited on page 5, line 96 report K values for\nd\ndpFOXO4 rather than dpFOXO3a, we used them as a valid reference because FOXO4 is a close\nhomologue of FOXO3a. The sequence identities of P1, P2, and the DBD between FOXO3a and\nFOXO4 are 100% (7/7 residues), 86% (6/7), and 83% (78/94), respectively, suggesting highly\nsimilar interaction modes with both DNA and 14-3-3ζ. Therefore, the FOXO4 K values provide a\nd\nreasonable benchmark for evaluating our FOXO3a measurements. We have clarified this point in the\nrevised text (pages 3–4, lines 64–66; page 5, lines 102–104), as follows:\nOn pages 3–4, lines 64–66\n“FOXO4 has high sequence identity with FOXO3a at P1 (100%), P2 (86%), and the DBD (83%),\nsupporting the assumption that their interaction modes with DNA and 14-3-3ζ are highly similar, and\nthus that K values of FOXO4 provide a reliable reference for FOXO3a.”\nd\nOn page 5, lines 102–104\n“These K values, together with binding stoichiometry, are consistent with previous reports for\nd\ndpFOXO4, which has the similar binding mode, indicating that the samples we prepared possessed\nappropriate binding activities.”\n3. Limited Evidence for Direct DBD Interaction:\nThe proposed direct binding of 14-3-3 to the DBD of Foxo3a and the suggested hybrid binding\nmodel are currently supported solely by NMR data, which were obtained using the truncated\nprotein and peptides. This weakens the overall conclusion. Crosslinking experiments and site-\nspecific mutagenesis would provide more robust support for these claims.\nThank you for your comment. We agree that additional evidence beyond CSP-based NMR\nmapping is important for supporting the direct interaction between the DBD of FOXO3a and 14-3-3ζ.\nAs noted in our response to Major Point Review 1-2 from Reviewer #2, we have already included\nnew NMR cross-saturation experiments, which directly identified residues on 14-3-3ζ in close\nspatial contact (<5 Å) with dpFOXO3a, as well as site-directed mutagenesis of selected residues\n26\npredicted to interact with the DBD. These data are now incorporated into the Results section (page\n11, lines 240–255) and illustrated in Fig. 7. Together, they provide both direct structural and\nfunctional evidence for the DBD–14-3-3ζ interaction, thereby supporting the proposed hybrid\nbinding model.\n“However, CSP identifies residues whose local chemical environment changes upon binding, which\ncan result from either direct contact or conformational changes. To obtain direct evidence for DBD –\n14-3-3ζ interaction beyond CSP mapping, we performed NMR cross-saturation experiments, which\nselectively detect residues in close spatial proximity (~5 Å) to dpFOXO3a. This analysis identified\nspecific contact residues on 14-3-3ζ, several of which are located outside the canonical phospho-\nmotif binding groove and positioned to interact with the DBD (Fig. 7).\nQuadruple-alanine mutants targeting these sites (D197A/E198A/E202A/D204A or\nT215A/L216A/Q219A/R222A) showed no reduction in DNA displacement ability in FSEC assays\n(Supplementary Fig.13), consistent with a broad interaction interface in which partial disruption does\nnot abolish binding. In contrast, NMR spectral changes of [2H, 15N]-CTD titrated with the\nT215A/L216A/Q219A/R222A 14-3-3ζ mutant demonstrated that signals, mainly in helix 3, shifted\nin a different manner or decreased in intensity compared with the titration using wildtype 14-3-3ζ.\nThe titration with the D197A/E198A/E202A/D204A mutant was not possible because the sample\nprecipitated and could not be concentrated sufficiently for NMR analysis. Together, these NMR data\nstrongly suggest that the helix 3 of DBD directly interacts with the residues around\nT215/L216/Q219/R222 of 14-3-3ζ (Supplementary Fig. 14).”\n27\n4. Inadequate Comparison with Existing Studies:\nThe authors only mentioned the similar mechanism of FOXO4 but did not conduct a detailed\ncomparison of the phosphorylation site specificity, DBD structural differences, or functional\nregulatory similarities and differences between FOXO3a and FOXO4, leading to limited\ninnovation in this study. It is recommended to include a structural and functional comparison\nbetween FOXO3a and FOXO4 in the Discussion section and to discuss other subtypes of the 14-3-\n3 family.\nThank you for your suggestion. We agree that a broader comparison with other FOXO isoforms\nand 14-3-3 family members is important. Among FOXO isoforms (FOXO1, FOXO3a, FOXO4, and\nFOXO6), the amino acid sequences of P1, P2, and the DBD are highly conserved, as noted in our\nresponse to Minor Point 2 (Cell Death Dis. 14, 702, 2023). Similarly, 14-3-3ζ has six paralogues (β,\nγ, ε, η, θ, and σ), whose phosphopeptide-binding grooves are highly conserved (Supplementary Fig.\n17) and recognize similar motifs (Cell 91, 7, 1997). Notably, the DBD-binding region on 14-3-3 is\nalso conserved (Supplementary Fig. 17). These observations suggest that the binding mode we\nidentified between phosphorylated FOXO3a and 14-3-3ζ may be shared by other FOXO–14-3-3\ncombinations.\nAlthough subtle structural differences exist among FOXO isoforms, particularly in regions\nadjacent to the DBD and phosphorylation motifs, their high conservation strongly supports the\ngenerality of the binding mode observed here.\nFurthermore, FOXO3a is the most ubiquitously expressed FOXO isoform (Cell Death Dis. 14,\n702, 2023), and 14-3-3ζ is frequently overexpressed in diverse cancers (Expert Opin. Ther. Targets,\n14, 12, 2010), highlighting the relevance of our findings for therapeutic strategies targeting FOXO–\n14-3-3 interactions, particularly in cancer. We have added this comparison to the Discussion section\n(page 18, lines 394–403), as follows:\n“Finally, we discuss other FOXO and 14-3-3 isoforms. The high sequence conservation of P1, P2,\nand the DBD among FOXO isoforms (FOXO1, FOXO3a, FOXO4, and FOXO6) (Cell Death Dis. 14,\n702, 2023) and the conserved phosphopeptide-binding grooves and DBD-binding region among 14-\n3-3 paralogues (β, γ, ε, η, θ, and σ) (Supplementary Fig. 17) suggest that the dpFOXO3a–14-3-3ζ\nbinding mode identified in this study may be a general feature of FOXO–14-3-3 interactions.\nAlthough subtle structural differences exist among FOXO isoforms, particularly in regions\nadjacent to the DBD and phosphorylation motifs, their high conservation strongly supports the\ngenerality of the binding mode observed here. Given that FOXO3a is the most ubiquitously expressed FOXO\nisoform (Cell Death Dis. 14, 702, 2023) and that 14-3-3ζ is frequently overexpressed in diverse\ncancers (Expert Opin. Ther. Targets, 14, 12, 2010), these findings may have broad implications for\ntherapeutic strategies targeting FOXO–14-3-3 complexes, particularly in disease contexts, especially\nin oncology.”\n28\n29\na Amino acid sequence alignment using CLUSTALW. Asterisk (*) and colon (:) indicate the fully\nconserved residue and the partially conserved residue, respectively. Residues of 14-3-3ζ affected by\nCTD binding, as detected by CSP analysis, are highlighted in yellow. Black squares mark residues of\n14-3-3ζ directly contacting dpFOXO3a, as identified by cross-saturation analysis. b Structural\nalignment of 14-3-3 isoforms: ζ (orange), β (yellow), θ (light green), γ (cyan), η (magenta), σ (gray),\nand ε (black). The corresponding PDB entries are 1QJA (ζ), 2C23 (β), 2BTP (θ), 2B05 (γ), 2C63 (η),\n1YWT (σ), and 7V9B (ε).\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nWe appreciate Reviewer #3’s time and effort.\nAdditional revisions from the authors:\n1) The figure titled “Spectral overlay of 14-3-3ζ bound to dpFOXO3a, pNTD, and P2-peptide.”\nwas relocated to Supplementary Fig. 12 and the related text was shortened.\nThe cross-saturation NMR experiment was more direct to demonstrate the direct interaction sites\nbetween dpFOXO3a and 14-3-3ζ than the spectral overlay of 14-3-3ζ bound to dpFOXO3a, pNTD,\nand P2-peptide. We relocated the figure titled “Spectral overlay of 14-3-3ζ bound to dpFOXO3a,\npNTD, and P2-peptide.” to Supplementary Fig. 12 and the related text was shortened. In addition,\nwe newly performed NMR cross-saturation experiments to directly map the dpFOXO3a–14-3-3ζ\ncontact interface and added these data as Fig. 7. Accordingly, we renumbered the figures in\nmanuscript and supplementary, and revised the text about Supplementary Fig. 12 (page 10, lines\n221–233), as follows:\n“In addition, the spectral overlay of 14-3-3ζ upon binding to dpFOXO3a with those upon binding to\nthe N-terminal unstructured region upstream of the DBD that is phosphorylated at T32 (referred to\n31\nas pNTD containing the P1 site) and with those upon binding to the P2-peptide, showed that many\nsignals with pNTD or the P2-peptide overlapped those with dpFOXO3a but 18 and 44 signals in the\npNTD-bound state and P2-peptide-bound state, respectively, did not overlap with those in the\ndpFOXO3a-bound state (Supplementary Fig. 12a). These residues clustered on the 14-3-3ζ surface\nformed by its helices 8–9 (Supplementary Fig. 12b and 12c). These spectral differences were likely\ncaused by the DBD that exists only in dpFOXO3a, neither in pNTD nor in the P2-peptide.\nBased on these results, we hypothesized that this upper region of 14-3-3ζ interacts with the\nDBD of dpFOXO3a. To confirm the hypothesis, we analyzed the changes in 1H-15N TROSY spectra\nof uniformly 2H, 15N-labeled 14-3-3ζ, upon the sequential additions of the unlabeled CTD, which\nconsists of the DBD and the C-terminal tail (Fig. 6).”\n2) Page 15, lines 339–340, we have changed “As shown in this study, 14-3-3ζ binds to\ndpFOXO3a at three sites, P1, P2, and the DBD, while DNA mainly binds to the DBD.”.\nWe newly referred that the DNA also interacted with P2 but its interaction makes minor contribution\nto the DNA – DBD interaction, as noted in our response to Major Point Review 1-1 from Reviewer\n#2, and hence we added “mainly” to the sentence to clarify our recognition.\n3) Page 16, line 344, we have corrected the grammatical error, changing \"exists\" to \"exist\".\n4) Pages 19–25, Methods section, we have removed the numbering of subheadings.\n5) Page 23, Methods section, lines 510–511, we have added a statement on technical replicates\nof FSEC analysis, as follows:\n“Each experiment was repeated independently 3–4 times on the same sample (technical replicates).”\n6) Pages 19–25, Methods section, we have added descriptions about additional experiments for\nrevision, as follows:\nOn page 19, lines 419–421\n“The DNA sequence encoding full-length human 14-3-3ζ (residues 1-245),\nthe pGEX-6P-1 (Cytiva).”\nOn page 20, lines 431–433\n“14-3-3ζ mutants were additionally purified by gel filtration using SuperdexTM 200 increase 10/300\nGL (Cytiva) in a buffer containing 20 mM NaPi (pH 7.4), 150 mM NaCl and 1 mM DTT.”\nOn page 21, lines 467–470\n“Sense and anti-sense were mixed in 1 equivalent each and annealed by decreasing the temperature\nfrom 100 ℃ to 25 ℃ at 1 ℃/min in a buffer containing 20 mM NaPi (pH 6.4) and 150 mM NaCl for\nITC experiments, 20 mM NaPi (pH 6.4) and 100 mM NaCl for NMR experiments, or 10 mM Tris-\nHCl (pH 7.5), 100 mM NaCl, and 1 mM EDTA for FSEC experiments.”\nOn page 23, lines 514–517\n“The full FSEC-based competition analysis for pS253-FOXO3a (T32A) (10 μM) and DNA (15 μM)\ntitrated 0, 2.5, 5, 7,5, 10, 12.5, 20, 40 μM 14-3-3ζ dimer. Measurements were performed in triplicate\non the same sample (technical replicates). Competition analyses using 14-3-3ζ mutants were\nconducted with dpFOXO3a (10 μM), DNA (11 μM), and 14-3-3ζ mutants dimer (10 μM).”\nOn page 24, lines 536–537\n“Data were collected on Bruker Avance 600 and 800, or Ascend Evo 1.0 GHz spectrometers\n(Billerica) equipped with TXI or TCI probes.”\nOn page 24, lines 547–551\n“2H, 15N-labeled dpFOXO3a (169 μM) and 2H, 15N-labeled CTD (150 μM) were titrated with\nunlabeled 14-3-3ζ dimer and 2H, 15N-labeled CTD (150 μM) was also titrated with\nT215A/L216A/Q219A/R222A 14-3-3ζ mutant. 2H, 15N-labeled 14-3-3ζ dimer (82 μM, 100 μM, 128\nμM, 92 μM, and 123 μM) was titrated with unlabeled dpFOXO3a, pNTD, pCTD, P2-peptide, and\nCTD, respectively. The experiments were monitored using a 1H-15N TROSY spectrum.”\nOn page 25, lines 567–578\n“In the cross-saturation experiment, we prepared 222 μM 2H, 15N-labeled 14-3-3ζ with 1.25\nequivalent of dpFOXO3a containing 20% H O / 80% 2H O and selectively saturated dpFOXO3a by\n2 2\nirradiating the aliphatic proton resonances, using the mlevsp180.p30 decoupling program. The\nsaturation frequency was set at 1.0 ppm and the saturation time was set to 1.0 s.\nCompetition analysis of 126 μM 2H, 15N-labeled dpFOXO3a with 1.1 equivalents of DNA and\n33\n1.5 equivalents of 14-3-3ζ dimer were monitored using 1H-15N TROSY spectrum at 600 and 800\nMHz. The diluted sample (16 μM dpFOXO3a with 18 μM DNA plus 24 μM 14-3-3ζ) was also\nmonitored using 1H-15N TROSY spectrum at 600 MHz. NMR samples were analyzed by gel\nfiltration using SuperdexTM 200 increase 10/300 GL (Cytiva).\nIn the magnetic field- and temperature-dependent analyses, we prepared 209 μM 2H, 15N-\nlabeled 14-3-3ζ with 1.25 equivalent of dpFOXO3a and measured 1H-15N TROSY spectra at 1 GHz\nat different temperatures: 303 K, 298 K, and 293 K.”\n7) Page 26, Data availability section, we have added the BMRB accession codes for chemical\nshift assignments and mention of source data, as follows:\n“The chemical shift assignments are available from the BMRB data bank: accession codes 26346\n(CTD), 26350 (dpFOXO3a), and 14-3-3ζ (26348). Source data are provided with the paper.”\n8) Page 28, Acknowledgements section, we additionally thank “Prof. Koh Takeuchi, Dr. Yutaka\nKofuku, and Dr. Shunsuke Imai” for the NMR machine time to conduct further experiments\nin response to the reviewers' suggestions.\n9) Page 28–29, Author contributions, we have distinguished between T.Kuwayama and\nT.Kimura by using their family names as they share the same initials.\nIn accordance with the reviewers’ comments, we have revised our manuscript and prepared a point-\nby-point response. We are grateful for Reviewer #1’s critical but constructive evaluation regarding\nthe novelty of our work and for Reviewer #2’s positive endorsement. In this response, reviewers’\ncomments and our replies are shown in italic and normal text, respectively. The revisions in the\nmanuscript are highlighted in yellow. We believe that these revisions sufficiently address all of the\nreviewers’ concerns.\nReviewer #1 (Remarks to the Author):\nI have studied the revised version of this manuscript, together with the authors’ rebuttal letter. I\nwant to thank the authors for the significant additional work that they have put into this revised\nversion, which consisted of additional experimental work and analysis.\nI have also received the report from referee 2 and the corresponding authors’ responses.\nUnfortunately, I have to say that the 2022 ACS Omega publication (now ref 28), which was\nunfortunately not cited in the original submission, does impact the novelty of the current\nmanuscript. It is clearly not identical to the current manuscript, and the current manuscript offers\nadditional insights. However, I am afraid that the novelty is not as strong as would be required for\npublication in Nature Communications. I am sorry to say this, given the significant work that the\nauthors have put into this revised version, and I am fine with the editor or referee 2 having a\ndifferent opinion. However, in my opinion, this manuscript is better suited for a more specialized\njournal.\nWe greatly appreciate Reviewer #1’s careful evaluation and the substantial time devoted to our\nmanuscript. We fully understand the concern that the 2022 ACS Omega study (now cited as ref. 28)\nbears conceptual relevance to our work. We have therefore revised the manuscript to more clearly\narticulate the distinct and novel aspects of our findings.\nOn page 13, line 11 from bottom – page 14, line 2, the revised text reads:\n“The novelty of our study represents a significant advance beyond prior investigations, defined by\ntwo key points. First, we identify a direct interaction between 14-3-3ζ and the FOXO3a-DBD, which\ncompetitively inhibits DNA binding. This is an interaction mode fundamentally distinct from the\nconventionally recognized binding at the P1 and P2 phosphomotifs. Second, our date reveal a two-\nstep displacement mechanism involving sequential tethering at P1 and P2 that markedly increases\n1\nthe effective local concentration of 14-3-3ζ around the DBD, thereby enabling efficient DNA\ndisplacement. Although previous work showed that the P2 motif contributes to DNA binding\n(Nucleic Acids Research, Vol. 35, No. 20, 2007) and interacts with 14-3-3ζ (ACS Omega, Vol. 7, No.\n28, 2022), its contribution to the total DNA-binding free energy is minor relative to helix 3 of the\nDBD (Nucleic Acids Research, Vol. 35, No. 20, 2007). Importantly, our NMR competition analysis\ndemonstrated that P2 engagement alone does not dissociate DNA, revealing the existence of a\nternary dpFOXO3a–DNA–14-3-3ζ complex. This key observation indicates that P2-centric\nmechanism is insufficient to explain DNA displacement. Together, these results establish that direct\nocclusion of the DBD by 14-3-3ζ, rather than P2-mediated competition alone, is the dominant\nmechanism governing displacement of DNA by 14-3-3ζ.”\nReviewer #2 (Remarks to the Author):\nThe authors have made substantial revisions and conducted additional experiments that\neffectively address all of the concerns I raised in my previous review. I am satisfied with the\nrevisions and would like to recommend the manuscript for publication in Nature Communications.\nWe greatly appreciate Reviewer #2’s approval. Your encouraging evaluation and constructive\nsuggestions have provided us with valuable insights that significantly improved the quality and\nclarity of our study. We sincerely thank Reviewer #2 again for your time and thoughtful\nconsideration.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nWe appreciate Reviewer #3’s contribution and time spent in reviewing our work.\n1) According to author check list, we have revised verbs to the present tense, as follows:\nIn Abstract section, on page 1, 2nd line from the bottom, from “found” to “find”\nIn Abstract section, on page 2, 2nd line, from “identified” to “identify”\nIn Introduction section, page 4, 6th line, from “analyzed” to “analyze”\nIn Introduction section, page 4, 8th line, from “executed” to “execute”\n2) According to author check list, we have deleted punctuation, as follows:\nPage 4, 4th line, from “ “dimer” ” to “dimer”\nPage 6, 2nd line, from “ “100-fold” ” to “100-fold”\nPage 7, 13th line, from “ “unphosphorylated” ” to “unphosphorylated”\nIn the legend of figure 7b, from “N”, “C”, and “X” to “[N]”, “[C]”, and “[X]”, respectively\n3) According to author check list, we have labeled equations sequentially in the main\nmanuscript and supplementary discussion.\n4) In the figure legend of Fig. 2, we have revised the description of a precise value of ‘n’ in the\naccording to author check list, as follows:\n“The mean values and standard deviations are calculated from three separate experiments, except for\nthe one equivalent of 14-3-3ζ dimer condition (n = 4).”\n5) In the figure legend of Fig. 7, we have clearly defined error bars and revised the description\nof them according to author check list, as follows:\n“The bars represent the experimental errors, calculated as √(1⁄𝑆𝑁 )2+(1⁄𝑆𝑁 )2, where\n𝑖𝑟𝑟(+) 𝑖𝑟𝑟(−)\n6) In the figure legend of Fig. 7, we have added the PDB code of the phosphorylated peptide in\nFig. 7d, as follows:\n“Cyan sticks represent P2-peptide extracted from the crystal structure with 14-3-3ε (PDB: 7V9B)\nafter aligned with 14-3-3ζ structure (PDB: 5JM4).“\n7) In Data availability section, on page 24, we have hyperlinked the BMRB accession codes\naccording to author check list, as follows:\n“The chemical shift assignments are available from the BMRB data bank: accession codes 26346\n(CTD) [https://bmrb.io/data_library/summary/index.php?bmrbId=26346], 26350 (dpFOXO3a)\n[https://bmrb.io/data_library/summary/index.php?bmrbId=26350], and 14-3-3ζ (26348)\n[https://bmrb.io/data_library/summary/index.php?bmrbId=26348].”\n8) In the figure legend of Supplementary Fig. 14b, we corrected a typographical error,\nchanging \"Zommed\" to \"Zoomed\".\n9) In the source data for Fig. 5, we corrected the equivalent ratio of dpFOXO3a, changing\n\"0.75 eq dpFOXO3a added (relative to the 14-3-3 monomer)\" to \"1.5 eq dpFOXO3a added\n(relative to the 14-3-3 dimer)\".\n4"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I have studied the revised version of this manuscript, together with the authors’ rebuttal letter. I want to thank the authors for the significant additional work that they have put into this revised version, which consisted of additional experimental work and analysis.\nI have also received the report from referee 2 and the corresponding authors’ responses. Unfortunately, I have to say that the 2022 ACS Omega publication (now ref 28), which was unfortunately not cited in the original submission, does impact the novelty of the current manuscript. It is clearly not identical to the current manuscript, and the current manuscript offers additional insights. However, I am afraid that the novelty is not as strong as would be required for publication in Nature Communications. I am sorry to say this, given the significant work that the authors have put into this revised version, and I am fine with the editor or referee 2 having a different opinion. However, in my opinion, this manuscript is better suited for a more specialized journal.", "reviewer_2": "The authors have made substantial revisions and conducted additional experiments that effectively address all of the concerns I raised in my previous review. I am satisfied with the revisions and would like to recommend the manuscript for publication in Nature Communications.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-by-point response to REVIEWER COMMENTS\nIn accordance with the reviewers’ comments, we have revised our manuscript and prepared a point-\nby-point response. We are grateful for Reviewer #1’s critical but constructive evaluation regarding\nthe novelty of our work and for Reviewer #2’s positive endorsement. In this response, reviewers’\ncomments and our replies are shown in italic and normal text, respectively. The revisions in the\nmanuscript are highlighted in yellow. We believe that these revisions sufficiently address all of the\nreviewers’ concerns.\nReviewer #1 (Remarks to the Author):\nI have studied the revised version of this manuscript, together with the authors’ rebuttal letter. I\nwant to thank the authors for the significant additional work that they have put into this revised\nversion, which consisted of additional experimental work and analysis.\nI have also received the report from referee 2 and the corresponding authors’ responses.\nUnfortunately, I have to say that the 2022 ACS Omega publication (now ref 28), which was\nunfortunately not cited in the original submission, does impact the novelty of the current\nmanuscript. It is clearly not identical to the current manuscript, and the current manuscript offers\nadditional insights. However, I am afraid that the novelty is not as strong as would be required for\npublication in Nature Communications. I am sorry to say this, given the significant work that the\nauthors have put into this revised version, and I am fine with the editor or referee 2 having a\ndifferent opinion. However, in my opinion, this manuscript is better suited for a more specialized\njournal.\nWe greatly appreciate Reviewer #1’s careful evaluation and the substantial time devoted to our\nmanuscript. We fully understand the concern that the 2022 ACS Omega study (now cited as ref. 28)\nbears conceptual relevance to our work. We have therefore revised the manuscript to more clearly\narticulate the distinct and novel aspects of our findings.\nOn page 13, line 11 from bottom – page 14, line 2, the revised text reads:\n“The novelty of our study represents a significant advance beyond prior investigations, defined by\ntwo key points. First, we identify a direct interaction between 14-3-3ζ and the FOXO3a-DBD, which\ncompetitively inhibits DNA binding. This is an interaction mode fundamentally distinct from the\nconventionally recognized binding at the P1 and P2 phosphomotifs. Second, our date reveal a two-\nstep displacement mechanism involving sequential tethering at P1 and P2 that markedly increases\n1\nthe effective local concentration of 14-3-3ζ around the DBD, thereby enabling efficient DNA\ndisplacement. Although previous work showed that the P2 motif contributes to DNA binding\n(Nucleic Acids Research, Vol. 35, No. 20, 2007) and interacts with 14-3-3ζ (ACS Omega, Vol. 7, No.\n28, 2022), its contribution to the total DNA-binding free energy is minor relative to helix 3 of the\nDBD (Nucleic Acids Research, Vol. 35, No. 20, 2007). Importantly, our NMR competition analysis\ndemonstrated that P2 engagement alone does not dissociate DNA, revealing the existence of a\nternary dpFOXO3a–DNA–14-3-3ζ complex. This key observation indicates that P2-centric\nmechanism is insufficient to explain DNA displacement. Together, these results establish that direct\nocclusion of the DBD by 14-3-3ζ, rather than P2-mediated competition alone, is the dominant\nmechanism governing displacement of DNA by 14-3-3ζ.”\nReviewer #2 (Remarks to the Author):\nThe authors have made substantial revisions and conducted additional experiments that\neffectively address all of the concerns I raised in my previous review. I am satisfied with the\nrevisions and would like to recommend the manuscript for publication in Nature Communications.\nWe greatly appreciate Reviewer #2’s approval. Your encouraging evaluation and constructive\nsuggestions have provided us with valuable insights that significantly improved the quality and\nclarity of our study. We sincerely thank Reviewer #2 again for your time and thoughtful\nconsideration.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nWe appreciate Reviewer #3’s contribution and time spent in reviewing our work.\n1) According to author check list, we have revised verbs to the present tense, as follows:\nIn Abstract section, on page 1, 2nd line from the bottom, from “found” to “find”\nIn Abstract section, on page 2, 2nd line, from “identified” to “identify”\nIn Introduction section, page 4, 6th line, from “analyzed” to “analyze”\nIn Introduction section, page 4, 8th line, from “executed” to “execute”\n2) According to author check list, we have deleted punctuation, as follows:\nPage 4, 4th line, from “ “dimer” ” to “dimer”\nPage 6, 2nd line, from “ “100-fold” ” to “100-fold”\nPage 7, 13th line, from “ “unphosphorylated” ” to “unphosphorylated”\nIn the legend of figure 7b, from “N”, “C”, and “X” to “[N]”, “[C]”, and “[X]”, respectively\n3) According to author check list, we have labeled equations sequentially in the main\nmanuscript and supplementary discussion.\n4) In the figure legend of Fig. 2, we have revised the description of a precise value of ‘n’ in the\naccording to author check list, as follows:\n“The mean values and standard deviations are calculated from three separate experiments, except for\nthe one equivalent of 14-3-3ζ dimer condition (n = 4).”\n5) In the figure legend of Fig. 7, we have clearly defined error bars and revised the description\nof them according to author check list, as follows:\n“The bars represent the experimental errors, calculated as √(1⁄𝑆𝑁 )2+(1⁄𝑆𝑁 )2, where\n𝑖𝑟𝑟(+) 𝑖𝑟𝑟(−)\n6) In the figure legend of Fig. 7, we have added the PDB code of the phosphorylated peptide in\nFig. 7d, as follows:\n“Cyan sticks represent P2-peptide extracted from the crystal structure with 14-3-3ε (PDB: 7V9B)\nafter aligned with 14-3-3ζ structure (PDB: 5JM4).“\n7) In Data availability section, on page 24, we have hyperlinked the BMRB accession codes\naccording to author check list, as follows:\n“The chemical shift assignments are available from the BMRB data bank: accession codes 26346\n(CTD) [https://bmrb.io/data_library/summary/index.php?bmrbId=26346], 26350 (dpFOXO3a)\n[https://bmrb.io/data_library/summary/index.php?bmrbId=26350], and 14-3-3ζ (26348)\n[https://bmrb.io/data_library/summary/index.php?bmrbId=26348].”\n8) In the figure legend of Supplementary Fig. 14b, we corrected a typographical error,\nchanging \"Zommed\" to \"Zoomed\".\n9) In the source data for Fig. 5, we corrected the equivalent ratio of dpFOXO3a, changing\n\"0.75 eq dpFOXO3a added (relative to the 14-3-3 monomer)\" to \"1.5 eq dpFOXO3a added\n(relative to the 14-3-3 dimer)\".\n4"}]

Decision letter