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

[Image: see text] The transcriptional activity of Forkhead Box O3 (FOXO3a) is inactivated by AKT-mediated phosphorylation on Serine 253 (S253), which enables FOXO3a binding to 14-3-3. Phosphorylated FOXO3a binding to 14-3-3 facilitates the nuclear exclusion of FOXO3a, causing cancer cell proliferation. The FOXO3a/14-3-3 interaction has, therefore, emerged as an important therapeutic target. Here, we report a comprehensive analysis using fluorescence polarization, isothermal titration calorimetry, small-angle X-ray scattering, X-ray crystallography, and molecular dynamics simulations to gain molecular-level insights into the interaction of FOXO3a(pS253) phosphopeptide with 14-3-3ε. A high-resolution structure of the fluorophore-labeled FOXO3a(pS253):14-3-3ε complex revealed a distinct mode of interaction compared to other 14-3-3 phosphopeptide complexes. FOXO3a(pS253) phosphopeptide showed significant structural difference in the positions of the -3 and -4 Arg residues relative to pSer, compared to that of a similar phosphopeptide, FOXO1(pS256) bound to 14-3-3σ. Moreover, molecular dynamics studies show that the significant structural changes and molecular interactions noticed in the crystal structure of FOXO3a(pS253):14-3-3ε are preserved over the course of the simulation. Thus, this study reveals structural differences between the binding to 14-3-3 isoforms of FOXO1(pS256) versus FOXO3a(pS253), providing a framework for the rational design of isoform-specific FOXO/14-3-3 protein–protein interaction inhibitors for therapy.