NMR investigation of conformational heterogeneity in FOXO4 CR2C binding to the MLL site of CBP KIX☆

Abstract

Forkhead box O (FOXO) transcription factors regulate metabolism, DNA damage repair, cell cycle, and apoptosis. FOXO4 contains Phi XX Phi Phi motifs in its intrinsically disordered conserved regions (CR2C and CR3) that mediate interactions with the KIX domain of CREB-binding protein (CBP), a transcriptional coactivator and histone acetyltransferase. While FOXO3a binds to CBP KIX through both CR2C and CR3, the interaction between FOXO4 CR2C and CBP KIX has remained uncharacterized, despite FOXO4 CR2C uniquely carrying four consecutive prolines immediately following its Phi XX Phi Phi motif. Here, we characterize this interaction by solution NMR spectroscopy. Chemical-shift perturbation identifies the binding interfaces on both proteins and reveals splitting of several amide cross-peaks in CR2C into two sets. Systematic proline mutagenesis and truncation support the presence of distinct conformational subpopulations, for which apparent Kd values were estimated independently. Circular dichroism analysis shows that CR2C remains disordered upon binding, and docking models suggest that FOXO4 CR2C binds in multiple disordered conformations on the KIX MLL site. These results illustrate how an adjacent poly-proline tract modulates the KIX binding behavior of CR2C through conformational heterogeneity.