NMR and biophysical investigation of selective recognition of FOXO4 CR3 by CBP TAZ1 domain

Abstract

Forkhead box protein O4 (FOXO4) is a transcription factor that plays a pivotal role in various cellular processes, such as metabolism, cell cycle progression, DNA damage response, and apoptosis. cAMP-response element-binding protein (CREB) binding protein (CBP) is a coactivator recruited by several transcription factors, including FOXO4. The FOXO4-CBP interaction is essential for regulating FOXO4 target gene expression. Previous studies have shown that the C-terminal region of FOXO4, which contains common NXXNN motifs, interacts with the transcriptional adapter zinc-binding domain 1 (TAZ1) of CBP. However, the detailed structural information of the FOXO4-CBP TAZ1 interaction remains unknown. Among the FOXO4 conserved regions (CRs) that contain the NXXNN motif, our isothermal titration calorimetry (ITC) analysis demonstrated that only CR3 binds to CBP TAZ1 with micromolar Kd, whereas CR2C shows no detectable binding. Chemical shift perturbation (CSP) analysis revealed that the CR3-binding interface corresponds to the surface formed by alpha 1 and alpha 3 of CBP TAZ1. Furthermore, circular dichroism (CD) spectroscopy and C alpha chemical shift difference analysis suggest that FOXO4 CR3 undergoes partial alpha-helix formation upon binding. These results indicate that FOXO4 CR3 adopts an alpha-helical conformation within the TAZ1 binding pocket. This study provides structural and biophysical insights into the FOXO4-CBP interaction at the molecular level.