NMR investigation of FOXO4-DNA interaction for discriminating target and non-target DNA sequences

Forkhead box O4 (FOXO4), a human transcription factor, recognizes target DNA through its forkhead domain (FHD) while maintaining comparable binding affinity to non-target DNA. The conserved region 3 (CR3), a transactivation domain, modulates DNA binding kinetics to FHD and contributes to target DNA selection, but the underlying mechanism of this selection remains elusive. Using paramagnetic relaxation enhancement analysis, we observed a minor state of CR3 close to FHD in the presence of non-target DNA, a state absent when FHD interacts with target DNA. This minor state suggests that CR3 effectively masks the non-target DNA-binding interface on FHD. The interaction weakens significantly under high salt concentration, implying that CR3 or high salt concentrations can modulate electrostatic interactions with non-target DNA. Our 15N relaxation measurements revealed FHD’s flexibility with non-target DNA and increased rigidity with target DNA binding. Our findings offer insights into the role of FOXO4 as a transcription initiator. FOXO4 CR3 masks the non-target DNA binding interface of FHD. FHD increases flexibility with non-target DNA and rigidity with target DNA, releasing CR3. Salt modulates the rate of FHD-DNA binding, discriminating between target and non-target DNA.