LAUSR

The fine-tuning of transcription factor DNA-binding activity is often governed by transient intramolecular interactions between the transactivation domain and the DNA-binding domain. An example of such interaction is found in the transcription factor ATF4, a central regulator of the Integrated Stress Response. In ATF4, dynamic coupling between the transactivation domain and the basic-leucine zipper (bZip) domain modulates the phosphorylation levels of the disordered transactivation domain by casein kinase 2. However, the structural and molecular basis of these interdomain interactions remains poorly understood. This study focuses on a secondary basic motif at the C-terminus of ATF4, which is shared exclusively with its closest paralogue, ATF5. Through a combination of solution NMR spectroscopy, fluorescence polarization assays, and long-timescale molecular simulations, we demonstrate that this secondary basic motif is the primary driver of interdomain coupling between the transactivation and bZip domains of ATF4. Moreover, this motif enhances ATF4′s DNA-binding specificity via interaction with the transactivation domain while also potentially facilitating rapid DNA scanning. Our findings reveal the pivotal role of a conserved motif in establishing disorder-mediated interactions that critically modulate ATF4′s DNA-binding activity.