Inhibition of HMGA2 binding to AT-rich DNA by its negatively charged C-terminus

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a small DNA-binding protein that specifically targets AT-rich DNA sequences. Structurally, HMGA2 is an intrinsically disordered protein (IDP), comprising three positively charged 'AT-hooks' and a negatively charged C-terminus. HMGA2 can form homodimers through electrostatic interactions between its 'AT-hooks' and C-terminus. This suggests that the negatively charged C-terminus may inhibit DNA binding by interacting with the positively charged 'AT-hooks.' In this paper, we demonstrate that the C-terminus significantly influences HMGA2's DNA-binding properties. For example, the C-terminal deletion mutant HMGA2Δ95-108 binds more tightly to the AT-rich DNA oligomer FL814 than wild-type HMGA2. Additionally, a synthetic peptide derived from the C-terminus (the C-terminal motif peptide or CTMP) strongly inhibits HMGA2's binding to FL814, likely by interacting with the 'AT-hooks,' as shown by various biochemical and biophysical assays. Molecular modeling demonstrates that electrostatic interactions and hydrogen bonding are the primary forces driving CTMP's binding to the 'AT-hooks.' Intriguingly, we found that hydration does not play a role in HMGA2-DNA binding. These results suggest that the highly negatively charged C-terminus of HMGA2 plays a critical role in regulating its DNA-binding capacity through autoinhibition, likely facilitating the target search process for AT-rich DNA sequences.