A critical role in structure-specific DNA binding for the acetylatable lysine residues in HMGB1

The structure-specific DNA-binding protein HMGB1 (high-mobility group protein B1) which comprises two tandem HMG boxes (A and B) and an acidic C-terminal tail, is acetylated in vivo at Lys² and Lys¹¹ in the A box. Mutation to alanine of both residues in the isolated A domain, which has a strong preference for pre-bent DNA, abolishes binding to four-way junctions and 88 bp DNA minicircles. The same mutations in full-length HMGB1 also abolish its binding to four-way junctions, and binding to minicircles is substantially impaired. In contrast, when the acidic tail is absent (AB di-domain) there is little effect of the double mutation on four-way junction binding, although binding to minicircles is reduced ∼15-fold. Therefore it appears that in AB the B domain is able to substitute for the non-functional A domain, whereas in full-length HMGB1 the B domain is masked by the acidic tail. In no case does single substitution of Lys² or Lys¹¹ abolish DNA binding. The double mutation does not significantly perturb the structure of the A domain. We conclude that Lys² and Lys¹¹ are critical for binding of the isolated A domain and HMGB1 to distorted DNA substrates.