Structural Insights into the Mechanism of Negative Regulation of Single-box High Mobility Group Proteins by the Acidic Tail Domain

Background: HMG-box proteins bind and bend DNA, regulated by their acidic C-terminal tails. Results: The acidic tails of HMG-D (Drosophila) and ZmHMGB1 (maize) occlude the DNA-binding regions; the strength of the interaction is phosphorylation-dependent. Conclusion: Phosphorylation provides an additional mode of negative regulation. Significance: Phosphorylation of the tails of insect and plant HMG-box proteins may modulate chromatin structure and accessibility. The Drosophila and plant (maize) functional counterparts of the abundant vertebrate chromosomal protein HMGB1 (HMG-D and ZmHMGB1, respectively) differ from HMGB1 in having a single HMG box, as well as basic and acidic flanking regions that vary greatly in length and charge. We show that despite these variations, HMG-D and ZmHMGB1 exist in dynamic assemblies in which the basic HMG boxes and linkers associate with their intrinsically disordered, predominantly acidic, tails in a manner analogous to that observed previously for HMGB1. The DNA-binding surfaces of the boxes and linkers are occluded in auto-inhibited forms of the protein, which are in equilibrium with transient, more open structures that are binding-competent. This strongly suggests that the mechanism of auto-inhibition may be a general one. HMG-D and ZmHMGB1 differ from HMGB1 in having phosphorylation sites in their tail and linker regions. In both cases, in vitro phosphorylation of serine residues within the acidic tail stabilizes the assembled form, suggesting another level of regulation for interaction with DNA, chromatin, and other proteins that is not possible for the uniformly acidic (hence unphosphorylatable) tail of HMGB1.