Distinct domains in HMGB1 are involved in specific intramolecular and nucleosomal interactions.

HMGB1 is composed of two DNA-binding domains and a long acidic tail at the C-terminus. The acidic tail interacts with the DNA-binding domains of HMGB1 and with core histone H3 in the nucleosome. These interactions are important for modulation of the DNA and chromatin binding activities of HMGB1, as well as biological functions of HMGB1. However, the interactions are not fully characterized, because the tertiary structure of full-length HMGB1 is still unknown. Here we use chemical cross-linking, mass spectrometry, and epitope masking analysis to perform a detailed characterization of the inter- and intramolecular protein interactions of the acidic tail of HMGB1. We show that specific regions of the acidic tail participate in intramolecular interactions with Lys2 of HMGB1 and in intermolecular interactions with Lys36 and Lys37 of histone H3. The acidic tail is oriented by its location adjacent to the C-terminus of helix III of DNA-binding HMG box A in the HMGB1 molecule. These results suggest that the acidic tail modulates the biological functions of HMGB1 through these specific interactions.