The HMGB1 C-Terminal Tail Regulates DNA Bending.

High mobility group box protein 1 (HMGB1) is an architectural protein that facilitates the formation of protein-DNA assemblies involved in transcription, recombination, DNA repair, and chromatin remodeling. Important to its function is the ability of HMGB1 to bend DNA non-sequence specifically. HMGB1 contains two HMG boxes that bind and bend DNA (the A box and the B box) and a C-terminal acidic tail. We investigated how these domains contribute to DNA bending by HMGB1 using single-molecule fluorescence resonance energy transfer (FRET), which enabled us to resolve heterogeneous populations of bent and unbent DNA. We found that full-length (FL) HMGB1 bent DNA more than the individual A and B boxes. Removing the C-terminal tail resulted in a protein that bent DNA to a greater extent than the FL protein. These data suggest that the A and B boxes simultaneously bind DNA in the absence of the C-terminal tail, but the tail modulates DNA binding and bending by one of the HMG boxes in the FL protein. Indeed, a construct composed of the B box and the C-terminal tail only bent DNA at higher protein concentrations. Moreover, in the context of the FL protein, mutating the A box such that it could not bend DNA resulted in a protein that bent DNA similar to a single HMG box and only at higher protein concentrations. We propose a model in which the HMGB1 C-terminal tail serves as an intramolecular damper that modulates the interaction of the B box with DNA.