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. [Display omitted] •HMGB1 is a multidomain protein that binds and bends DNA non-sequence specifically.•DNA bending by HMGB1 domains was monitored using single-molecule FRET.•FL HMGB1 bent DNA slightly more than the A box and B box individually.•Deleting the HMGB1 C-terminal tail caused DNA to be bent into a higher FRET state.•Data support the model that the C-terminal tail impedes DNA binding by the B box.