Mechanism of DNA binding of an unusual bacterial transcription regulator.

GabR is a chimeric transcription factor from Bacillus subtilis belonging to the GntR family of transcription factors and controls genes involved in the metabolism of gamma-aminobutyric acid (GABA). The crystal structure of GabR reveals a head-to-tail domain swap homodimer whereby the C-terminal aminotransferase I-like domains form a dimeric core with the N-terminal winged helix-turn-helix DNA (wHTH) binding domains located at opposing sides of the dimer. This unusual structure has raised the questions about the structural changes required for DNA binding and the switch from transcription repressor state to activator state upon binding of the effector molecule GABA. Here, we show that GabR binds to its cognate direct repeat sequence in the promoter region as a dimer to form both the repressive and the activating complex and ruled out changes in stoichiometry as part of regulatory mechanism. Small angle X-ray scattering (SAXS) showed that the solution structure of the GabR dimer is consistent with the crystal structure and that binding of GABA resulted in small structural changes contrary to models predicting large conformational changes for DNA binding. The SAXS data of the GabR-DNA complex were consistent with a model in which the DNA duplex undergoes an unexpected conformational change during complex formation: The DNA molecule bends around the dimeric core such that the direct repeat sequences can simultaneously contact the helix-turn-helix domains on opposite sides of the GabR dimer without the need for major conformational changes of the protein. Analysis of the binding and unbinding kinetics of the GabR to wild type DNA and a range of mutants showed that (1) the complex is only stable when both helix-turn-helix domains can bind to their cognate direct repeat sequences and (2) the sequence of DNA in between the repeat sequences has evolved to contain a particular pattern of alternating regions of high and low bendability that generates intrinsic curvature in the DNA in the correct direction to facilitate GabR binding. Thus, both direct readout mechanisms involving the contacts of the helix-turn-helix domains with the bases in the repeat sequences and indirect readout mechanisms relying on the shape complementarity of the DNA to the protein are essential for GabR-DNA complex formation