Study of DNA binding and bending by Bacillus subtilis GabR, a PLP-dependent transcription factor

• Published in: Biochimica et biophysica acta. General subjects Vol. 1861; no. 1; pp. 3474 - 3489
• Main Authors: Amidani, Davide, Tramonti, Angela, Canosa, Andrea Valeria, Campanini, Barbara, Maggi, Stefano, Milano, Teresa, di Salvo, Martino L., Pascarella, Stefano, Contestabile, Roberto, Bettati, Stefano, Rivetti, Claudio
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2017
• Subjects: Atomic Force Microscopy; Bacillus subtilis; Bacillus subtilis - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Base Sequence; Circular Dichroism; DNA; DNA, Bacterial - chemistry; DNA, Bacterial - metabolism; DNA-binding domains; electrophoresis; GABA metabolism; gamma-aminobutyric acid; gamma-Aminobutyric Acid - metabolism; genes; in vitro studies; mechanism of action; Microscopy, Atomic Force; MocR; Models, Molecular; Mutant Proteins - metabolism; mutants; nitroprusside; Nucleic Acid Conformation; oligomerization; PLP enzyme; Promoter Regions, Genetic - genetics; Protein Binding; Protein Domains; pyridoxal; pyridoxal phosphate; Pyridoxal Phosphate - metabolism; Repetitive Sequences, Nucleic Acid - genetics; Sequence Alignment; Spectrophotometry, Ultraviolet; Static Electricity; stoichiometry; transaminases; Transcription factor; transcription factors; Transcription Factors - metabolism; Atomic Force Microscopy; Transcription factor; MocR; PLP enzyme; GABA metabolism
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2016.09.013

GabR is a transcriptional regulator belonging to the MocR/GabR family, characterized by a N-terminal wHTH DNA-binding domain and a C-terminal effector binding and/or oligomerization domain, structurally homologous to aminotransferases (ATs). In the presence of γ-aminobutyrate (GABA) and pyridoxal 5′-phosphate (PLP), GabR activates the transcription of gabT and gabD genes involved in GABA metabolism. Here we report a biochemical and atomic force microscopy characterization of Bacillus subtilis GabR in complex with DNA. Complexes were assembled in vitro to study their stoichiometry, stability and conformation. The fractional occupancy of the GabR cognate site suggests that GabR binds as a dimer with Kd of 10nM. Upon binding GabR bends the DNA by 80° as measured by anomalous electrophoretic mobility. With GABA we observed a decrease in affinity and conformational rearrangements compatible with a less compact nucleo-protein complex but no changes of the DNA bending angle. By employing promoter and GabR mutants we found that basic residues of the positively charged groove on the surface of the AT domain affect DNA affinity. The present data extend current understanding of the GabR-DNA interaction and the effect of GABA and PLP. A model for the GabR-DNA complex, corroborated by a docking simulation, is proposed. Characterization of the GabR DNA binding mode highlights the key role of DNA bending and interactions with bases outside the canonical direct repeats, and might be of general relevance for the action mechanism of MocR transcription factors. [Display omitted] •GabR binds two DNA direct repeats as a dimer and bends the cognate site by ~80°.•Proper phasing of direct repeats is crucial for GabR binding.•PLP is not required for the GabR-DNA interaction.•GABA reduces GabR-DNA affinity and alters slightly the shape of the complexes.•Basic residues of the AT domain affect GabR-DNA binding affinity.