Purification, crystallization and X-ray crystallographic analysis of ATPase domain in transcription regulator DmpR

DmpR is a Pseudomonas-derived σ54-dependent transcription regulator in the presence of aromatic compounds. As a single component regulator and a bacterial enhancer binding protein in the AAA+ ATPase family, the activation mechanism of the DmpR protein has long remained elusive. The AAA+ ATPase domain of the protein is known to be essential in its oligomerization and interaction with the RNA polymerase sigma factor σ54. However, the structure of the ATPase domain of DmpR is still unknown. Thus, we performed a purification, crystallization and preliminary X-ray crystallographic study of the ATPase domain (residues 232−480) of DmpR from Pseudomonas putida KCTC 1452. The ATPase domain was crystallized using hanging-drop vapor diffusion from a reservoir solution containing 20% w/v PEG 3350, 280 mM Ammonium sulfate, and 100 mM Tris pH 8.5 and 30% 1,6-Hexanediol. The diffraction data to a ~3.0 Å resolution show that the crystal belongs to the space group P622 with unit cell parameters of a = 104.366 Å, b = 104.366 Å, c = 46.865 Å, α = β = 90° and γ = 120°. The structure of the ATPase would help to understand the DmpR oligomerization mechanism and interaction with σ54 in transcriptional initiation.