Purification, crystallization and X-ray crystallographic analysis of ATPase domain in transcription regulator DmpR = 전사 조절자 DmpR ATPase 도메인의 정제, 결정화 및 X선 결정학적 분석

Abstract

DmpR is a Pseudomonas-derived σ54-dependent transcription regulator in the presence of aromatic compounds. As asingle component regulator and a bacterial enhancer binding protein in the AAA+ ATPase family, the activation mechanismof the DmpR protein has long remained elusive. The AAA+ ATPase domain of the protein is known to be essential in itsoligomerization and interaction with the RNA polymerase sigma factor σ54. However, the structure of the ATPase domain ofDmpR is still unknown. Thus, we performed a purification, crystallization and preliminary X-ray crystallographic study of theATPase domain (residues 232？480) of DmpR from Pseudomonas putida KCTC 1452. The ATPase domain was crystallizedusing 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 A resolution show that the crystal belongsto the space group P622 with unit cell parameters of a = 104.366 A, b = 104.366 A, c = 46.865 A, α = β = 90° and γ = 120°. The structure of the ATPase would help to understand the DmpR oligomerization mechanism and interaction with σ54 intranscriptional initiation.