Crystallization and preliminary X-ray diffraction analysis of a fatty-acid metabolism regulatory protein, FadR, fromBacillus halodurans

Abstract

FadR is an acyl-CoA-dependent transcription factor which regulates genes encoding proteins involved in fatty-acid degradation and synthesis in order to maintain lipid homeostasis. FadR from the alkaliphilic bacterium Bacillus halodurans was cloned and overexpressed in Escherichia coli. The FadR (Bh3102) protein from B. halodurans is composed of 195 amino-acid residues with a molecular mass of 22 378 Da. Crystals were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.05 Å resolution. FadR was crystallized at 296 K using polyethylene glycol 3350 as a precipitant. The crystal belonged to the apparent trigonal space group P3 2 21, with unit-cell parameters a = b = 56.34, c = 199.73 Å . The Matthews coefficient and solvent content were estimated to be 2.0 Å 3 Da À1 and 39.8%, respectively, assuming that the asymmetric unit contained two molecules of FadR, which was subsequently confirmed by molecular-replacement calculations.

Structural basis of operator sites recognition and effector binding in the TetR family transcription regulator FadR

“…Gene cloning, expression and purification of B. halodurans FadR were conducted as previously described ( 31 ). Native FadR was expressed in E. coli BL21 (DE3) Star pLysS cells.…”

“…A 1% NP-40 solution was added during lysis to remove lipid molecules from native FadR, and the remainder of the purification procedure was the same as that for native FadR. The insoluble fraction including cellular debris was removed by centrifugation at 31 000 g for 60 min at 277 K, and the recombinant protein in the supernatant fraction was purified using three chromatographic steps described previously in detail ( 31 ). The purified proteins were concentrated to 70 mg/ml using the Centricon YM-10 (Millipore, USA) and stored at 193 K.…”

“…Native FadR crystals were obtained by the sitting-drop vapor diffusion method in a reservoir containing 0.1 M Tris–HCl at pH 8.5, 0.3 M MgCl 2 and 25% PEG 3350. The detailed crystallization procedure for native FadR has been described previously ( 31 ). Native FadR crystals contained fatty acids derived from E. coli during purification and verified as a mixture of three fatty acids (myristic acid, palmitic acid and stearic acid) by gas chromatography-mass spectrometry (GC-MS).…”

Structural characterization of a ligand‐bound form of Bacillus subtilis FadR involved in the regulation of fatty acid degradation

“…The amino acid sequence identity between FadR Bs and FadR Tt is 19%, and that between the ligand‐binding domain of the two regulators is only 15%. Crystallization of a FadR Bs homologue from Bacillus halodurans, whose amino acid sequence is 65% identical to that of FadR Bs , has been reported, although its structure has not been determined to date. To elucidate the structural mechanism underlying the regulation of FadR Bs , we performed a structure analysis of FadR Bs with and without stearoyl‐CoA as the ligand molecule.…”