Current Research Summaries

  • [Crystallization] Expression, purification, crystallization, and X-ray diffraction studies on a PadR-like protein from Bacillus cereus

    Transcriptional regulators that belong to the PadR family play a critical role in the regulation of various biological processes, such as detoxification, catabolism, toxin production, and antibiotic resistance. The structural basis of effector and DNA recognition by PadR has recently been provided through our previous structural study. However, it is unclear whether the mechanism is universally used by other PadR family members. As a first step to reveal the transcription regulatory mechanism of Bacillus cereus PadR-like protein (bcPLP), we expressed the bcPLP protein in Escherichia coli cells and purified it to homogeneity by chromatographic methods. bcPLP was crystallized in PEG 3350 solutions. A bcPLP crystal diffracted X-ray to 1.95Å resolution. Our analysis of the X-ray diffraction data indicates that the crystal belongs to space group P21212 and has two bcPLP chains in the asymmetric unit.

  • [Crystallization] Purification, crystallization and X-ray crystallographic analysis of Csm1/Csm4 subcomplex in CRISPR/Cas Type III-A system

    The CRISPR/Cas system is an adaptive prokaryotic immune response that defends against exogenous genetic elements. The CRISPR/Cas Type III-A system targets RNA and DNA that is coupled with transcription. The effector complex of the Type III-A CRISPR-Cas system has five Csm components, Csm1~Csm5. The Csm1/Csm4 sub-complex is placed in the crRNA 5' ends of the effector RNP complex and likely involved in the discriminative function for self vs non-self DNA. Here, we prepared Csm1/Csm4 proteins from Thermococcus onnurineus NA1 and crystallized the sub-complex. The Csm1/Csm4 was crystallized using hanging-drop vapor diffusion from a reservoir solution containg 200 mM sodium chloride, 100 mM sodium acetate trihydrate, pH 4.6, 26% (+/-)-2-methyl-2,4-pentanediol. The diffraction data to 2.8 Å resolution show that the crystal belongs to the space group P6522 with unit cell parameters of a=154.91Å, b=154.91Å, c=182.25Å, α=β=90° and γ=120°.

  • [Crystallization] Purification and preliminary analysis of the regulatory domain of MexT from Pseudomonas aeruginosa, a LysR-type transcriptional activator of the MexEF-OprN multidrug efflux pump

    Pseudomonas aeruginosa is an opportunistic pathogen, and have multiple multidrug efflux pumps. The MexEF-OprN multidrug efflux system is overexpressed in nfxC-type mutants and give resistance to quinolones, chloramphenicol and trimethoprim. A LysR-type transcriptional activator, MexT, is the major activator of this efflux system. Although the activity of MexT is regulated in response to the cellular redox state, the ligand and activation mechanism remain unknown yet. MexT consists of the N-terminal DNA binding domain and the C-terminal regulatory domain that contains the ligand binding site. In this study, we overproduced and purified the regulatory domain of MexT from P. aeruginosa and obtained the crystals suitable for the structural study. The crystal diffracted X-ray to 2.3 Å resolution, revealing that the crystals belong to space group P212121, with unit cell parameters a = 65.7, b = 108.5, and c = 109.1 Å. The cell content analysis suggested that 3 or 4 molecules are contained in the asymmetric unit. To solve the phasing problem, growing of the Se- Met substituted crystals are underway. This structure will give insight into the molecular mechanism of the activation of MexT in the presence of antibiotics.

  • [Crystallization] Purification, crystallization and X-ray crystallographic analysis of enoyl-CoA hydratase/isomerase-family protein from Cupriavidus necator H16

    H16_B0756 from Cupriavidus necator H16 is a putative enoyl-coenzyme A (CoA) hydratase/isomerase-family protein. Enoyl-CoA hydratase (ECH) in β-oxidation might provide hydroxyalkanoate monomers for PHA production. However, β-oxidation pathway and ECH family enzymes in C. necator have not been fully characterized. To identify the function of H16_B0756, the protein was overexpressed and purified to homogeneity by affinity and size-exclusion chromatography. The H16_B0756 protein was crystallized using hanging-drop vapor-diffusion method in the presence of 30% polyethylene glycol 550 monomethyl ether, 0.1 M sodium chloride and 0.1 M bicine, pH 9.0 at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.0 Å. The crystal belonged to space group P3, with unit cell parameters a = b = 132.94 Å, c = 44.16, α = β = 90.0°, γ = 120.0°. With two molecules per asymmetric unit, the crystal volume per unit protein mass was 1.94 Å3 Da-1, which correspond to a solvent content of approximately 36.80%.