Current Research Summaries

  • [Crystallization] Crystallization and X-ray crystallographic analysis of the C-terminal domain of Bacillus subtilis GabR in complex with pyridoxal 5'-phosphate and γ-aminobutyric acid

    Bacillus subtilis GabR (BsGabR) functions as a transcriptional activator that regulates the expression of gabTD operon involved in the γ-aminobutyric acid (GABA) catabolism. Most studies of BsGabR have been focused on the structurefunctional relationship regarding the Cterminal aminotransferase-like domain of BsGabR (BsGabR-CTD), but it still remains unclear due to lack of the structural information containing the GABA. To better understand the role of this domain, BsGabR-CTD was purified and crystallized in complex with pyridoxal 5'-phosphate and GABA. The crystal of ternary complex diffracted to a resolution of 2.0 Å and belonged to the tetragonal space group P41, with unit-cell parameters a = b = 118.497, c = 75.862 Å. Preliminary molecular replacement further confirmed the presence of one dimer in the asymmetric unit with a Matthews coefficient (VM) of 2.86 Å3 Da-1, corresponding to a solvent content of 56.9%.

  • [Crystallization] Crystallization and preliminary crystallographic analysis of a zincdependent alcohol dehydrogenase from Streptococcus pneumoniae

    Alcohol dehydrogenase (ADH) is one of essential enzymes for all living organisms, and conserved from archaea to mammals. The most abundant type of ADH is zinc-containing one. All the zinc containing ADHs possess the catalytic zinc site, while some have the second, structural zinc site. ADHs require either NAD(H) or NADP(H) as a cofactor. Despite lots of efforts to determine the key residues for cofactor specificity of ADHs, there is no general rule for cofactor specificity so far. To establish the general rules for the cofactor specificity of ADHs by structural analysis, we cloned and overexpressed the zinc dependent alcohol dehydrogenase from Streptococcus pneumoniae strain D39 (SpADH2) in Escherichia coli. Rod-shape crystals of SpADH2 were obtained by hanging-drop vapour diffusion from a reservoir solution containing 50 mM Tris-HCl pH 8.0, 50 mM NaCl, PEG 3350 15% (w/v) and diffracted to 2.19 Å resolution. Crystal belonged to orthorhombic space group P212121 with unit cell parameters a = 88.2 Å, b = 123.2 Å, c = 130.4 Å and contained four molecules in the asymmetric unit.

  • [Crystallization] Crystallization and X-ray crystallographic analysis of the PH-like domain of lipid transfer protein anchored at membrane contact sites from Saccharomyces cerevisiae

    Lam6 is a member of sterol-specific lipid transfer proteins anchored at membrane contact sites (LAMs). Lam6 localizes to the ER-mitochondria contact sites by its PH-like domain and the C-terminal transmembrane helix. Here, we purified and crystallized the Lam6 PH-like domain from Saccharomyces cerevisiae. To aid crystallization of the Lam6 PH-like domain, T4 lysozyme was fused to the N-terminus of the Lam6 PH-like domain with a short dipeptide linker, GlySer. The fusion protein was crystallized under the condition of 0.1 M HEPES-HCl pH 7.0, 10% (w/v) PEG 8000, and 0.1 M Na3Citrate at 293K. X-ray diffraction data of the crystals were collected to 2.4 Å resolution using synchrotron radiation. The crystals belong to the orthorhombic space group P212121 with unit cell parameters a = 59.5 Å, b = 60.1 Å, and c = 105.6 Å. The asymmetric unit contains one T4L-Lam6 molecule with a solvent content of 58.7%. The initial attempt to solve the structure by molecular replacement using the T4 lysozyme structure was successful.

  • [Crystallization] Crystallization and preliminary diffraction analysis of DUSP28 through identification of a pseudo-thrombin cleavage site

    Dual specificity protein phosphatases (DUSPs) belong to the protein tyrosine phosphatase (PTP) family. DUSPs dephosphorylate both phospho-serine/threonine and phospho-tyrosine of mitogen activated protein kinases (MAPKs) and play important roles in cell growth, regulation and signaling. DUSP28, a member of the atypical DUSPs, has dephosphorylation activity towards proteins involved in cellular signaling processes. DUSP28 is also implicated in the development of pancreatic cancer and liver cancer. The atomic resolution structure of DUSP28 should help the structurebased design of specific and potent therapeutics. However, the structure and detailed function of DUSP28 have not been elucidated yet. Here, we prepared a large quantity of DUSP28 protein and crystallized the protein. During the protein preparation, we encountered an unexpected proteolytic cleavage in the middle of the protein domain and overcame the problem by identifying and mutating the pseudo-thrombin cleavage site. By using the purified protein, we were able to grow diffraction quality crystals and collected a 2.1 Å resolution diffraction data. The preliminary diffraction analysis revealed that the crystal is in the space group P3121 with unit cell parameters of a = 78.85 Å, b = 78.85 Å, c = 90.26 Å, α = 90.00°, β = 90.00° and γ = 120.00°