Acta Crystallographica Section F-structural Biology and Crystallization Communications最新文献

α(1)-Microglobulin (α(1)m) is one of the phylogenetically most widespread lipocalins and is distributed in various organs and tissues, including liver, heart, eye, kidney, brain, lung, pancreas and skeletal muscle. α(1)m has been found to exert multifarious functions, including interacting with IgA, albumin and prothrombin, binding strongly to haem and exhibiting reductase activity. Nevertheless, little structural information is available regarding these functions of α(1)m. Since determination of three-dimensional structure is a powerful means of functional characterization, X-ray crystallography was used to accomplish this task. Here, the expression, purification, crystallization and preliminary crystallographic analysis of human α(1)m are reported. The crystal belonged to space group P4(3), with unit-cell parameters a = b = 36.45, c = 112.68 Å, and diffracted to a resolution of 2.0 Å. The crystals are most likely to contain one molecule in the asymmetric unit, with a V(M) value of 1.63 Å(3) Da(-1).

The core domain of a human histidine decarboxylase mutant was purified and cocrystallized with the inhibitor L-histidine methyl ester. Using synchrotron radiation, a data set was collected from a single crystal at 100 K to 1.8 Å resolution. The crystal belonged to space group C2, with unit-cell parameters a = 215.16, b = 112.72, c = 171.39 Å, β = 110.3°. Molecular replacement was carried out using the structure of aromatic L-amino-acid decarboxylase as a search model. The crystal contained three dimers per asymmetric unit, with a Matthews coefficient (V(M)) of 3.01 Å(3) Da(-1) and an estimated solvent content of 59.1%.

Bag2, an atypical member of the Bag family of Hsp70 co-chaperones, acts as both an Hsp70 nucleotide-exchange factor and an inhibitor of the Hsp70-binding E3 ubiquitin ligase CHIP (carboxyl-terminus of Hsp70-interacting protein). The amino-terminal domain of Bag2 (Bag2-NTD), which is required for inhibition of CHIP, has no sequence homologs in the PDB. Native and selenomethionyl (SeMet) forms of Bag2-NTD were crystallized by hanging-drop vapor diffusion. Native Bag2-NTD crystals diffracted to 2.27 Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 75.5, b = 84.7, c = 114.1 Å. SeMet Bag2-NTD crystals diffracted to 3.10 Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 37.2, b = 53.3, c = 86.7 Å. Phases for the SeMet Bag2-NTD crystal were solved by single-wavelength anomalous diffraction. Initial phasing and model building using the 3.10 Å resolution SeMet Bag2-NTD data set suggested that Bag2-NTD forms a dimer and adopts a fold distinct from those of any domains annotated in the Pfam or SMART domain databases.

p-Hydroxyphenylacetate 3-hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) at the ortho position to yield 3,4-dihydroxyphenylacetate (DHPA). HPAH from A. baumannii is a two-component flavoprotein consisting of a smaller reductase (C(1)) component and a larger oxygenase (C(2)) component. The C(1) component supplies a reduced flavin in its free form to the C(2) counterpart for hydroxylation. In addition, HPA can bind to C(1) and enhance the flavin-reduction rate without becoming hydroxylated. The recombinant C(1) component was purified and crystallized using the microbatch method at 295 K. X-ray diffraction data were collected to 2.3 Å resolution using synchrotron radiation on the BL13B1 beamline at NSRRC, Taiwan. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 47.78, b = 59.92, c = 211.85 Å, and contained two molecules of C(1) per asymmetric unit.

Haloalkane dehalogenases are enzymes that catalyze the hydrolytic reaction of a wide variety of haloalkyl substrates to form the corresponding alcohol and hydrogen halide products. DatA from Agrobacterium tumefaciens C58 is a haloalkane dehalogenase that has a unique pair of halide-binding residues, asparagine (Asn43) and tyrosine (Tyr109), instead of the asparagine and tryptophan that are conserved in other members of the subfamily. DatA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with a reservoir solution consisting of 0.1 M CHES pH 8.6, 1.0 M potassium sodium tartrate, 0.2 M lithium sulfate, 0.01 M barium chloride. X-ray diffraction data were collected to 1.70 Å resolution. The space group of the crystal was determined as the primitive tetragonal space group P422, with unit-cell parameters a = b = 123.7, c = 88.1 Å. The crystal contained two molecules in the asymmetric unit.

Cerebral cavernous malformation 2 (CCM2) is a novel two-domain adaptor protein which participates in multiple cellular signalling pathways. Loss-of-function mutations in the gene encoding CCM2 are the cause of common human vascular lesions called cerebral cavernous malformations. Here, the purification, crystallization and preliminary X-ray crystallographic studies of the C-terminal domain of CCM2 (CCM2-Ct) are reported. Diffraction data were collected from native and selenomethionine-substituted crystals of CCM2-Ct to resolutions of 2.9 and 2.7 Å, respectively. Both crystals belonged to space group I4(1)22 with similar unit-cell parameters. The native crystals had unit-cell parameters a = b = 113.29, c = 101.62 Å.

Aspergillus fumigatus UDP-galactopyranose mutase (AfUGM) is a potential drug target involved in the synthesis of the cell wall of this fungal pathogen. AfUGM was recombinantly produced in Escherichia coli, purified and crystallized by the sitting-drop method, producing orthorhombic crystals that diffracted to a resolution of 3.25 Å. The crystals contained four molecules per asymmetric unit and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 127.72, b = 134.30, c = 173.84 Å. Incorporation of selenomethionine was achieved, but the resulting crystals did not allow solution of the phase problem.

Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNA(Sec)) is first ligated with serine by seryl-tRNA synthetase (SerRS). To elucidate the tertiary structure of bacterial tRNA(Sec) and its specific interaction with SerRS, the bacterial tRNA(Sec) from Aquifex aeolicus was crystallized as the heterologous complex with the archaeal SerRS from Methanopyrus kandleri. Although X-ray diffraction by crystals of tRNA(Sec) in complex with wild-type SerRS was rather poor (to 5.7 Å resolution), the resolution was improved by introducing point mutations targeting the crystal-packing interface. Heavy-atom labelling also contributed to resolution improvement. A 3.2 Å resolution diffraction data set for phase determination was obtained from a K(2)Pt(CN)(4)-soaked crystal.

The expression, purification, crystallization and preliminary X-ray diffraction characterization of malate dehydrogenase (MDH) from the malarial parasite Plasmodium falciparum (PfMDH) are reported. In order to gain a deeper understanding of the function and role of PfMDH, the protein was purified to homogeneity. The purified protein crystallized in space group P1, with unit-cell parameters a = 72, b = 157, c = 159 Å, α = 105, β = 101, γ = 95°. The resulting crystals diffracted to a maximal resolution of 2.24 Å and the structure has been solved by molecular replacement, with 16 monomers in the asymmetric unit. The 16 monomers are arranged into four independent tetramers, in agreement with previous reports demonstrating the tetrameric solution state of PfMDH. The X-ray structure of PfMDH is expected to clarify the differences in catalysis by PfMDH compared with other MDH family members and to provide a basis for the structure-based design of specific PfMDH inhibitors as well as general MDH inhibitors.

The removal of chemically damaged DNA bases such as 3-methyladenine (3-MeA) is an essential process in all living organisms and is catalyzed by the enzyme 3-MeA DNA glycosylase I. A key question is how the enzyme selectively recognizes the alkylated 3-MeA over the much more abundant adenine. The crystal structures of native and Y16F-mutant 3-MeA DNA glycosylase I from Staphylococcus aureus in complex with 3-MeA are reported to 1.8 and 2.2 Å resolution, respectively. Isothermal titration calorimetry shows that protonation of 3-MeA decreases its binding affinity, confirming previous fluorescence studies that show that charge-charge recognition is not critical for the selection of 3-MeA over adenine. It is hypothesized that the hydrogen-bonding pattern of Glu38 and Tyr16 of 3-MeA DNA glycosylase I with a particular tautomer unique to 3-MeA contributes to recognition and selection.