Search

The smu.1420 gene from the cariogenic pathogen Streptococcus mutans encodes a putative protein which has sequence homology to NQO [NAD(P)H:quinone oxidoreductase] family members, including mammalian NQO and bacterial MdaB (modulator of drug activity B). NQO can detoxify quinones by converting them to hydroquinones and prevent the generation of reactive oxygen species. Thus, comprehensive studies on Smu.1420 will be important for uncovering the antioxidation and antidrug mechanisms of S. mutans. Here, the catalytic properties of Smu.1420 have been characterized, and its structure was determined in complexes with NADP+ and menadione, respectively. Smu.1420 binds menadione directly and exhibits a pronounced preference for NADPH over NADH as a substrate, demonstrating that it is an NADPH- specific quinone oxidoreductase. The structure of Smu.1420 shows a compact homodimer with two substrate pockets located in the cleft of the dimer interface. The nicotinamide moiety of NADP+ is bound on top of the isoalloxazine moiety of the FAD cofactor and overlaps with the binding site of menadione, suggesting a hydride-transfer process from NADPH to FAD and then to menadione. Two strongly basic patches near the substrate pocket are expected to confer the preference for NADPH over NADH. These studies shed light on future drug development against the cariogenic pathogen S. mutans. [ABSTRACT FROM AUTHOR]

Caspase 6 (CASP6) is a neuron degeneration-related protease and is widely considered to be a potential drug-design target against neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. The N-terminal pro-peptide of CASP6, also referred to as the pro-domain, contains 23 residues and its functional role remains elusive. In this study, the crystal structure of a full-length CASP6 zymogen mutant, proCASP6H121A, was solved. Although the pro-domain was flexible in the crystal, without visible electron density, structural analyses combined with biochemical assays revealed that the pro-domain inhibited CASP6 auto-activation by inhibiting intramolecular cleavage at the intersubunit cleavage site TEVD193 and also by preventing this site from intermolecular cleavage at low protein concentration through a so-called 'suicide-protection' mechanism. Further experiments showed that the length of the pro-domain and the side chain of Asn18 played critical roles in suicide protection. These results disclosed a new inhibitory mechanism of CASP6 and shed light on the pathogenesis and therapeutically relevant study of CASP6-related neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Sulfur single-wavelength anomalous dispersion (S-SAD) and halide-soaking methods are increasingly being used for a,b initio phasing. With the introduction of in-house Cr X-ray sources, these methods benefit from the enhanced anomalous scattering of S and halide atoms, respectively. Here, these methods were combined to determine the crystal structure of BsDegV, a DegV protein-family member from Bacillus subtilis. The protein was cocrystallized with bromide and low-redundancy data were collected to 2.5 Å resolution using Cr Kα radiation. 17 heavy-atom sites (ten sulfurs and seven bromides) were located using standard methods. The anomalous scattering of some of the BsDegV S atoms and Br atoms was weak, thus neither sulfurs nor bromides could be used alone for structure determination using the collected data. When all 17 heavy-atom sites were used for SAD phasing, an easily interpretable electron-density map was obtained after density modification. The model of BsDegV was built automatically and a palmitate was found tightly bound in the active site. Sequence alignment and comparisons with other known DegV structures provided further insight into the specificity of fatty-acid selection and recognition within this protein family. [ABSTRACT FROM AUTHOR]

As part of a structural genomics platform in a university laboratory, a low-cost in-house-developed automated imaging system for SBS microplate experiments has been designed and constructed. The imaging system can scan a microplate in 2–­6 min for a 96-well plate depending on the plate layout and scanning options. A web-based crystallization database system has been developed, enabling users to follow their crystallization experiments from a web browser. As the system has been designed and built by students and crystallographers using commercially available parts, this report is aimed to serve as a do-it-yourself example for laboratory robotics. [ABSTRACT FROM AUTHOR]

A large-scale, high-efficiency and low-cost platform based on a Beckman Coulter Biomek FX and custom-made automation systems for structural genomics has been set up at Peking University, Beijing, People's Republic of China. This platform has the capacity to process up to 2000 genes per year for structural and functional analyses. Bacillus subtilis, a model organism for Gram-positive bacteria, and Streptococcus mutans, a major pathogen of dental caries, were selected as the main targets. To date, more than 470 B. subtilis and 1200 S. mutans proteins and hundreds of proteins from other sources, including human liver proteins, have been selected as targets for this platform. The selected genes are mainly related to important metabolism pathways and/or have potential relevance for drug design. To date, 40 independent structures have been determined; of these 11 are in the category of novel structures by the criterion of having less than 30% sequence identity to known structures. More than 13 structures were determined by SAD/MAD phasing. The macromolecular crystallography beamline at the Beijing Synchrotron Radiation Facility and modern phasing programs have been crucial components of the operation of the platform. The idea and practice of the genomic approach have been successfully adopted in a moderately funded structural biology program and it is believed this adaptation will greatly improve the production of protein structures. The goal is to be able to solve a protein structure of moderate difficulty at a cost about US $10 000. [ABSTRACT FROM AUTHOR]

54 human genes were selected as test targets for parallel cloning, expression, purification and crystallization. Proteins from these genes were selected to have a molecular weight of between 14 and 50 kDa, not to have a high percentage of hydrophobic residues (i.e. more likely to be soluble) and to have no known crystal structures and were not known to be subunits of heterocomplexes. Four proteins containing transmembrane regions were selected for comparative tests. To date, 44 expression clones have been constructed with the Gateway™ cloning system (Invitrogen, The Netherlands). Of these, 35 clones were expressed as recombinant proteins in Escherichia coli strain BL21 (DE3)-pLysS, of which 12 were soluble and four have been purified to homogeneity. Crystallization conditions were screened for the purified proteins in 96-well plates under oil. After further refinement with the same device or by the hanging-drop method, crystals were grown, with needle, plate and prism shapes. A 2.12 Å data set was collected for protein NCC27. The results provide insights into the high-throughput target selection, cloning, expression and crystallization of human genomic proteins. [ABSTRACT FROM AUTHOR]

The adrenal gland protein AD-004 was identified in the human adrenal gland. Full-length AD-004 contains 172 amino acids, with a predicted molecular weight of about 20 kDa. In attempts to crystallize human AD-004, the gene was subcloned into a modified pET vector, pET21-DEST, with an N-terminal His5 tag using the Gateway cloning system, followed by protein expression in Escherichia coli strain BL21(DE3). The protein was purified in two steps to near-homogeneity and was crystallized. The crystals belong to space group P61 or P65, with unit-cell parameters a = b = 99.56, c = 57.19 Å. A complete 2.0 Å data set has been collected at a rotating-anode X-ray source and structure determination is under way. [ABSTRACT FROM AUTHOR]

Describes the expression, purification and crystallization of an extended-spectrum Β-lactamase from Klebsiella oxytoca. Complete data sets collected from both crystal forms; Unit cell parameters of orthorhombic crystals; Asymmetric unit of the orthorhombic crystal estimated to contain one OXY-1a molecule.

Native and His-tagged mutant (L1601) hyoxanthine-guanine phosphoribosyltransferase (HGPRT) from Thermoanaerobacter tengcongensis were cloned. Expressed in Escherichia coli and purified. Both proteins were crystallized with polyethylene glycol as the mainprecipitant at 293 K using the hanging-drop vapour-diffusion method. The crystal of native HGPRT belongs to space group C222[SUB1], with unit-cell parameters a = 65.77, b = 137.73, c = 95.27 Å, and diffracted to 2.2 Å resolution on an inhouse X-ray generator. The crystal of the His-tagged mutant (L1601) HGPRT belongs to the space group I222, with unit-cell parameters a = 52.21, b = 88.36, c = 93.03 Å and diffracted to 1.7 Å resolution in-house. [ABSTRACT FROM AUTHOR]

A novel calcium-independent serine protease from an alkaliphilic bacterium, Nesterenkonia sp. AL20, has been purified and crystallized at 296 K using sodium formate as the main precipitant. This enzyme is optimally active at pH 10, exhibits high stability towards autolytic digestion and its stability is not affected by the presence of EDTA or detergents. The triangular prism-shaped crystals diffracted X-rays to beyond 1.5 Å at a synchrotron beamline, with space group R3 and unit-cell parameters a = b = 92.26, c = 137.88 Å. A complete data set has been collected to 1.39 Å resolution. The asymmetric unit is estimated and confirmed by self-rotation function calculation to contain two molecules, giving a crystal volume per protein mass (V[sub M]) of 2.68 ų Da[sup -1] and a solvent content of 54%. [ABSTRACT FROM AUTHOR]

SelB is a bacterial elongation factor required for the decoding of a UGA stop codon together with a specific mRNA hairpin to selenocysteine. In attempts to crystallize Moorella therrnoacetica SelB, a proteolysis process occurred and crystals of a proteolytic fragment were observed. The crystals, which appeared after a year, contained a C-terminal 30 kDa fragment containing the mRNAbinding domain. This fragment was reproduced through recloning. Crystals diffracting to 2.7 Å were obtained. [ABSTRACT FROM AUTHOR]

Zm-p60.1, a cytokinin glucoside specific β-glucosidase from maize, is a key enzyme involved in plant development and growth. It has been overexpressed in soluble form from Escherichia coli with a His tag at its N-terminus. The recombinant protein has been purified and crystallized at room temperature using PEG 4000 as the main precipitant. At least three crystal forms have been observed from very similar growth conditions. A flash-annealed monoclinic crystal diffracted to high resolution (beyond 2 Å) with space group P21 and unit-cell parameters a = 55.66, b = 110.72, c = 72.94 Å β = 92.10°.The asymmetric unit is estimated and confirmed by molecular- replacement solution to contain one Zm-p60.1 dimer, giving a crystal volume per protein mass (VM) of 1.89 Å3 Da-1 and a solvent content of 35%. [ABSTRACT FROM AUTHOR]

Information about several papers discussed at a symposium on protein structure determination is presented. The participants introduce and describe the new methods in biological macromolecular crystallography and the recent development in the field of structural biology. The conference was attended by remarkable persons in crystallography and structural biology including Paul Adams, Gerard Bricogne and Axel Brunger.