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26.  Crystallization and preliminary X-ray characterization of the tetrapyrrole-biosynthetic enzyme porphobilinogen deaminase from Arabidopsis thaliana  
The enzyme porphobilinogen deaminase (PBGD) catalyses a key early step in the biosynthesis of haem in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. PBGD from the model plant organism A. thaliana has been expressed and the enzyme was crystallized in a form that diffracted synchrotron radiation to high resolution.
The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step of the haem-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor which is covalently linked by a thioether bridge to an invariant cysteine residue. Since PBGD catalyses a reaction which is common to the biosynthesis of both haem and chlorophyll, structural studies of a plant PBGD enzyme offer great potential for the discovery of novel herbicides. Until recently, structural data have only been available for the Escherichia coli and human forms of the enzyme. Expression in E. coli of a codon-optimized gene for Arabidopsis thaliana PBGD has permitted for the first time the crystallization and preliminary X-ray analysis of the enzyme from a plant species at high resolution.
doi:10.1107/S1744309112042212
PMCID: PMC3509971  PMID: 23192030
tetrapyrrole biosynthesis; porphobilinogen deaminase; dipyrromethane complex
27.  Cloning, expression, purification and crystallization of an endotoxin-biosynthesis enzyme from Neisseria meningitidis  
The cloning, expression, purification and crystallization of phosphoethanolamine transferase A, an endotoxin-modifying enzyme from N. meningitidis, are reported.
The enzyme phosphoethanolamine transferase A is involved in the addition of phosphoethanolamine moieties to lipid A in Neisseria meningitidis. The enzyme is composed of an N-terminal transmembrane domain and a C-terminal soluble domain that is present in the periplasm of the bacteria. A membrane-deletion construct of the enzyme was designed and expressed in Escherichia coli. Well ordered crystals that diffracted to 1.7 Å resolution were obtained by carrying out a limited trypsin digestion of the protein to remove a predicted N-­terminal disordered portion. The crystals belonged to space group P21, with unit-cell parameters a = 44.3, b = 71.6, c = 49.9 Å, β = 109.2°, and contained one molecule in the asymmetric unit.
doi:10.1107/S1744309112042236
PMCID: PMC3509972  PMID: 23192031
endotoxin biosynthesis; LptA; phosphoethanolamine transferase
28.  Crystallization and preliminary X-ray crystallographic analysis of the oxysterol-binding protein Osh3 from Saccharomyces cerevisiae  
The PH domain and ORD of the oxysterol-binding protein Osh3 from S. cerevisae were crystallized and X-ray diffraction data were collected.
Oxysterol-binding protein (OSBP) related proteins (ORPs) are conserved from yeast to humans and are implicated in regulation of sterol homeostasis and in signal transduction pathways. Osh3 of Saccharomyces cerevisiae is a pleckstrin-homology (PH) domain-containing ORP member that regulates phosphoinositide metabolism at endoplasmic reticulum–plasma membrane contact sites. The N-terminal PH domain of Osh3 was purified and crystallized as a lysozyme fusion and the resulting crystal diffracted to 2.3 Å resolution. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 98.03, b = 91.31, c = 84.13 Å, β = 81.41°. With two molecules in the asymmetric unit, the Matthews coefficient was 3.13 Å3 Da−1. Initial attempts to solve the structure by molecular-replacement techniques using T4 lysozyme as a search model were successful. The C-terminal OSBP-related domain (OBD) of Osh3 was crystallized by the vapour-diffusion method and the resulting crystal diffracted to 1.5 Å resolution. The crystal was orthorhombic, belonging to space group P212121, with unit-cell parameters a = 41.57, b = 87.52, c = 100.58 Å. With one molecule in the asymmetric unit, the Matthews coefficient was 2.01 Å3 Da−1. Initial attempts to solve the structure by the single-wavelength anomalous dispersion technique using bromine were successful.
doi:10.1107/S1744309112042510
PMCID: PMC3509973  PMID: 23192032
oxysterol-binding protein; Osh3; Saccharomyces cerevisiae
29.  Purification, crystallization and X-ray diffraction analysis of Trypanosoma congolense insect-stage surface antigen (TcCISSA) 
A novel stage-specific surface protein, T. congolense insect-stage surface antigen (TcCISSA), from the African trypanosome T. congolense was recently identified by mass-spectrometric differential protein-expression analysis. To gain structure–function insight into this protein, the extracellular domain of TcCISSA was expressed, purified and crystallized and X-ray diffraction data were collected and processed to 2.7 Å resolution.
Trypanosoma congolense is a major contributor to the vast socioeconomic devastation in sub-Saharan Africa caused by animal African trypanosomiasis. These protozoan parasites are transmitted between mammalian hosts by tsetse-­fly vectors. A lack of understanding of the molecular basis of tsetse–trypanosome interactions stands as a barrier to the development of improved control strategies. Recently, a stage-specific T. congolense protein, T. congolense insect-stage surface antigen (TcCISSA), was identified that shows considerable sequence identity (>60%) to a previously identified T. brucei insect-stage surface molecule that plays a role in the maturation of infections. TcCISSA has multiple di-amino-acid and tri-amino-acid repeats in its extracellular domain, making it an especially interesting structure–function target. The predicted mature extracellular domain of TcCISSA was produced by recombinant DNA techniques, purified from Escherichia coli, crystallized and subjected to X-ray diffraction analysis; the data were processed to 2.7 Å resolution.
doi:10.1107/S1744309112042686
PMCID: PMC3509974  PMID: 23192033
Trypanosoma congolense; procyclic form; animal African trypanosomiasis; insect stage; vector–pathogen interactions; surface proteins
30.  Purification, crystallization and preliminary X-ray analysis of two hydrogen sulfide-producing enzymes from Fusobacterium nucleatum  
Two homologous hydrogen sulfide-producing enzymes, Fn1220 and Cdl, from F. nucleatum (which actively produces hydrogen sulfide) were overproduced, purified and crystallized. The crystals obtained were characterized by X-ray diffraction.
Hydrogen sulfide produced by oral bacteria is responsible for oral malodour. Two homologous hydrogen sulfide-producing enzymes, Fn1220 and Cdl, from Fusobacterium nucleatum (which actively produces hydrogen sulfide) were overproduced, purified and crystallized. X-ray diffraction data were collected from the crystals using a synchrotron-radiation source. The Fn1220 crystal belonged to tetragonal space group P41212 or P43212 (unit-cell parameters a = b = 116.8, c = 99.2 Å) and the Cdl crystal belonged to monoclinic space group P21 (unit-cell parameters a = 84.9, b = 70.9, c = 87.6 Å, β = 90.3°).
doi:10.1107/S1744309112042546
PMCID: PMC3509975  PMID: 23192034
hydrogen sulfide; Fn1220; Cdl; Fusobacterium nucleatum
31.  Expression, crystallization and preliminary X-ray crystallographic analysis of cystathionine γ-synthase (XometB) from Xanthomonas oryzae pv. oryzae  
Cystathionine γ-synthase (CGS) catalyzes the γ-replacement reaction of O-succinylhomoserine and l-cysteine to form l-cystathionine and succinate. As an antibacterial drug target, CGS from X. oryzae pv. oryzae (XometB) was cloned, purified and crystallized, and a preliminary X-ray crystallography analysis of the XometB crystal was performed.
Cystathionine γ-synthase (CGS) catalyzes the first step in the transsulfuration pathway leading to the formation of cystathionine from O-succinylhomoserine and l-­cysteine through a γ-replacement reaction. As an antibacterial drug target against Xanthomonas oryzae pv. oryzae (Xoo), CGS from Xoo (XometB) was cloned, expressed, purified and crystallized. The XometB crystal diffracted to 2.4 Å resolution and belonged to the tetragonal space group I41, with unit-cell parameters a = b = 165.4, c = 241.7 Å. There were four protomers in the asymmetric unit, with a corresponding solvent content of 73.9%.
doi:10.1107/S1744309112042819
PMCID: PMC3509977  PMID: 23192036
cystathionine γ-synthase; XometB; antibacterial drug targets; Xanthomonas oryzae pv. oryzae
32.  Crystallization and preliminary X-ray crystallographic analysis of human Apaf-1-interacting protein 
Apaf-1-interacting protein (APIP) inhibits cell death involving caspase-1 and caspase-9. APIP was crystallized and X-ray diffraction data were collected to 2.40 Å resolution.
Apaf-1-interacting protein (APIP) is known to inhibit two different types of cell death: caspase-1-dependent pyroptosis and caspase-9-dependent apoptosis. APIP is also involved in the methionine-salvage pathway, where it is called 5-­methylthioribulose-1-phosphate dehydratase (MtnB). The enzyme activity seems to be essential for inhibition of pyroptosis by APIP, but not for inhibition of apoptosis. In this study, human APIP was overproduced in Escherichia coli, purified and crystallized. An X-ray diffraction data set was collected to 2.40 Å resolution and the crystals belonged to space group C2221, with unit-cell parameters a = 106.61, b = 107.50, c = 189.76 Å. Given that four APIP molecules exist in the asymmetric unit, the Matthews coefficient is 2.70 Å3 Da−1 and the corresponding solvent content is 54.4%.
doi:10.1107/S1744309112042832
PMCID: PMC3509978  PMID: 23192037
Apaf-1; APIP; MtnB
33.  Crystallization and preliminary X-ray characterization of Epstein–Barr virus BHRF1 in complex with a benzoylurea peptidomimetic 
The expression and purification of Epstein–Barr virus BHRF1 as well as its co-crystallization with a peptidomimetic are described.
BHRF1 is a pro-survival Bcl-2 homologue encoded by Epstein–Barr virus (EBV) that plays a key role in preventing premature host cell death during viral infection and may contribute to the development of malignancies associated with chronic EBV infections. The anti-apoptotic action of BHRF1 is based on its ability to sequester pro-apoptotic Bcl-2 family proteins, in particular Bim and Bak. These interactions have been previously studied in three dimensions by determining crystal structures of BHRF1 in complex with both Bim and Bak BH3 domains. Screening of a library of peptidomimetic compounds based on the benzoylurea scaffold that mimics critical Bim BH3 domain side chains against BHRF1 led to the identification of an inhibitor of BHRF1 that displays micromolar affinity. Single crystals were obtained from the co-crystallization of recombinant BHRF1 protein with this peptidomimetic compound. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 66.8, b = 91.1, c = 151.9 Å. Diffraction data were collected to 2.11 Å resolution on the MX2 beamline at the Australian Synchrotron.
doi:10.1107/S1744309112043333
PMCID: PMC3509979  PMID: 23192038
BHRF1; apoptosis; peptidomimetic; intrinsic pathway
34.  Molecular cloning, expression, purification and crystallographic analysis of zebrafish THEM2 
The THEM2 protein from zebrafish has successfully been expressed, purified and crystallized. Diffraction data were collected to a resolution of 1.8 Å.
Thioesterase superfamily member 2 (THEM2) is essential for cell proliferation of mammalian cells. It belongs to the hotdog-fold thioesterase superfamily and catalyzes the hydrolysis of the thioester bonds of acyl-CoA in vitro. In this study, THEM2 protein from zebrafish (fTHEM2) was expressed in Escherichia coli and purified by Ni-affinity and gel-filtration chromatography. fTHEM2 crystals were obtained using the sitting-drop vapour-diffusion method with PEG 10 000 as precipitant. X-ray diffraction data were collected to 1.80 Å resolution using a synchrotron-radiation source. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 77.1, b = 74.4, c = 96.6 Å, β = 93.7°.
doi:10.1107/S1744309112043813
PMCID: PMC3509980  PMID: 23192039
THEM2; thioesterase superfamily member 2; Danio rerio
35.  Purification, crystallization and preliminary X-ray crystallographic analysis of the CIDE-N domain of Fsp27 
The CIDE-N domain of Fsp27 expressed in E. coli was purified as a monomer and identified by LC/MS/MS. Crystals of the Fsp27 CIDE-N domain were grown in sitting-drop mode and diffracted to 1.92 Å resolution.
Fsp27, a member of the CIDE protein family which is selectively expressed in adipocytes, has emerged as a novel regulator for unilocular lipid droplet (LD) formation, lipid metabolism, differentiation of adipocytes and insulin sensitivity. An LD is a subcellular compartment that is used by adipocytes for the efficient storage of fats. The CIDE-N domain of Fsp27 functions as a recruitment platform that induces the correct configuration of the Fsp27 CIDE-C domain to facilitate LD fusion. This study reports the high-yield expression of the mouse Fsp27 CIDE-N domain in Escherichia coli; a two-step purification protocol with high efficiency was established and crystallographic analysis was performed. The purity of the recombinant Fsp27 was >95% as assessed by SDS–PAGE. Crystals were obtained at 291 K using 28% polyethylene glycol 4000 as a precipitant. Diffraction data were collected to 1.92 Å resolution and the crystal belonged to space group P65, with unit-cell parameters a = b = 63.3, c = 37.4 Å, α = β = 90, γ = 120°. The components of the crystal were identified by ion-trap LC/MS/MS spectrometric analysis. The structure has been solved by molecular replacement and refinement is in progress.
doi:10.1107/S1744309112043989
PMCID: PMC3509981  PMID: 23192040
CIDEC; Fsp27; obesity; prokaryotic expression; unilocular lipid droplets
36.  Crystallization and preliminary X-ray diffraction analysis of the invertase from Saccharomyces cerevisiae  
The invertase from S. cerevisiae was overexpressed, purified and crystallized, and its preliminary X-ray diffraction analysis is reported at 3.3 Å resolution.
Saccharomyces cerevisiae invertase (ScInv) is an enzyme encoded by the SUC2 gene that releases β-fructose from the nonreducing termini of various β-­d-fructofuranoside substrates. Its ability to produce 6-kestose by transglycosylation makes this enzyme an interesting research target for applications in industrial biotechnology. The native enzyme, which presents a high degree of oligomerization, was crystallized by vapour-diffusion methods. The crystals belonged to space group P3121, with unit-cell parameters a = 268.6, b = 268.6, c = 224.4 Å. The crystals diffracted to 3.3 Å resolution and gave complete data sets using a synchrotron X-ray source.
doi:10.1107/S1744309112044417
PMCID: PMC3509983  PMID: 23192042
invertases; Saccharomyces cerevisiae
37.  Cloning, purification, crystallization and preliminary crystallographic study of calcium-binding protein 5 from Entamoeba histolytica  
Calcium-binding protein 5 from E. histolytica was cloned, expressed in E. coli and purified. The purified protein crystallized in space group C222 and the crystals diffracted to 2 Å resolution.
Entamoeba histolytica is the causative agent of human amoebiasis. Phagocytosis is the major route of food intake by this parasite and is responsible for its virulence. Calcium and calcium-binding proteins play major roles in its phagocytosis. Calcium-binding protein 5 from E. histolytica (EhCaBP5) is a cytoplasmic protein; its expression is very sensitive to serum starvation and it seems to be involved in binding to myosin I. In this study, EhCaBP5 was cloned, expressed in Escherichia coli and purified using affinity and size-exclusion chromatography. The purified protein crystallized in space group C222 and the crystals diffracted to 2 Å resolution. The Matthews coefficient indicated the presence of one molecule in the asymmetric unit, with a V M of 2.35 Å3 Da−1 and a solvent content of 47.7%.
doi:10.1107/S1744309112044612
PMCID: PMC3509984  PMID: 23192043
calcium-binding protein 5; Entamoeba histolytica
38.  Crystallization and preliminary X-ray analysis of the open form of human ecto-5′-nucleotidase (CD73) 
The crystallization of human ecto-5′-nucleotidase (CD73) paves the way for detailed studies of the domain motion between the open and closed forms. It will also enable the structure-based design of inhibitors targeting the open form.
Eukaryotic ecto-5′-nucleotidase (e5NT) catalyses the hydrolysis of extracellular AMP to adenosine and plays a pivotal role in switching on adenosine signalling via the P1 receptors of the purinergic signalling pathway. With such an important regulatory role, e5NT has become an appealing new drug target, with potential applications in the treatment of inflammation, chronic pain, hypoxia and cancer. In order to gain insight into the structure and function of the eukaryotic e5NT enzymes and to assist in structure-based drug design, the crystal structure of human e5NT has been solved. Recombinant human e5NT comprising four asparagine-to-aspartate surface mutations targeting potential glycosylation sites was refolded from bacterial inclusion bodies. Refolded and purified human e5NT crystallized in space group P4332 and a data set to 1.85 Å resolution was obtained. The structure could be solved by molecular replacement using a polyalanine model generated from Thermus thermophilus 5′-nucleotidase (5NT). An anomalous data set revealed the presence of a metal-ion binding site, as well as calcium and chloride ion-binding sites. Structural comparisons with bacterial 5NT homologues showed that the human e5NT crystal structure has an open conformation in which the metal- and substrate-binding sites are distant from each other. Here, the crystallization and preliminary X-ray crystallo­graphic analysis of an open structural conformation of human e5NT are described.
doi:10.1107/S1744309112045447
PMCID: PMC3509985  PMID: 23192044
e5NT; CD73; ecto-enzyme; AMP hydrolysis; membrane proteins
39.  Crystallization and preliminary X-ray data analysis of the pXO1 plasmid-partitioning factor TubZ from Bacillus cereus  
B. cereus TubZ was cocrystallized with GDP and diffraction data were collected to 2.1 Å resolution.
TubZ is a structural homologue of tubulin and FtsZ GTPases, which are involved in the type III plasmid-partitioning system. TubZ assembles into polymers in a GTP-dependent manner and drives plasmid segregation as ‘cytomotive’ filaments. In this study, C-terminally truncated TubZ from Bacillus cereus was crystallized in the presence or absence of GDP by the hanging-drop vapour-diffusion method. The crystal of TubZ in complex with GDP belonged to the monoclinic space group P21, with unit-cell parameters a = 67.05, b = 84.49, c = 67.66 Å, β = 92.92°, and was non-isomorphous with GDP-bound TubZ previously crystallized in the presence of the slowly hydrolysable GTP analogue GTPγS. TubZ was also crystallized in the free form and the crystal belonged to space group P21, with unit-cell parameters a = 53.91, b = 65.54, c = 58.18 Å, β = 106.19°. Data were collected to 1.7 and 2.1 Å resolution for the free and GDP-bound forms, respectively.
doi:10.1107/S1744309112045551
PMCID: PMC3509986  PMID: 23192045
TubZ; Bacillus cereus
40.  A preliminary X-ray study of transketolase from Burkholderia pseudomallei  
The transketolase TktA from B. pseudomallei has been cloned, expressed, purified and crystallized. Synchrotron X-ray data were collected to 2.0 Å resolution.
TktA is the most critical enzyme in the nonoxidative pentose phosphate pathway. It catalyzes the conversion of xylulose 5-phosphate and ribose 5-­phosphate into sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate, and its products are used in the biosynthesis of acetyl-CoA, aromatic amino acids, nucleic acids and ADP-l-glycero-β-d-manno-heptose. TktA also has an unexpected role in chromosome structure that is independent of its metabolic responsibilities. Therefore, it is a new potent antibiotic target. In this study, TktA from Burkholderia pseudomallei has been cloned, expressed, purified and crystallized. Synchrotron X-ray data were also collected to 2.0 Å resolution. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 146.2, b = 74.6, c = 61.6 Å, β = 113.0°. A full structural determination is under way in order to provide insight into the structure–function relationship of this protein.
doi:10.1107/S1744309112044375
PMCID: PMC3509987  PMID: 23192046
transketolase; Burkholderia pseudomallei; TktA
41.  Preliminary crystallographic analysis of the Megavirus superoxide dismutase 
The preliminary crystallographic analysis of the Megavirus chilensis superoxide dismutase Mg277 is reported. The crystals belonged to space group P41212 or P43212, with one dimer per asymmetric unit.
Megavirus chilensis, a close relative of the Mimivirus giant virus, is able to replicate in Acanthamoeba castellanii. The first step of viral infection involves the internalization of the virions in host vacuoles. It has been experimentally demonstrated that Mimivirus particles contain many proteins capable of resisting oxidative stress, as encountered in the phagocytic process. These proteins are conserved in Megavirus, which has an additional gene (Mg277) encoding a putative superoxide dismutase. The Mg277 ORF product was overexpressed in Escherichia coli, purified and crystallized. A SAD data set was collected to 2.24 Å resolution at the selenium peak wavelength on the BM30 beamline at the ESRF from a single crystal of selenomethionine-substituted recombinant superoxide dismutase protein.
doi:10.1107/S174430911204657X
PMCID: PMC3509988  PMID: 23192047
Megavirus chilensis; Megaviridae; giant virus; superoxide dismutase
42.  Crystallization and preliminary X-ray diffraction analysis of mevalonate kinase from Methanosarcina mazei  
Recombinant mevalonate kinase from M. mazei has been crystallized. Diffraction data were collected to 2.08 Å resolution.
Mevalonate kinase (MVK), which plays an important role in catalysing the biosynthesis of isoprenoid compounds derived from the mevalonate pathway, transforms mevalonate to 5-phosphomevalonate using ATP as a cofactor. Mevalonate kinase from Methanosarcina mazei (MmMVK) was expressed in Escherichia coli, purified and crystallized for structural analysis. Diffraction-quality crystals of MmMVK were obtained by the vapour-diffusion method using 0.32 M MgCl2, 0.08 M bis-tris pH 5.5, 16%(w/v) PEG 3350. The crystals belonged to space group P21212, with unit-cell parameters a = 97.11, b = 135.92, c = 46.03 Å. Diffraction data were collected to 2.08 Å resolution.
doi:10.1107/S1744309112047070
PMCID: PMC3509989  PMID: 23192048
mevalonate kinase; Methanosarcina mazei; multiple-wavelength anomalous dispersion
43.  Cloning, expression, purification, crystallization and preliminary X-ray analysis of a fructokinase from Vibrio cholerae O395 
A fructokinase from V. cholerae O395 has been cloned, expressed, purified and crystallized in the apo form; the crystals belonged to the orthorhombic space group P21212 and diffracted to 2.45 Å resolution. Fructokinase with ADP and fructose bound has also been crystallized and the crystals diffracted to a resolution of 1.75 Å.
Fructokinase (FK), one of the crucial enzymes for sugar metabolism in bacterial systems, catalyses the unidirectional phosphorylation reaction from fructose to fructose 6-phosphate, thereby allowing parallel entry of fructose into glycolysis beside glucose. The cscK gene from Vibrio cholerae O395 coding for the enzyme FK has been cloned, overexpressed in Escherichia coli BL21 (DE3) and purified using Ni–NTA affinity chromatography. Crystals of V. cholerae FK (Vc-FK) and its cocrystal with fructose, adenosine diphosphate (ADP) and Mg2+ were grown in the presence of polyethylene glycol 6000 and diffracted to 2.45 and 1.75 Å resolution, respectively. Analysis of the diffraction data showed that both crystal forms have symmetry consistent with space group P21212, but with different unit-cell parameters. Assuming the presence of two molecules in the asymmetric unit, the Matthews coefficient for the apo Vc-FK crystals was estimated to be 2.4 Å3 Da−1, which corresponds to a solvent content of 48%. The corresponding values for the ADP- and sugar-bound Vc-FK crystals were 2.1 Å3 Da−1 and 40%, respectively, assuming the presence of one molecule in the asymmetric unit.
doi:10.1107/S1744309112047598
PMCID: PMC3509990  PMID: 23192049
Vibrio cholerae; fructokinase; fructose; ATP
44.  Expression, purification, crystallization and preliminary X-ray analysis of NAD(P)H-dependent carbonyl reductase specifically expressed in thyroidectomized chicken fatty liver 
An NAD(P)H-dependent carbonyl reductase specifically expressed in thyroidectomized chicken fatty liver was successfully isolated and crystallized.
An NAD(P)H-dependent carbonyl reductase specifically expressed in thyroid­ectomized chicken fatty liver was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 300 as the precipitant. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 104.26, b = 81.32, c = 77.27 Å, β = 119.43°, and diffracted to 1.86 Å resolution on beamline NE3A at the Photon Factory. The overall R merge was 5.4% and the data completeness was 99.4%.
doi:10.1107/S1744309112046453
PMCID: PMC3509991  PMID: 23192050
thyroidectomized chicken fatty liver; NAD(P)H-dependent carbonyl reductases; short-chain dehydrogenase/reductase family
45.  Coiled-coil dimerization of the LOV2 domain of the blue-light photoreceptor phototropin 1 from Arabidopsis thaliana  
The X-ray structure of the light, oxygen or voltage domain 2 of phototropin 1 from A. thaliana (AtLOV2) in its dark-adapted state has been determined. The N-terminal flanking A′α helix of AtLOV2 plays a role in its dimerization.
A key role in signal transduction and dimerization mediated by Per–Arnt–Sim (PAS) domains is played by α-helical linkers that flank the structurally similar α/β cores of these domains. However, crystal-packing forces and the different construct lengths and sequences of the PAS domains influence the final length and orientation of the linkers relative to the core and create uncertainty in the exact mechanism of the linker function. Thus, structural characterization and comparison of the linkers within isolated PAS-domain constructs and/or full-length PAS-containing proteins is important for clarification of the mechanism. The plant blue-light photoreceptors phototropins possess two N-terminal flavin mononucleotide-based light, oxygen or voltage (LOV) domains (LOV1 and LOV2) that comprise a subclass of the PAS family and one C-terminal serine/threonine kinase domain whose enzymatic activity is regulated by blue light. The dark-adapted state crystal structures of the Arabidopsis thaliana phototropin 1 and phototropin 2 LOV1-domain constructs flanked by an N-terminal A′α helix and the structure of the phototropin 2 core LOV2 domain are known. Here, the crystal structure of the A. thaliana phototropin 1 LOV2 domain has been determined in its dark-adapted state. The core is flanked by an N-terminal A′α helix and a C-terminal Jα helix similar to those in the previously reported structure of Avena sativa phototropin 1 LOV2. In contrast to the monomeric A. sativa LOV2, A. thaliana LOV2 is a dimer in which two A′α helices adopt a scissor-like orientation at the dimer interface and form a short α-helical coiled coil. The Jα helix predominantly interacts with the β-sheet and plays a role in coiled-coil formation and dimerization.
doi:10.1107/S1744309113029199
PMCID: PMC3855711  PMID: 24316821
LOV domains; N-terminal A′α helix; C-terminal Jα helix; coiled coil; dimerization; light-mediated signal transduction
46.  Crystallization and preliminary X-ray analysis of the ATPase domain of the σ54-dependent transcription activator NtrC1 from Aquifex aeolicus bound to the ATP analog ADP–BeFx  
This study reports the crystallization of a new nucleotide state of the ATPase domain of a bacterial transcription activator NtrC1 from the hyperthermophilic bacterium Aquifex aeolicus. Wild-type NtrC1 ATPase domain was crystallized in the presence of the ATP analog ADP–BeFx–Mg and the crystals diffracted anisotropically to at best 3.2, 5.2 and 3.2 Å resolution in the a*, b* and c* directions, respectively.
One way that bacteria regulate the transcription of specific genes to adapt to environmental challenges is to use different σ factors that direct the RNA polymerase holoenzyme to distinct promoters. Unlike σ70 RNA polymerase (RNAP), σ54 RNAP is unable to initiate transcription without an activator: enhancer-binding protein (EBP). All EBPs contain one ATPase domain that belongs to the family of ATPases associated with various cellular activities (AAA+ ATPases). AAA+ ATPases use the energy of ATP hydrolysis to remodel different target macromolecules to perform distinct functions. These mechanochemical enzymes are known to form ring-shaped oligomers whose conformations strongly depend upon nucleotide status. Here, the crystallization of the AAA+ ATPase domain of an EBP from Aquifex aeolicus, NtrC1, in the presence of the non-hydrolyzable ATP analog ADP–BeFx is reported. X-ray diffraction data were collected from two crystals from two different protein fractions of the NtrC1 ATPase domain. Previously, this domain was co-crystallized with ADP and ATP, but the latter crystals were grown from the Walker B substitution variant E239A. Therefore, the new data sets are the first for a wild-type EBP ATPase domain co-crystallized with an ATP analog and they reveal a new crystal form. The resulting structure(s) will shed light on the mechanism of EBP-type transcription activators.
doi:10.1107/S174430911302976X
PMCID: PMC3855726  PMID: 24316836
enhancer-binding protein; σ54 transcription activator; AAA+ ATPases; NtrC1; Aquifex aeolicus
47.  Liquid–liquid diffusion crystallization improves the X-ray diffraction of EndoS, an endo-β-N-acetyl­glucosaminidase from Streptococcus pyogenes with activity on human IgG 
A comparative study of vapor diffusion versus liquid–liquid diffusion methods used for the crystallization of EndoS is reported. X-ray diffraction data to 2.6 and 1.9 Å resolution were collected for wild-type endoglycosidase and glycosynthase constructs of EndoS, respectively.
Endoglycosidase S (EndoS) is an enzyme secreted by Streptococcus pyogenes that specifically hydrolyzes the β-1,4-di-N-acetylchitobiose core glycan on immunoglobulin G (IgG) antibodies. One of the most common human pathogens and the cause of group A streptococcal infections, S. pyogenes secretes EndoS in order to evade the host immune system by rendering IgG effector mechanisms dysfunctional. On account of its specificity for IgG, EndoS has also been used extensively for chemoenzymatic synthesis of homogeneous IgG glycoprotein preparations and is being developed as a novel therapeutic for a wide range of autoimmune diseases. The structural basis of its enzymatic activity and substrate specificity, however, remains unknown. Here, the purification and crystallization of EndoS are reported. Using traditional hanging-drop and sitting-drop vapor-diffusion crystallization, crystals of EndoS were grown that diffracted to a maximum of 3.5 Å resolution but suffered from severe anisotropy, the data from which could only be reasonably processed to 7.5 Å resolution. When EndoS was crystallized by liquid–liquid diffusion, it was possible to grow crystals with a different space group to those obtained by vapor diffusion. Crystals of wild-type endoglycosidase and glycosynthase constructs of EndoS grown by liquid–liquid diffusion diffracted to 2.6 and 1.9 Å resolution, respectively, with a greatly diminished anisotropy. Despite extensive efforts, the failure to reproduce these liquid–liquid diffusion-grown crystals by vapor diffusion suggests that these crystallization methods each sample a distinct crystallization space.
doi:10.1107/S1744309113030650
PMCID: PMC3855731  PMID: 24316841
endoglycosidase S; liquid–liquid diffusion; Streptococcus pyogenes
48.  Expression, purification, crystallization and preliminary X-ray diffraction analysis of the aspartate transcarbamoylase domain of human CAD 
The recombinant aspartate transcarbamoylase domain of human CAD was expressed in E. coli, purified and crystallized in the presence and absence of the inhibitor PALA. X-ray diffraction data sets were collected for both crystal forms at 2.1 Å resolution.
Aspartate transcarbamoylase (ATCase) catalyzes the synthesis of N-carbamoyl-l-aspartate from carbamoyl phosphate and aspartate in the second step of the de novo biosynthesis of pyrimidines. In prokaryotes, the first three activities of the pathway, namely carbamoyl phosphate synthetase (CPSase), ATCase and dihydroorotase (DHOase), are encoded as distinct proteins that function independently or in noncovalent association. In animals, CPSase, ATCase and DHOase are part of a 243 kDa multifunctional polypeptide named CAD. Up-regulation of CAD is essential for normal and tumour cell proliferation. Although the structures of numerous prokaryotic ATCases have been determined, there is no structural information about any eukaryotic ATCase. In fact, the only detailed structural information about CAD is that it self-assembles into hexamers and trimers through interactions of the ATCase domains. Here, the expression, purification and crystallization of the ATCase domain of human CAD is reported. The recombinant protein, which was expressed in bacteria and purified with good yield, formed homotrimers in solution. Crystallization experiments both in the absence and in the presence of the inhibitor PALA yielded small crystals that diffracted X-rays to 2.1 Å resolution using synchrotron radiation. The crystals appeared to belong to the hexagonal space group P6322, and Matthews coefficient calculation indicated the presence of one ATCase subunit per asymmetric unit, with a solvent content of 48%. However, analysis of the intensity statistics suggests a special case of the P21 lattice with pseudo-symmetry and possibly twinning.
doi:10.1107/S1744309113031114
PMCID: PMC3855736  PMID: 24316846
CAD; aspartate transcarbamoylase domain; de novo pyrimidine synthesis; PALA
49.  Complex of myoglobin with phenol bound in a proximal cavity 
Acta Crystallographica Section F  2012;68(Pt 12):1465-1471.
The structure of a complex of sperm whale myoglobin with phenol places this inhibitor of the haloperoxidase activity in a proximal cavity that is unlikely to be the halophenol binding site. The absence of phenol binding at the heme edge, where dehalogenation is likely to take place, indicates that the inhibitor and the halophenol substrates only bind in the active site in the presence of hydrogen peroxide.
Sperm whale myoglobin (Mb) has weak dehaloperoxidase activity and catalyzes the peroxidative dehalogenation of 2,4,6-trichlorophenol (TCP) to 2,6-dichloroquinone. Crystals of Mb and of its more active G65T variant were used to study the binding of TCP, 4-iodophenol (4-IP) and phenol. The structures of crystals soaked overnight in a 10 mM solution of phenol revealed that a phenol molecule binds in the proximal cavity, forming a hydrogen bond to the hydroxyl of Tyr146 and hydrophobic contacts which include interactions with Cβ and Cγ of the proximal histidine His93. The phenol position corresponds to the strongest xenon binding site, Xe1. It appears that the ligand enters the proximal cavity through a gate formed by the flexible loops 79–86 and 93–103. TCP and 4-­IP do not bind to Mb in this manner under similar conditions; however, it appears to be likely that dimethyl sulfoxide (DMSO), which was used at a concentration of 0.8 M to facilitate 4-IP dissolution, binds in the phenol/Xe1 binding site. In this structure, a water molecule coordinated to the heme iron was replaced by an oxygen molecule, reflecting the reduction of the heme. Crystals of Mb and G65T Mb soaked for 5–10 min did not show bound phenol. Kinetic studies of TCP dechlorination showed that phenol has a dual effect: it acts as a competitive inhibitor that is likely to interfere with TCP binding at the heme edge and as a weak activator, likely through binding in the proximal cavity. The lack of phenol bound at the heme edge in the crystal structures suggests that its inhibitory binding only takes place when the heme is activated by hydrogen peroxide.
doi:10.1107/S1744309112045514
PMCID: PMC3509966  PMID: 23192025
myoglobin; dehalogenases; heme proteins; peroxidases; DMSO
50.  Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118 
Acta Crystallographica Section F  2012;68(Pt 12):1427-1433.
The crystal structure of ribose 5-phosphate isomerase has been determined to 1.72 Å resolution and is presented with a brief comparison to other known ribose 5-phosphate isomerase A structures.
The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β d-­ribose 5-­phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography.
doi:10.1107/S174430911204273X
PMCID: PMC3509960  PMID: 23192019
ribose 5-phosphate isomerase; in situ diffraction; Lactobacillus salivarius

Results 26-50 (2828)