The ‘Nucleobase-Cation-Symport-1’, NCS1, transporters are essential components of salvage pathways for nucleobases and related metabolites. Here, we report the 2.85 Å resolution structure of the NCS1 benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefaciens. Mhp1 contains 12 transmembrane helices, ten of which are arranged in two inverted repeats of 5 helices. The structures of the outward-facing open and substrate-bound occluded conformations were solved showing how the outward-facing cavity closes upon binding of substrate. Comparisons with the leucine (LeuTAa) and the galactose (vSGLT) transporters reveal that the outward- and inward-facing cavities are symmetrically arranged on opposite sides of the membrane. The reciprocal opening and closing of these cavities is synchronised by the inverted repeat helices 3 and 8, providing the structural basis of the ‘alternating access’ model for membrane transport.
Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members.
The substrate binding protein (SP_0149) of an ABC transporter from Streptococcus pneumoniae was molecularly cloned, overexpressed and purified. Diffraction quality crystals were grown using the hanging-drop vapour-diffusion technique.
A truncated (29 residues from the N-terminus) and N-terminal His-tagged form of SP_0149 from pneumococcal strain ATCC BAA-334 was overexpressed and purified to homogeneity using affinity and gel-filtration chromatography. Diffraction quality crystals were grown at 293 K using the hanging-drop vapour-diffusion technique. X-ray diffraction data were collected to 2.3 Å resolution from a single-crystal that belonged to the orthorhombic space group P212121 with the unit-cell parameters a = 54.56, b = 75.61, c = 75.52 Å. The calculated values of the Matthews coefficient assuming one molecule (with calculated molecular weight of 30 400 Da) in the crystal asymmetric unit and the corresponding solvent content were 2.56 Å3 Da−1 and 52.0%, respectively.
ABC transporter; Streptococcus pneumoniae; SP_0149; ATCC BAA-334
The structure of the sodium-benzylhydantoin transport protein, Mhp1, from Microbacterium liquefaciens comprises a 5-helix inverted repeat, which is widespread amongst secondary transporters. Here we report the crystal structure of an inward-facing conformation of Mhp1 at 3.8 Å resolution, complementing its previously-described structures in outward-facing and occluded states. From analyses of the three structures and molecular dynamics simulations we propose a mechanism for the transport cycle in Mhp1. Switching from the outward- to the inward- facing state, to effect the inward release of sodium and benzylhydantoin, is primarily achieved by a rigid body movement of transmembrane helices 3, 4, 8 and 9 relative to the rest of the protein. This forms the basis of an alternating access mechanism applicable to many transporters of this emerging superfamily.
Dextran glucosidase from S. mutans was crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.2 Å resolution.
Dextran glucosidase from Streptococcus mutans is an exo-hydrolase that acts on the nonreducing terminal α-1,6-glucosidic linkage of oligosaccharides and dextran with a high degree of transglucosylation. Based on amino-acid sequence similarity, this enzyme is classified into glycoside hydrolase family 13. Recombinant dextran glucosidase was purified and crystallized by the hanging-drop vapour-diffusion technique using polyethylene glycol 6000 as a precipitant. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 72.72, b = 86.47, c = 104.30 Å. A native data set was collected to 2.2 Å resolution from a single crystal.
dextran glucosidase; Streptococcus mutans; α-amylase family
A novel family GH16 β-agarase from the marine bacterium Zobellia galactanivorans was expressed, purified and crystallized. Hexagonal crystals belonging to space group P3121 diffracted to 2.2 Å resolution, whereas orthorhombic crystals belonging to space group P212121 diffracted to 1.5–Å resolution.
Marine bacteria secrete specific glycoside hydrolases such as agarases to access polysaccharides from algal cell walls as a carbon and energy source. In an attempt to identify agarases with variable degradation patterns, a novel family GH16 β-agarase from the marine bacterium Zobellia galactanivorans was expressed, purified and crystallized. The purified enzyme crystallized in two distinct forms that were grown by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. Hexagonal crystals belonging to space group P3121 diffracted to 2.2 Å resolution, whereas orthorhombic crystals belonging to space group P212121 diffracted to 1.5 Å resolution.
β-agarases; glycoside hydrolases; heterologous expression; Zobellia galactanivorans
Phosphoglucose isomerase from P. falciparum has been crystallized. Diffraction data to 1.8 Å resolution have been collected using synchrotron radiation.
Phosphoglucose isomerase (PGI) is a key enzyme in glycolysis and glycogenesis that catalyses the interconversion of glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). For crystallographic studies, PGI from the human malaria parasite Plasmodium falciparum (PfPGI) was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data to 1.5 Å resolution were collected from an orthorhombic crystal form belonging to space group P212121 with unit-cell parameters a = 103.3, b = 104.1, c = 114.6 Å. Structural analysis by molecular replacement is in progress.
glucose 6-phosphate isomerase; malaria; phosphoglucose isomerase; phosphohexose isomerase
The first crystallization of deoxyuridine triphosphate nucleotidohydrolase from plant, Arabidopsis thaliana, has been performed. An additive, taurine, was effective in producing the single crystal.
The deoxyuridine triphosphate nucleotidohydrolase gene from Arabidopsis thaliana was expressed and the gene product was purified. Crystallization was performed by the hanging-drop vapour-diffusion method at 298 K using 2 M ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.2 Å resolution using Cu Kα radiation. The crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 69.90, b = 70.86 Å, c = 75.55 Å. Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a V
M of 1.8 Å3 Da−1.
deoxyuridine triphosphate nucleotidohydrolase; Arabidopsis thaliana
The arginine repressor of the hyperthermophile T. neapolitana was crystallized with and without its corepressor arginine. Both crystals diffracted to high resolution and belong to the orthorhombic space group P212121, with similar unit-cell parameters.
The arginine repressor of Thermotoga neapolitana (ArgRTnp) is a member of the family of multifunctional bacterial arginine repressors involved in the regulation of arginine metabolism. This hyperthermophilic repressor shows unique DNA-binding features that distinguish it from its homologues. ArgRTnp exists as a homotrimeric protein that assembles into hexamers at higher protein concentrations and/or in the presence of arginine. ArgRTnp was crystallized with and without its corepressor arginine using the hanging-drop vapour-diffusion method. Crystals of the aporepressor diffracted to a resolution of 2.1 Å and belong to the orthorhombic P212121 space group, with unit-cell parameters a = 117.73, b = 134.15, c = 139.31 Å. Crystals of the repressor in the presence of its corepressor arginine diffracted to a resolution of 2.4 Å and belong to the same space group, with similar unit-cell parameters.
arginine repressor; regulation of transcription; hyperthermophiles
The catalytic domain of an alkaline mannanase from the alkaliphilic Bacillus sp. N16-5 was expressed in E. coli and purified. Crystallization and preliminarily X-ray crystallographic analysis were performed for the recombinant enzyme.
The catalytic domain of an alkaline β-mannanase from the alkaliphilic Bacillus sp. N16-5 has been expressed and purified. The recombinant enzyme was crystallized using the hanging-drop vapour-diffusion method at 298 K. X-ray diffraction data were collected to 1.6 Å resolution. The crystal belonged to the orthorhombic space group P212121, with unit-cell parameters a = 59.03, b = 63.31, c = 83.34 Å. Initial phasing was carried out by molecular replacement using the three-dimensional structure of a mannanase from the alkaliphilic Bacillus sp. JAMB602 as a search model.
β-mannanases; Bacillus sp. N16-5; alkaliphiles
A low-molecular-weight cellulase from an alkalothermophilic Thermomonospora sp. has been crystallized. A diffraction data set has been collected to 2.3 Å resolution.
Cellulases catalyze the hydrolysis of β-1,4-glycosidic linkages within cellulose, the most abundant organic polymer on earth. The cellulase (TSC; EC 18.104.22.168) from an alkalothermophilic Thermomonospora sp. has a low molecular weight of 14.2 kDa. It is optimally active at 323 K and stable over the wide pH range of 5–9. Moreover, it has bifunctional activity against cellulose and xylan polymers. In this study, TSC was purified from the native source and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 49.9, b = 79.5, c = 99.7 Å, and diffract to better than 2.3 Å resolution.
cellulases; 1,4-β-d-glucan glucohydrolase
Crystals of the helicase domain from a tomato mosaic virus replication protein obtained using the hanging-drop vapour-diffusion method at 285 K diffracted X-rays to 2.05 Å resolution. They belonged to the orthorhombic space group P212121, with unit-cell parameters a = 85.8, b = 128.3, c = 40.7 Å.
Tomato mosaic virus belongs to the genus Tobamovirus in the alphavirus-like superfamily of positive-strand RNA viruses. The alphavirus-like superfamily includes many plant and animal viruses of agronomical and clinical importance. These viruses encode replication-associated proteins that contain a putative superfamily 1 helicase domain. No three-dimensional structures for this domain have been determined to date. Here, the crystallization and preliminary X-ray diffraction analysis of the 130K helicase domain are reported. Diffraction data were collected and processed to 2.05 and 1.75 Å resolution from native and selenomethionine-labelled crystals, respectively. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 85.8, b = 128.3, c = 40.7 Å.
tomato mosaic virus; replicase protein; helicase domain
A novel haemagglutinin from Jatropha curcas seeds is purified and crystallized. X-ray diffraction data collected from the rod-shaped crystals were processed in the orthorhombic space group P212121 and the crystals diffracted to 2.8 Å resolution at 103 K.
The plant Jatropha curcas (Euphorbiaceae) is an important source of biofuel from the inedible oil present in its toxic seeds. The toxicity arises from the presence of curcin, a ribosome-inactivating protein showing haemagglutination activity. In this communication, the purification, crystallization and preliminary X-ray characterization are reported of a small protein isolated from J. curcas seeds with a molecular mass of ∼10 kDa that agglutinates rabbit erythrocytes. The protein was crystallized using the hanging-drop vapour-diffusion method and also by the microbatch method in 72-well HLA plates, using PEG 8000 as the precipitant in both conditions. X-ray diffraction data collected from the rod-shaped crystals were processed in the orthorhombic space group P212121. The crystals diffracted to 2.8 Å resolution at 103 K.
Jatropha curcas; haemagglutinins
The complex of NADH and 3α-HSD from Pseudomonas sp. B-0831 has been crystallized and X-ray diffraction data have been collected to 1.8 Å resolution.
The NAD(P)+-dependent enzyme 3α-hydroxysteroid dehydrogenase (3α-HSD) catalyzes the reversible interconversion of hydroxyl and oxo groups at position 3 of the steroid nucleus. The complex of NADH and 3α-HSD from Pseudomonas sp. B-0831 was crystallized by the hanging-drop vapour-diffusion method. Refinement of crystallization conditions with microseeding improved the quality of the X-ray diffraction data to a resolution of 1.8 Å. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 62.46, b = 82.25, c = 86.57 Å, and contained two molecules, reflecting dimer formation of 3α-HSD, in the asymmetric unit.
3α-hydroxysteroid dehydrogenase; NADH; Pseudomonas sp. B-0831
The phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv was crystallized and diffraction data were collected to 2.8 Å resolution.
Phosphoglucose isomerase is a ubiquitous enzyme that catalyzes the isomerization of d-glucopyranose-6-phosphate to d-fructofuranose-6-phosphate. The present investigation reports the expression, purification, crystallization and preliminary crystallographic studies of the phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv, which shares 46% sequence identity with that of its human host. The recombinant protein, which was prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.8 Å and belonged to the orthorhombic space group I212121, with unit-cell parameters a = 109.0, b = 119.8, c = 138.9 Å.
phosphoglucose isomerase; Mycobacterium tuberculosis
The secondary alcohol dehydrogenase mutant I86A from Thermoanaerobacter ethanolicus (TeSADH) was crystallized in novel crystallization conditions. Diffraction data to 3.2 Å were collected at the Canadian Light Source.
The Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase I86A mutant is stereospecific for (R)-alcohols instead of (S)-alcohols. Pyramidal crystals grown in the presence of (R)-phenylethanol via the hanging-drop vapour-diffusion method diffracted to 3.2 Å resolution at the Canadian Light Source. The crystal belonged to the orthorhombic space group P212121, with unit-cell parameters a = 80.23, b = 124.90, c = 164.80 Å. The structure was solved by molecular replacement using the structure of T. brockii SADH (PDB entry 1ykf).
secondary alcohol dehydrogenase; Thermoanaerobacter ethanolicus
The MaoC-like dehydratase from P. capsici was cloned, expressed and purified to homogeneity. Crystals were obtained that diffracted to 1.93 Å resolution.
MaoC-like dehydratase (MaoC) plays an important role in supplying (R)-3-hydroxyacyl-CoA from the fatty-acid oxidation pathway to polyhydroxyalkanoate (PHA) biosynthetic pathways. PHAs have been attracting much attention as they can be used in the biosynthesis of synthetic plastics. Crystals of MaoC from Phytophora capsici were grown by the hanging-drop vapour-diffusion method at 289 K in a number of screening conditions. An MaoC crystal diffracted to 1.93 Å resolution using X-ray radiation and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 81.458, b = 82.614, c = 124.228 Å, α = β = γ = 90°.
MaoC-like dehydratases; Phytophora capsici; enoyl-CoA hydratases; polyhydroxyalkanoates
P. aeruginosa CobE, a protein implicated in vitamin B12 biosynthesis, has been crystallized and data on the native and SeMet forms recorded to resolutions of 1.9 and 1.7 Å, respectively. The anomalous measurements will be used for phasing.
CobE, a protein implicated in vitamin B12 biosynthesis, from Pseudomonas aeruginosa has been overexpressed in Escherichia coli, purified and crystallized using hanging-drop vapour diffusion. The crystals belong to the primitive orthorhombic space group P212121, with unit-cell parameters a = 31.86, b = 41.07, c = 87.41 Å. The diffraction extends to a resolution of 1.9 Å. There is one molecule per asymmetric unit and the estimated solvent content is 35%. SeMet-labelled CobE has been prepared and crystallizes under the same conditions as the native protein with diffraction to 1.7 Å. The anomalous measurements will be used for phasing.
CobE; vitamin B12 biosynthesis
Recombinant cryptochrome 3 from A. thaliana with FAD and MTHF cofactors has been crystallized using the hanging-drop vapour-diffusion technique in the orthorhombic space group P212121 and X-ray diffraction data were collected to 1.9 Å resolution.
Cryptochromes are flavoproteins which serve as blue-light receptors in plants, animals, fungi and prokaryotes and belong to the same protein family as the catalytically active DNA photolyases. Cryptochrome 3 from the plant Arabidopsis thaliana (cry3; 525 amino acids, 60.7 kDa) is a representative of the novel cryDASH subfamily of UV-A/blue-light receptors and has been expressed as a mature FAD-containing protein in Escherichia coli without the signal sequence that directs the protein into plant organelles. The purified cryptochrome was found to be complexed to methenyltetrahydrofolate as an antenna pigment. Crystals of the cryptochrome–antenna pigment complex were obtained by vapour diffusion and display orthorhombic symmetry, with unit-cell parameters a = 76.298, b = 116.782, c = 135.024 Å. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The asymmetric unit comprises a cry3 dimer, the physiological role of which remains to be elucidated.
cryptochrome 3; light receptors
D. rostrata lectin was crystallized by hanging-drop vapor diffusion. The crystal belongs to the orthorhombic space group I222 and diffracted to 1.87 Å resolution.
Lectins from the Diocleinae subtribe (Leguminosae) are highly similar proteins that promote various biological activities with distinctly differing potencies. The structural basis for this experimental data is not yet fully understood. Dioclea rostrata lectin was purified and crystallized by hanging-drop vapour diffusion at 293 K. The crystal belongs to the orthorhombic space group I222, with unit-cell parameters a = 61.51, b = 88.22, c = 87.76 Å. Assuming the presence of one monomer per asymmetric unit, the solvent content was estimated to be about 47.9%. A complete data set was collected at 1.87 Å resolution.
lectins; Dioclea rostrata
Crystals of an active-site mutated hydantoin racemase from S. meliloti have been obtained in the presence and absence of d,l-5-isopropyl-hydantoin and characterized by X-ray diffraction.
A recombinant active-site mutant of hydantoin racemase (C76A) from Sinorhizobium meliloti CECT 4114 (SmeHyuA) has been crystallized in the presence and absence of the substrate d,l-5-isopropyl hydantoin. Crystals of the SmeHyuA mutant suitable for data collection and structure determination were grown using the counter-diffusion method. X-ray data were collected to resolutions of 2.17 and 1.85 Å for the free and bound enzymes, respectively. Both crystals belong to space group R3 and contain two molecules of SmeHyuA per asymmetric unit. The crystals of the free and complexed SmeHyuA have unit-cell parameters a = b = 85.43, c = 152.37 Å and a = b = 85.69, c = 154.38 Å, crystal volumes per protein weight (V
M) of 1.94 and 1.98 Å3 Da−1 and solvent contents of 36.7 and 37.9%, respectively.
hydantoin racemase; Sinorhizobium meliloti; d,l-5-isopropyl hydantoin
The hatching enzyme of zebrafish, ZHE1, was expressed, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal belonged to space group P212121 and diffracted X-rays to a resolution of 1.14 Å.
The hatching enzyme of the zebrafish, ZHE1 (29.3 kDa), is a zinc metalloprotease that catalyzes digestion of the egg envelope (chorion). ZHE1 was heterologously expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. Two diffraction data sets with resolution ranges 50.0–1.80 and 50.0–1.14 Å were independently collected from two crystals and were merged to give a highly complete data set over the full resolution range 50.0–1.14 Å. The space group was assigned as primitive orthorhombic P212121, with unit-cell parameters a = 32.9, b = 62.5, c = 87.4 Å. The crystal contained one ZHE1 molecule in the asymmetric unit.
ZHE1; hatching enzymes; zebrafish
The crystallization and preliminary X-ray diffraction analysis of a red marine alga lectin isolated from H. musciformis is reported.
HML, a lectin from the red marine alga Hypnea musciformis, defines a novel lectin family. Orthorhombic crystals of HML belonging to space group P212121 grew within three weeks at 293 K using the hanging-drop vapour-diffusion method. A complete data set was collected at 2.4 Å resolution. HML is the first marine alga lectin to be crystallized.
red marine algal lectin; Hypnea musciformis; novel lectin family
XynX, a family 10 xylanase from A. punctata ME-1, was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to beyond 1.8 Å resolution.
Xylanases catalyze the hydrolysis of β-1,4-glycosidic linkages within the xylan backbone. XynX is a xylanase from Aeromonas punctata ME-1 and belongs to glycoside hydrolase family 10. While most xylanases show endo-type catalytic activities, XynX shows exo-like catalytic activities, selectively producing xylobiose from birchwood xylan. In this study, XynX was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 79.0, b = 88.6, c = 93.2 Å, and diffracted to beyond 1.8 Å resolution.
Aeromonas punctata ME-1; glycoside hydrolase family 10; xylanases; xylobioses
ϕ29 bacteriophage scaffolding protein (gp7) has been overproduced in E. coli, purified, crystallized and characterized by X-ray diffraction. Two distinct crystal forms were obtained and a diffraction data set was collected to 1.8 Å resolution.
The Bacillus subtilis bacteriophage ϕ29 scaffolding protein (gp7) has been crystallized by the hanging-drop vapour-diffusion method at 293 K. Two new distinct crystal forms that both differed from a previously crystallized and solved scaffolding protein were grown under the same conditions. Form I belongs to the primitive tetragonal space group P41212, with unit-cell parameters a = b = 77.13, c = 37.12 Å. Form II crystals exhibit an orthorhombic crystal form, with space group C222 and unit-cell parameters a = 107.50, b = 107. 80, c = 37.34 Å. Complete data sets have been collected to 1.78 and 1.80 Å for forms I and II, respectively, at 100 K using Cu Kα X-rays from a rotating-anode generator. Calculation of a V
M value of 2.46 Å3 Da−1 for form I suggests the presence of one molecule in the asymmetric unit, corresponding to a solvent content of 50.90%, whereas form II has a V
M of 4.80 Å3 Da−1 with a solvent content of 48.76% and two molecules in the asymmetric unit. The structures of both crystal forms are being determined by the molecular-replacement method using the coordinates of the published crystal structure of gp7.
scaffolding protein; bacteriophage ϕ29