PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (30)
 

Clipboard (0)
None

Select a Filter Below

Journals
more »
Year of Publication
Document Types
1.  Crystallization and preliminary X-ray crystallographic analysis of UDP-glucuronic acid:flavonol-3-O-glucuronosyltransferase (VvGT5) from the grapevine Vitis vinifera  
The UDP-glucuronic acid:flavonol-3-O-glucuronosyltransferase (VvGT5) from the grapevine V. vinifera was purified and crystallized. The best crystal diffracted X-rays to 2.2 Å resolution and belonged to space group P6122.
Grapevine (Vitis vinifera) glycosyltransferase 5 (VvGT5) is a UDP-glucuronic acid:flavonol-3-O-glucuronosyltransferase that catalyses the 3-O-specific glucuronosylation of flavonols using UDP-glucuronic acid as a sugar donor to produce flavonol 3-O-glucosides, which are important bioactive phytochemicals. Recombinant VvGT5 expressed in Escherichia coli cells was purified and crystallized by the sitting-drop vapour-diffusion method. A full set of X-ray diffraction data was collected to 2.2 Å Bragg spacing from a single crystal using a synchrotron-radiation source. The crystal was hexagonal, belonging to space group P6122, with unit-cell parameters a = b = 102.70, c = 535.92 Å. The initial phases were determined by the molecular-replacement method.
doi:10.1107/S1744309112045095
PMCID: PMC3539707  PMID: 23295490
VvGT5; UGT; glycosyltransferase; flavonol; flavonoid
2.  Direct Evidence for Pitavastatin Induced Chromatin Structure Change in the KLF4 Gene in Endothelial Cells 
PLoS ONE  2014;9(5):e96005.
Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is mediated by Kruppel-like factor (KLF) family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs) treated with pitavastatin, and KLF4 was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (NOS3) and thrombomodulin (THBD), were suppressed by KLF4 knockdown. Myocyte enhancer factor-2 (MEF2) family activation is reported to be involved in pitavastatin-dependent KLF4 induction. We focused on MEF2C among the MEF2 family members and identified a novel functional MEF2C binding site 148 kb upstream of the KLF4 gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq) followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET), and real time chromosome conformation capture (3C) assay}, we observed that the MEF2C-bound enhancer and transcription start site (TSS) of KLF4 came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH) imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells.
doi:10.1371/journal.pone.0096005
PMCID: PMC4010393  PMID: 24797675
3.  Cross-enhancement of ANGPTL4 transcription by HIF1 alpha and PPAR beta/delta is the result of the conformational proximity of two response elements 
Genome Biology  2014;15(4):R63.
Background
Synergistic transcriptional activation by different stimuli has been reported along with a diverse array of mechanisms, but the full scope of these mechanisms has yet to be elucidated.
Results
We present a detailed investigation of hypoxia-inducible factor (HIF) 1 dependent gene expression in endothelial cells which suggests the importance of crosstalk between the peroxisome proliferator-activated receptor (PPAR) β/δ and HIF signaling axes. A migration assay shows a synergistic interaction between these two stimuli, and we identify angiopoietin-like 4 (ANGPTL4) as a common target gene by using a combination of microarray and ChIP-seq analysis. We profile changes of histone marks at enhancers under hypoxia, PPARβ/δ agonist and dual stimulations and these suggest that the spatial proximity of two response elements is the principal cause of the synergistic transcription induction. A newly developed quantitative chromosome conformation capture assay shows the quantitative change of the frequency of proximity of the two response elements.
Conclusions
To the best of our knowledge, this is the first report that two different transcription factors cooperate in transcriptional regulation in a synergistic fashion through conformational change of their common target genes.
doi:10.1186/gb-2014-15-4-r63
PMCID: PMC4053749  PMID: 24721177
4.  Crystallization and preliminary X-ray diffraction analysis of orotate phosphoribosyltransferase from the human malaria parasite Plasmodium falciparum  
Orotate phosphoribosyltransferase from Plasmodium falciparum produced in Escherichia coli was crystallized by the sitting-drop vapour-diffusion method in complex with OA and PRPP in the presence of Mg2+.
Orotate phosphoribosyltransferase (OPRT) catalyzes the Mg2+-dependent condensation of orotic acid (OA) with 5-α-d-phosphorylribose 1-diphosphate (PRPP) to yield diphosphate (PPi) and the nucleotide orotidine 5′-monophos­phate. OPRT from Plasmodium falciparum produced in Escherichia coli was crystallized by the sitting-drop vapour-diffusion method in complex with OA and PRPP in the presence of Mg2+. The crystal exhibited tetragonal symmetry, belonging to space group P41 or P43, with unit-cell parameters a = b = 49.15, c = 226.94 Å. X-ray diffraction data were collected to 2.5 Å resolution at 100 K using a synchrotron-radiation source.
doi:10.1107/S1744309111043247
PMCID: PMC3274414  PMID: 22298010
orotate phosphoribosyltransferase; Plasmodium falciparum
5.  Crystallization and preliminary X-ray diffraction analysis of mouse prostaglandin F2α synthase, AKR1B3 
Aldo-keto reductase 1B3 (AKR1B3) produced in Escherichia coli has been crystallized in complex with NADPH by the sitting-drop vapour-diffusion method.
Aldo-keto reductase 1B3 (AKR1B3) catalyzes the NADPH-dependent reduction of prostaglandin H2 (PGH2), which is a common intermediate of various prostanoids, to form PGF2α. AKR1B3 also reduces PGH2 to PGD2 in the absence of NADPH. AKR1B3 produced in Escherichia coli was crystallized in complex with NADPH by the sitting-drop vapour-diffusion method. The crystal was tetragonal, belonging to space group P41212 or P43212, with unit-cell parameters a = b = 107.62, c = 120.76 Å. X-ray diffraction data were collected to 2.4 Å resolution at 100 K using a synchrotron-radiation source.
doi:10.1107/S1744309111036165
PMCID: PMC3232157  PMID: 22139184
aldo-keto reductases; AKR1B3; prostaglandin F2α synthase
6.  Crystallization and preliminary X-ray crystallographic analysis of a helicase-like domain from a tomato mosaic virus replication protein 
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.
doi:10.1107/S174430911104231X
PMCID: PMC3232162  PMID: 22139189
tomato mosaic virus; replicase protein; helicase domain
7.  Structure of the human-heart fatty-acid-binding protein 3 in complex with the fluorescent probe 1-anilinonaphthalene-8-sulphonic acid 
Journal of Synchrotron Radiation  2013;20(Pt 6):923-928.
The crystal structure of human-heart-type fatty-acid-binding protein in complex with anilinonaphthalene-8-sulfonate was solved at 2.15 Å resolution revealing the detailed binding mechanism of the fluorescent probe 1-anilinonaphthalene-8-sulfonate.
Heart-type fatty-acid-binding protein (FABP3), which is a cytosolic protein abundantly found in cardiomyocytes, plays a role in trafficking fatty acids throughout cellular compartments by reversibly binding intracellular fatty acids with relatively high affinity. The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) is extensively utilized for examining the interaction of ligands with fatty-acid-binding proteins. The X-ray structure of FABP3 was determined in the presence of ANS and revealed the detailed ANS-binding mechanism. Furthermore, four water molecules were clearly identified in the binding cavity. Through these water molecules, the bound ANS molecule forms indirect hydrogen-bond interactions with FABP3. The adipocyte-type fatty-acid-binding protein (FABP4) exhibits 67% sequence identity with FABP3 and its crystal structure is almost the same as that of FABP3. However, FABP4 can bind with a higher affinity to ANS than FABP3. To understand the difference in their ligand specificities, a structural comparison was performed between FABP3–ANS and FABP4–ANS complexes. The result revealed that the orientation of ANS binding to FABP3 is completely opposite to that of ANS binding to FABP4, and the substitution of valine in FABP4 to leucine in FABP3 may result in greater steric hindrance between the side-chain of Leu115 and the aniline ring of ANS.
doi:10.1107/S0909049513021298
PMCID: PMC3795557  PMID: 24121341
X-ray structure; FABP3–ANS complex; human-heart fatty-acid-binding protein
8.  Cloning, expression, purification, crystallization and preliminary X-ray crystallo­graphic study of GK0767, the copper-containing nitrite reductase from Geobacillus kaustophilus  
The Cu-containing nitrite reductase from G. kaustophilus has been overexpressed, purified and crystallized in space group R3. The crystals diffracted to 1.3 Å resolution.
The soluble region (residues 32–354) of GK0767, a copper-containing nitrite reductase from the thermophilic Gram-positive bacterium Geobacillus kaustophilus HTA426, has been cloned and overexpressed in Escherichia coli. The purified recombinant protein was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected and processed to a maximum resolution of 1.3 Å. The crystals belonged to space group R3, with unit-cell parameters a = b = 115.1, c = 87.5 Å. Preliminary studies and molecular-replacement calculations reveal the presence of one subunit of the homotrimeric structure in the asymmetric unit; this corresponds to a V M value of 3.14 Å3 Da−1.
doi:10.1107/S1744309111013297
PMCID: PMC3107145  PMID: 21636914
nitrite reductases; copper; denitrification; Gram-positive bacteria
9.  Dynamic Change of Chromatin Conformation in Response to Hypoxia Enhances the Expression of GLUT3 (SLC2A3) by Cooperative Interaction of Hypoxia-Inducible Factor 1 and KDM3A 
Molecular and Cellular Biology  2012;32(15):3018-3032.
Hypoxia-inducible factor 1 (HIF1) is a master regulator of adaptive gene expression under hypoxia. However, a role for HIF1 in the epigenetic regulation remains unknown. Genome-wide analysis of HIF1 binding sites (chromatin immunoprecipitation [ChIP] with deep sequencing) of endothelial cells clarified that HIF1 mainly binds to the intergenic regions distal from transcriptional starting sites under both normoxia and hypoxia. Next, we examined the temporal profile of gene expression under hypoxic conditions by using DNA microarrays. We clarified that early hypoxia-responsive genes are functionally associated with glycolysis, including GLUT3 (SLC2A3). Acetylated lysine 27 of histone 3 covered the HIF1 binding sites, and HIF1 functioned as an enhancer of SLC2A3 by interaction with lysine (K)-specific demethylase 3A (KDM3A). Knockdown of HIF1α and KDM3A showed that glycolytic genes are regulated by both HIF1 and KDM3A and respond to hypoxia in a manner independent of cell type specificity. We elucidated that both the chromatin conformational structure and histone modification change under hypoxic conditions and enhance the expression of SLC2A3 based on the combined results of chromatin conformation capture (3C) and ChIP assays. KDM3A is recruited to the SLC2A3 locus in an HIF1-dependent manner and demethylates H3K9me2 so as to upregulate its expression. These findings provide novel insights into the interaction between HIF1 and KDM3A and also the epigenetic regulation of HIF1.
doi:10.1128/MCB.06643-11
PMCID: PMC3434521  PMID: 22645302
10.  Hematopoietic prostaglandin D synthase (HPGDS): a high stability, Val187Ile isoenzyme common among African Americans and its relationship to risk for colorectal cancer 
Intestinal tumors in ApcMin/+ mice are suppressed by over-production of HPGDS, which is a glutathione transferase that forms prostaglandin D2 (PGD2). We characterized naturally occurring HPGDS isoenzymes, to see if HPGDS variation is associated with human colorectal cancer risk. We used DNA heteroduplex analysis and sequencing to identify HPGDS variants among healthy individuals. HPGDS isoenzymes were produced in bacteria, and their catalytic activities were tested. To determine in vivo effects, we conducted pooled case-control analyses to assess whether there is an association of the isoenzyme with colorectal cancer. Roughly 8% of African Americans and 2% of Caucasians had a highly stable Val187lle isoenzyme (with isoleucine instead of valine at position 187). At 37 °C, the wild-type enzyme lost 15% of its activity in one hour, whereas the Val187Ile form remained >95% active. At 50 °C, the half life of native HPGDS was 9 minutes, compared to 42 minutes for Val187Ile. The odds ratio for colorectal cancer among African Americans with Val187Ile was 1.10 (95% CI, 0.75–1.62; 533 cases, 795 controls). Thus, the Val187Ile HPGDS isoenzyme common among African Americans is not associated with colorectal cancer risk. Other approaches will be needed to establish a role for HPGDS in occurrence of human intestinal tumors, as indicated by a mouse model.
doi:10.1016/j.prostaglandins.2011.07.006
PMCID: PMC3226866  PMID: 21821144
glutathione transferase; HPGDS; prostaglandin D2; colon cancer
11.  Crystal Structure of the Superfamily 1 Helicase from Tomato Mosaic Virus 
Journal of Virology  2012;86(14):7565-7576.
The genomes of the Tomato mosaic virus and many other plant and animal positive-strand RNA viruses of agronomic and medical importance encode superfamily 1 helicases. Although helicases play important roles in viral replication, the crystal structures of viral superfamily 1 helicases have not been determined. Here, we report the crystal structure of a fragment (S666 to Q1116) of the replication protein from Tomato mosaic virus. The structure reveals a novel N-terminal domain tightly associated with a helicase core. The helicase core contains two RecA-like α/β domains without any of the accessory domain insertions that are found in other superfamily 1 helicases. The N-terminal domain contains a flexible loop, a long α-helix, and an antiparallel six-stranded β-sheet. On the basis of the structure, we constructed deletion mutants of the S666-to-Q1116 fragment and performed split-ubiquitin-based interaction assays in Saccharomyces cerevisiae with TOM1 and ARL8, host proteins that are essential for tomato mosaic virus RNA replication. The results suggested that both TOM1 and ARL8 interact with the long α-helix in the N-terminal domain and that TOM1 also interacts with the helicase core. Prediction of secondary structures in other viral superfamily 1 helicases and comparison of those structures with the S666-to-Q1116 structure suggested that these helicases have a similar fold. Our results provide a structural basis of viral superfamily 1 helicases.
doi:10.1128/JVI.00118-12
PMCID: PMC3416300  PMID: 22573863
12.  Hyperpolarization-activated cyclic nucleotide gated channels: a potential molecular link between epileptic seizures and Aβ generation in Alzheimer’s disease 
Background
One of the best-characterized causative factors of Alzheimer’s disease (AD) is the generation of amyloid-β peptide (Aβ). AD subjects are at high risk of epileptic seizures accompanied by aberrant neuronal excitability, which in itself enhances Aβ generation. However, the molecular linkage between epileptic seizures and Aβ generation in AD remains unclear.
Results
X11 and X11-like (X11L) gene knockout mice suffered from epileptic seizures, along with a malfunction of hyperpolarization-activated cyclic nucleotide gated (HCN) channels. Genetic ablation of HCN1 in mice and HCN1 channel blockage in cultured Neuro2a (N2a) cells enhanced Aβ generation. Interestingly, HCN1 levels dramatically decreased in the temporal lobe of cynomolgus monkeys (Macaca fascicularis) during aging and were significantly diminished in the temporal lobe of sporadic AD patients.
Conclusion
Because HCN1 associates with amyloid-β precursor protein (APP) and X11/X11L in the brain, genetic deficiency of X11/X11L may induce aberrant HCN1 distribution along with epilepsy. Moreover, the reduction in HCN1 levels in aged primates may contribute to augmented Aβ generation. Taken together, HCN1 is proposed to play an important role in the molecular linkage between epileptic seizures and Aβ generation, and in the aggravation of sporadic AD.
doi:10.1186/1750-1326-7-50
PMCID: PMC3524764  PMID: 23034178
13.  Three-dimensional, non-invasive, cross-sectional imaging of protein crystals using ultrahigh resolution optical coherence tomography 
Biomedical Optics Express  2012;3(4):735-740.
Micro-scale, non-invasive, three-dimensional cross-sectional imaging of protein crystals was successfully accomplished using ultra-high resolution optical coherence tomography (UHR-OCT) with low noise, Gaussian like supercontinuum. This technique facilitated visualization of protein crystals even those in medium that also contained substantial amounts of precipitates. We found the enhancement of the scattered signal from protein crystal by inclusion of agarose gel in the crystallization medium. Crystals of a protein and a salt in the same sample when visualized by UHR-OCT showed distinct physical characteristics, suggesting that protein and salt crystals may, in general, be distinguishable by UHR-OCT. UHR-OCT is a nondestructive and rapid method, which should therefore find use in automated systems designed to visualize crystals.
doi:10.1364/BOE.3.000735
PMCID: PMC3345802  PMID: 22574261
(110.4500) Optical coherence tomography; (170.3880) Medical and biological imaging
14.  Structure of hyperthermophilic β-glucosidase from Pyrococcus furiosus  
Recombinant hyperthermophilic β-glucosidase from P. furiosus was crystallized. The crystal structure was solved to a resolution of 2.35 Å.
Three categories of cellulases, endoglucanases, cellobiohydrolases and β-glucosidases, are commonly used in the process of cellulose saccharification. In particular, the activity and characteristics of hyperthermophilic β-glucosidase make it promising in industrial applications of biomass. In this paper, the crystal structure of the hyperthermophilic β-glucosidase from Pyrococcus furiosus (BGLPf) was determined at 2.35 Å resolution in a new crystal form. The structure showed that there is one tetramer in the asymmetric unit and that the dimeric molecule exhibits a structure that is stable towards sodium dodecyl sulfate (SDS). The dimeric molecule migrated in reducing SDS polyacrylamide gel electrophoresis (SDS–PAGE) buffer even after boiling at 368 K. Energy calculations demonstrated that one of the two dimer interfaces acquired the largest solvation free energy. Structural comparison and sequence alignment with mesophilic β-glucosidase A from Clostridium cellulovorans (BGLACc) revealed that the elongation at the C-terminal end forms a hydrophobic patch at the dimer interface that might contribute to hyperthermostability.
doi:10.1107/S1744309111035238
PMCID: PMC3232120  PMID: 22139147
hyperthermophilic; cellulases; biomass; Pyrococcus furiosus
15.  Metabolic Control of Vesicular Glutamate Transport and Release 
Neuron  2010;68(1):99-112.
Fasting has been used to control epilepsy since antiquity, but the mechanism of coupling between metabolic state and excitatory neurotransmission remains unknown. Previous work has shown that the vesicular glutamate transporters (VGLUTs) required for exocytotic release of glutamate undergo an unusual form of regulation by Cl−. Using functional reconstitution of the purified VGLUTs into proteoliposomes, we now show that Cl− acts as an allosteric activator, and the ketone bodies that increase with fasting inhibit glutamate release by competing with Cl− at the site of allosteric regulation. Consistent with these observations, acetoacetate reduced quantal size at hippocampal synapses, and suppresses glutamate release and seizures evoked with 4-aminopyridine in the brain. The results indicate an unsuspected link between metabolic state and excitatory neurotransmission through anion-dependent regulation of VGLUT activity.
doi:10.1016/j.neuron.2010.09.002
PMCID: PMC2978156  PMID: 20920794
vesicular glutamate transporter; chloride; acetoacetate; epilepsy; ketone body; glutamatergic neurotransmission
16.  Crystallization and preliminary X-ray analysis of 2,3-diketo-5-methylthiopentyl-1-phosphate enolase from Bacillus subtilis  
Crystals of the 45.1 kDa functional form of 2,3-diketo-5-methylthiopentyl-1-phosphate enolase from B. subtilis diffracted to 2.30 Å resolution.
2,3-Diketo-5-methylthiopentyl-1-phosphate enolase (DK-MTP-1P enolase) from Bacillus subtilis was crystallized using the hanging-drop vapour-diffusion method. Crystals grew using PEG 3350 as the precipitant at 293 K. The crystals diffracted to 2.3 Å resolution at 100 K using synchrotron radiation and were found to belong to the monoclinic space group P21, with unit-cell parameters a = 79.3, b = 91.5, c = 107.0 Å, β = 90.8°. The asymmetric unit contained four molecules of DK-MTP-1P enolase, with a V M value of 2.2 Å3 Da−1 and a solvent content of 43%.
doi:10.1107/S174430910804311X
PMCID: PMC2635871  PMID: 19194007
methionine-salvage pathway; Bacillus subtilis; RuBisCO; RuBisCO-like proteins; 2,3-diketo-5-methylthiopentyl-1-phosphate enolase
17.  Crystallization and preliminary X-ray crystallographic analysis of Ca2+-free primary Ca2+-sensor of Na+/Ca2+ exchanger 
The plasma-membrane Na+/Ca2+ exchanger (NCX) regulates intracellular Ca2+ levels in cardiac myocytes. Two Ca2+-binding domains (CBD1 and CBD2) exist in the large cytosolic loop of NCX. Recombinant CBD1 (NCX1 372–508) with a molecular weight of 16 kDa has been crystallized by the sitting-drop vapour-diffusion method at 293 K.
The plasma-membrane Na+/Ca2+ exchanger (NCX) regulates intracellular Ca2+ levels in cardiac myocytes. Two Ca2+-binding domains (CBD1 and CBD2) exist in the large cytosolic loop of NCX. The binding of Ca2+ to CBD1 results in conformational changes that stimulate exchange to exclude Ca2+ ions, whereas CBD2 maintains the structure, suggesting that CBD1 is the primary Ca2+-sensor. In order to clarify the structural scaffold for the Ca2+-induced conformational transition of CBD1 at the atomic level, X-ray structural analysis of its Ca2+-free form was attempted; the structure of the Ca2+-bound form is already available. Recombinant CBD1 (NCX1 372–508) with a molecular weight of 16 kDa was crystallized by the sitting-drop vapour-diffusion method at 293 K. The crystals belonged to the hexagonal space group P6222 or P6422, with unit-cell parameters a = b = 56.99, c = 153.86 Å, β = 120°, and contained one molecule per asymmetric unit (V M = 2.25 Å3 Da−1) with a solvent content of about 55% (V S = 45.57%). Diffraction data were collected within the resolution range 27.72–3.00 Å using an R-AXIS detector and gave a data set with an overall R merge of 10.8% and a completeness of 92.8%.
doi:10.1107/S1744309108032934
PMCID: PMC2593703  PMID: 19052365
Na+/Ca2+ exchanger; Ca2+-sensors
18.  Structure of the inhibitor complex of old yellow enzyme from Trypanosoma cruzi  
Journal of Synchrotron Radiation  2010;18(Pt 1):66-69.
The structures of old yellow enzyme from Trypanosoma cruzi which produces prostaglandin F2α from PGH2 have been determined in the presence or absence of menadione.
Old yellow enzyme (OYE) is an NADPH oxidoreductase which contains flavin mononucleotide as prosthetic group. The X-ray structures of OYE from Trypanosoma cruzi (TcOYE) which produces prostaglandin (PG) F2α from PGH2 have been determined in the presence or absence of menadione. The binding motif of menadione, known as one of the inhibitors for TcOYE, should accelerate the structure-based development of novel anti-chagasic drugs that inhibit PGF2α production specifically.
doi:10.1107/S0909049510033595
PMCID: PMC3004258  PMID: 21169695
X-ray structure; inhibitor complex; prostaglandin synthase
19.  Approach for growth of high-quality and large protein crystals 
Journal of Synchrotron Radiation  2010;18(Pt 1):16-19.
Three crystallization methods, including crystallization in the presence of a semi-solid agarose gel, top-seeded solution growth (TSSG) and a large-scale hanging-drop method, have previously been presented. In this study, crystallization has been further evaluated in the presence of a semi-solid agarose gel by crystallizing additional proteins. A novel crystallization method combining TSSG and the large-scale hanging-drop method has also been developed.
Three crystallization methods for growing large high-quality protein crystals, i.e. crystallization in the presence of a semi-solid agarose gel, top-seeded solution growth (TSSG) and a large-scale hanging-drop method, have previously been presented. In this study the effectiveness of crystallization in the presence of a semi-solid agarose gel has been further evaluated by crystallizing additional proteins in the presence of 2.0% (w/v) agarose gel, resulting in complete gelification with high mechanical strength. In TSSG the seed crystals are hung by a seed holder protruding from the top of the growth vessel to prevent polycrystallization. In the large-scale hanging-drop method, a cut pipette tip was used to maintain large-scale droplets consisting of protein–precipitant solution. Here a novel crystallization method that combines TSSG and the large-scale hanging-drop method is reported. A large and single crystal of lysozyme was obtained by this method.
doi:10.1107/S090904951003445X
PMCID: PMC3004246  PMID: 21169683
semi-solid agarose gels; top-seeded solution growth; large-scale hanging-drop method; X-ray crystallography; neutron crystallography
20.  Crystallization and preliminary neutron diffraction studies of HIV-1 protease cocrystallized with inhibitor KNI-272 
In order to determine the protonation states of the residues within the active site of an HIV-1 protease–inhibitor complex, a crystal of HIV-1 protease complexed with inhibitor (KNI-272) was grown to a size of 1.4 mm3 for neutron diffraction study. The crystal diffracted to 2.3 Å resolution with sufficient quality for further structure determination.
This paper reports the crystallization and preliminary neutron diffraction measurements of HIV-1 protease, a potential target for anti-HIV therapy, complexed with an inhibitor (KNI-272). The aim of this neutron diffraction study is to obtain structural information about the H atoms and to determine the protonation states of the residues within the active site. The crystal was grown to a size of 1.4 mm3 by repeated macroseeding and a slow-cooling method using a two-liquid system. Neutron diffraction data were collected at room temperature using a BIX-4 diffractometer at the JRR-3 research reactor of the Japan Atomic Energy Agency (JAEA). The data set was integrated and scaled to 2.3 Å resolution in space group P21212, with unit-cell parameters a = 59.5, b = 87.4, c = 46.8 Å.
doi:10.1107/S1744309108029679
PMCID: PMC2581681  PMID: 18997326
HIV-1 protease; inhibitors; neutron diffraction
21.  Crystallization and preliminary X-ray diffraction studies of an RNA aptamer in complex with the human IgG Fc fragment 
An RNA aptamer in complex with the human IgG Fc fragment have been crystallized. The stirring technique with a rotary shaker was used to improve the crystals and to ensure that they were of high quality and single, resulting in crystals that diffracted to 2.2 Å resolution.
Aptamers, which are folded DNA or RNA molecules, bind to target molecules with high affinity and specificity. An RNA aptamer specific for the Fc fragment of human immunoglobulin G (IgG) has recently been identified and it has been demonstrated that an optimized 24-nucleotide RNA aptamer binds to the Fc fragment of human IgG and not to other species. In order to clarify the structural basis of the high specificity of the RNA aptamer, it was crystallized in complex with the Fc fragment of human IgG1. Preliminary X-ray diffraction studies revealed that the crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 83.7, b = 107.2, c = 79.0 Å. A data set has been collected to 2.2 Å resolution.
doi:10.1107/S1744309108028236
PMCID: PMC2564881  PMID: 18931441
RNA aptamers; Fc fragments; immunoglobulin G
22.  Conformational plasticity of RNA for target recognition as revealed by the 2.15 Å crystal structure of a human IgG–aptamer complex 
Nucleic Acids Research  2010;38(21):7822-7829.
Aptamers are short single-stranded nucleic acids with high affinity to target molecules and are applicable to therapeutics and diagnostics. Regardless of an increasing number of reported aptamers, the structural basis of the interaction of RNA aptamer with proteins is poorly understood. Here, we determined the 2.15 Å crystal structure of the Fc fragment of human IgG1 (hFc1) complexed with an anti-Fc RNA aptamer. The aptamer adopts a characteristic structure fit to hFc1 that is stabilized by a calcium ion, and the binding activity of the aptamer can be controlled many times by calcium chelation and addition. Importantly, the aptamer–hFc1 interaction involves mainly van der Waals contacts and hydrogen bonds rather than electrostatic forces, in contrast to other known aptamer–protein complexes. Moreover, the aptamer–hFc1 interaction involves human IgG-specific amino acids, rendering the aptamer specific to human IgGs, and not crossreactive to other species IgGs. Hence, the aptamer is a potent alternative for protein A affinity purification of Fc-fusion proteins and therapeutic antibodies. These results demonstrate, from a structural viewpoint, that conformational plasticity and selectivity of an RNA aptamer is achieved by multiple interactions other than electrostatic forces, which is applicable to many protein targets of low or no affinity to nucleic acids.
doi:10.1093/nar/gkq615
PMCID: PMC2995045  PMID: 20675355
23.  Preparation, crystallization and preliminary crystallographic analysis of old yellow enzyme from Trypanosoma cruzi  
Old yellow enzyme from Trypanosoma cruzi, has been crystallized using the hanging-drop vapour-diffusion method.
Old yellow enzyme (OYE) is an NADPH oxidoreductase that contains a flavin mononucleotide as a prosthetic group. The OYE from Trypanosoma cruzi, which produces prostaglandin F2α, a potent mediator of various physiological and pathological processes, from prostaglandin H2. The protein was recombinantly expressed and purified from Escherichia coli and was crystallized using the hanging-drop vapour-diffusion method. The crystal belongs to the monoclinic space group P21, with unit-cell parameters a = 56.3, b = 78.8, c = 78.8 Å, β = 93.4° and two molecules per asymmetric unit. The crystals were suitable for X-ray crystallographic studies and diffracted to 1.70 Å resolution. A Patterson search method is in progress using the structure of OYE from Pseudomonas putida as a starting model.
doi:10.1107/S1744309107044879
PMCID: PMC2339734  PMID: 17909300
old yellow enzyme; NADPH oxidoreductases
24.  Crystallization and preliminary crystallographic analysis of orotidine 5′-monophosphate decarboxylase from the human malaria parasite Plasmodium falciparum  
Orotidine 5′-monophosphate decarboxylase of human malaria parasite P. falciparum was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation.
Orotidine 5′-monophosphate (OMP) decarboxylase (OMPDC; EC 4.1.1.23) catalyzes the final step in the de novo synthesis of uridine 5′-monophosphate (UMP) and defects in the enzyme are lethal in the malaria parasite Plasmodium falciparum. Active recombinant P. falciparum OMPDC (PfOMPDC) was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation at the Swiss Light Source. The crystal exhibits trigonal symmetry (space group R3), with hexagonal unit-cell parameters a = b = 201.81, c = 44.03 Å. With a dimer in the asymmetric unit, the solvent content is 46% (V M = 2.3 Å3 Da−1).
doi:10.1107/S1744309106015594
PMCID: PMC2243097  PMID: 16754976
orotidine 5′-monophosphate decarboxylase; Plasmodium falciparum
25.  Crystallization and preliminary X-ray analysis of the tRNA thiolation enzyme MnmA from Escherichia coli complexed with tRNAGlu  
The RNA thiouridylase MnmA in complex with tRNA was crystallized with and without ATP in three different crystal forms, which may reflect distinct sulfuration-reaction stages.
MnmA catalyzes a sulfuration reaction to synthesize 2-thiouridine at the wobble positions of tRNAGlu, tRNAGln and tRNALys in Escherichia coli. The binary complex of MnmA and tRNAGlu was crystallized in two different crystal forms: forms I and II. Cocrystallization of MnmA–tRNAGlu with ATP yielded form III crystals. The three crystal forms diffracted to 3.1, 3.4 and 3.4 Å resolution, respectively, using synchrotron radiation at SPring-8. These crystals belong to space groups C2, I212121 and C2, with unit-cell parameters a = 225.4, b = 175.8, c = 53.0 Å, β = 101.6°, a = 101.5, b = 108.0, c = 211.2 Å and a = 238.1, b = 102.1, c = 108.2 Å, β = 117.0°, respectively. The asymmetric units of these crystals are expected to contain two, one and two MnmA–tRNAGlu complexes, respectively.
doi:10.1107/S174430910600738X
PMCID: PMC2222564  PMID: 16582487
MnmA; tRNA; ATP; Escherichia coli

Results 1-25 (30)