The adiponectin receptors (AdipoR1 and AdipoR2) are membrane proteins with seven transmembrane helices. These receptors regulate glucose and fatty acid metabolism, thereby ameliorating type 2 diabetes. The full-length human AdipoR1 and a series of N-terminally truncated mutants of human AdipoR1 and AdipoR2 were expressed in insect cells. In small-scale size exclusion chromatography, the truncated mutants AdipoR1Δ88 (residues 89–375) and AdipoR2Δ99 (residues 100–386) eluted mostly in the intact monodisperse state, while the others eluted primarily as aggregates. However, gel filtration chromatography of the large-scale preparation of the tag-affinity-purified AdipoR1Δ88 revealed the presence of an excessive amount of the aggregated state over the intact state. Since aggregation due to contaminating nucleic acids may have occurred during the sample concentration step, anion-exchange column chromatography was performed immediately after affinity chromatography, to separate the intact AdipoR1Δ88 from the aggregating species. The separated intact AdipoR1Δ88 did not undergo further aggregation, and was successfully purified to homogeneity by gel filtration chromatography. The purified AdipoR1Δ88 and AdipoR2Δ99 proteins were characterized by thermostability assays with 7-diethylamino-3-(4-maleimidophenyl)-4-methyl coumarin, thin layer chromatography of bound lipids, and surface plasmon resonance analysis of ligand binding, demonstrating their structural integrities. The AdipoR1Δ88 and AdipoR2Δ99 proteins were crystallized with the anti-AdipoR1 monoclonal antibody Fv fragment, by the lipidic mesophase method. X-ray diffraction data sets were obtained at resolutions of 2.8 and 2.4 Å, respectively.
Membrane protein; Adiponectin receptors AdipoR1 and AdipoR2; Purification; Antibody; Crystallization; Lipidic mesophase
Stenotrophomonas maltophilia (SM) is an important nosocomial pathogen that exhibits intrinsic resistance to various antimicrobial agents. However, the risk factors for SM bacteraemia have not been sufficiently evaluated. From January 2005 to September 2012, we retrospectively compared the clinical backgrounds and outcomes of SM bacteraemic patients (SM group) with those of bacteraemic patients due to Pseudomonas aeruginosa (PA group) or Acinetobacter species (AC group). DNA genotyping of the SM isolates using the Diversilab system was performed to investigate the genetic relationships among the isolates. The SM, PA, and AC groups included 54, 167, and 69 patients, respectively. Nine of 17 patients in the SM group receiving trimethoprim-sulfamethoxazole prophylaxis developed SM bacteraemia. Independent risk factors for SM bacteraemia were the use of carbapenems and antipseudomonal cephalosporins and SM isolation within 30 days prior to the onset of bacteraemia. Earlier SM isolation was observed in 32 of 48 patients (66.7%) with SM bacteraemia who underwent clinical microbiological examinations. Of these 32 patients, 15 patients (46.9%) had the same focus of bacteraemia as was found in the previous isolation site. The 30-day all-cause mortality rate among the SM group (33.3%) was higher than that of the PA group (21.5%, p = 0.080) and the AC group (17.3%, p = 0.041). The independent factor that was associated with 30-day mortality was the SOFA score. DNA genotyping of SM isolates and epidemiological data suggested that no outbreak had occurred. SM bacteraemia was associated with high mortality and should be considered in patients with recent use of broad-spectrum antibiotics or in patients with recent isolation of the organism.
Escherichia coli sequence type 131 (ST131) and ST405 are important clonal groups, because they are associated with the global increase of extended-spectrum-β-lactamase (ESBL) producers. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is emerging as a rapid, inexpensive, and accurate method for bacterial identification. We investigated the detection performance of MALDI-TOF for the ST131 and ST405 clonal groups using 41 ST131-O25b, 26 ST131-O16, and 41 ST405 ESBL-producing isolates and 41 ESBL-producing isolates frrom other STs. The main spectra representing each clonal group were used for classification with Biotyper (Bruker Daltonics GmbH, Bremen, Germany). The peak that had the highest area under the receiver-operating characteristic curve generated by ClinProTools (Bruker) was detected with FlexAnalysis (Bruker), and an optimal signal-to-noise ratio cutoff was determined. The optimal detection models were generated by ClinProTools. Classification by Biotyper could detect the ST131-whole (O25b and O16 together) group with a sensitivity of 98.5% and a specificity of 93.9%. With FlexAnalysis, a peak of 9,720 Da detected the ST131-whole group with a sensitivity of 97.0% and a specificity of 91.5% at a cutoff value of 8.0. The ClinProTools models exhibited good performance for the detection of the ST131-whole group (sensitivity and specificity, 94.0% and 92.7%, respectively), the ST131-O25b group (95.1% and 98.2%, respectively), and the ST405 group (90.2% and 96.3%, respectively). MALDI-TOF MS had high detection performance for the ST131-whole, ST131-O25b, and ST405 clonal groups. MALDI-TOF MS should be considered as an alternative method to monitor the epidemiology of the ESBL-producing E. coli ST131 and ST405 clonal groups.
The global increase of extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli is associated with the specific clonal group sequence type 131 (ST131). In order to understand the successful spread of ESBL-producing E. coli clonal groups, we characterized fluoroquinolone resistance determinants, virulence genotypes, and plasmid replicons of ST131 and another global clonal group, ST405. We investigated 41 ST131-O25b, 26 ST131-O16, 41 ST405, and 41 other ST (OST) ESBL-producing isolates, which were collected at seven acute care hospitals in Japan. The detection of ESBL types, fluoroquinolone resistance-associated mutations (including quinolone resistance-determining regions [QRDRs]), virulence genotypes, plasmid replicon types, and IncF replicon sequence types was performed using PCR and sequencing. blaCTX-M, specifically blaCTX-M-14, was the most common ESBL gene type among the four groups. Ciprofloxacin resistance was found in 90% of ST131-O25b, 19% of ST131-O16, 100% of ST405, and 54% of OST isolates. Multidrug resistance was more common in the ST405 group than in the ST131-O25 group (56% versus 32%; P = 0.045). All ST131-O25b isolates except one had four characteristic mutations in QRDRs, but most of the isolates from the other three groups had three mutations in common. The ST131-O25b and ST405 groups had larger numbers of virulence genes than the OST group. All of the ST131-O25b and ST405 isolates and most of the ST131-O16 and OST isolates carried IncF replicons. The most prevalent IncF replicon sequence types differed between the four clonal groups. Both the ST131-O25b and ST405 clonal groups had a fluoroquinolone resistance mechanism in QRDRs, multidrug resistance, high virulence, and IncF plasmids, suggesting the potential for further global expansion and a need for measures against these clonal groups.
We report two cases of graft migration during open stent grafting, detected by transesophageal echocardiography (TEE). The incidence was 3.7% in our series. The length of landing zone was reduced from 45 mm to 25 mm in case 1 and from 50 mm to 22 mm in case 2 before chest closure. Aneurysmal protrusion on the greater curvature with thin mural thrombus were findings common in both cases. Although additional intervention was not done based on the TEE findings of no endoleak and thrombus formation in the aneurysm, and postoperative course was uneventful, meticulous imaging check-up was needed.
stent graft; complication; echocardiography
We report a crystal structure of Hfq and catalase HPII from Escherichia coli. The post-transcriptional regulator Hfq plays a key role in the survival of bacteria under stress. A small non-coding RNA (sRNA) DsrA is required for translation of the stationary phase sigma factor RpoS, which is the central regulator of the general stress response. Hfq facilitates efficient translation of rpoS mRNA, which encodes RpoS. Hfq helps in the function of other specific proteins involved in RNA processing, indicating its versatility in the cell. However, structural information regarding its interactions with partners is missing. Here we obtained crystals of Hfq and HPII complexes from cell lysates following attempts to overexpress a foreign membrane protein. HPII is one of two catalases in E. coli and its mRNA is transcribed by an RNA polymerase holoenzyme containing RpoS, which in turn is under positive control of small non-coding RNAs and of the RNA chaperone Hfq. This sigma factor is known to have a pronounced effect on the expression of HPII. The crystal structure reveals that a Hfq hexamer binds each subunit of a HPII tetramer. Each subunit of the Hfq hexamer exhibits a unique binding mode with HPII. The hexamer of Hfq interacts via its distal surface. The proximal and distal surfaces are known to specifically bind different sRNAs, and binding of HPII could affect Hfq function. Hfq-HPII complexation has no effect on catalase HPII activity.
SPring-8 BL41XU provides a high-flux X-ray beam of size 10–50 µm, and enables high-quality diffraction data to be obtained from various types of protein crystals. Details of this beamline and an upgrade project are described.
SPring-8 BL41XU is a high-flux macromolecular crystallography beamline using an in-vacuum undulator as a light source. The X-rays are monochromated by a liquid-nitrogen-cooling Si double-crystal monochromator, and focused by Kirkpatrick–Baez mirror optics. The focused beam size at the sample is 80 µm (H) × 22 µm (V) with a photon flux of 1.1 × 1013 photons s−1. A pinhole aperture is used to collimate the beam in the range 10–50 µm. This high-flux beam with variable size provides opportunities not only for micro-crystallography but also for data collection effectively making use of crystal volume. The beamline also provides high-energy X-rays covering 20.6–35.4 keV which allows ultra-high-resolution data to be obtained and anomalous diffraction using the K-edge of Xe and I. Upgrade of BL41XU for more rapid and accurate data collection is proceeding. Here, details of BL41XU are given and an outline of the upgrade project is documented.
macromolecular crystallography; micro-crystallography; high-flux beam; high-energy beam; SPring-8
An online UV–visible microspectrophotometer has been developed for the macromolecular crystallography beamline at SPring-8. Details of this spectrophotometer are reported.
Measurement of the UV–visible absorption spectrum is a convenient technique for detecting chemical changes of proteins, and it is therefore useful to combine spectroscopy and diffraction studies. An online microspectrophotometer for the UV–visible region was developed and installed on the macromolecular crystallography beamline, BL38B1, at SPring-8. This spectrophotometer is equipped with a difference dispersive double monochromator, a mercury–xenon lamp as the light source, and a photomultiplier as the detector. The optical path is mostly constructed using mirrors, in order to obtain high brightness in the UV region, and the confocal optics are assembled using a cross-slit diaphragm like an iris to eliminate stray light. This system can measure optical densities up to a maximum of 4.0. To study the effect of radiation damage, preliminary measurements of glucose isomerase and thaumatin crystals were conducted in the UV region. Spectral changes dependent on X-ray dose were observed at around 280 nm, suggesting that structural changes involving Trp or Tyr residues occurred in the protein crystal. In the case of the thaumatin crystal, a broad peak around 400 nm was also generated after X-ray irradiation, suggesting the cleavage of a disulfide bond. Dose-dependent spectral changes were also observed in cryo-solutions alone, and these changes differed with the composition of the cryo-solution. These responses in the UV region are informative regarding the state of the sample; consequently, this device might be useful for X-ray crystallography.
UV–visible spectroscopy; protein crystallography; radiation damage; microspectroscopy; SPring-8
The latest revolution in macromolecular crystallography was incited by the development of dedicated, user friendly, micro-crystallography beamlines. Brilliant X-ray beams of diameter 20 microns or less, now available at most synchrotron sources, enable structure determination from samples that previously were inaccessible. Relative to traditional crystallography, crystals with one or more small dimensions have diffraction patterns with vastly improved signal-to-noise when recorded with an appropriately matched beam size. Structures can be solved from isolated, well diffracting regions within inhomogeneous samples. This review summarizes the technological requirements and approaches to producing micro-beams and how they continue to change the practice of crystallography.
The incidence of fungaemia has been increasing worldwide. It is important to distinguish non-Candida fungaemia from candidaemia because of their different antifungal susceptibilities. The aims of this study were to investigate the clinical characteristics of non-Candida fungaemia and identify the clinical factors that differentiate it from candidaemia.
We investigated the clinical manifestations and mortality of non-Candida fungaemia in Kyoto University Hospital from 2004 to 2009.
There were 110 episodes of fungaemia during the study period. There were 11 renal replacement therapy episodes of fungaemia due to non-Candida yeasts (10.0%), including 6 episodes with Cryptococcus neoformans, 4 with Trichosporon asahii, and 1 with Kodamaea ohmeri, in addition to 99 episodes of candidaemia (90.0%). The presence of collagen disease [odds ratio (OR) 9.00; 95% confidence interval (CI) 1.58-51.4; P = 0.01] or renal replacement therapy (OR 15.0; 95% CI 3.06-73.4; P < 0.01) was significantly more common in non-Candida fungaemia patients than in candidaemia patients. Prior colonisation by the species may be a predictor of non-Candida fungaemia. Non-Candida fungaemia had a higher mortality than candidaemia (54.5% versus 21.2%, P = 0.03).
Although Candida species frequently cause fungaemia, we should also be aware of non-Candida yeasts because of their high mortality, particularly among high-risk patients, such as those with collagen disease and those under renal replacement therapy. Prior colonisation by the causative organisms may be an important predictor of non-Candida fungaemia.
Fungaemia; Non-Candida yeast; Risk factor; Mortality; Colonisation
Biliverdin reductase (BVR) from Synechocystis sp. PCC6803 and its selenomethionine derivative were overexpressed and purified. X-ray diffraction data from an SeMet BVR microcrystal were collected to 3.0 Å resolution on microfocus beamline BL32XU at SPring-8.
Biliverdin reductase (BVR) catalyzes the conversion of biliverdin IX α to bilirubin IX α with concomitant oxidation of an NADH or NADPH cofactor. This enzyme also binds DNA and enhances the transcription of specific genes. Recombinant cyanobacterial BVR was overexpressed in Escherichia coli, purified and crystallized. A native data set was collected to 2.34 Å resolution on beamline BL38B1 at SPring-8. An SeMet data set was collected from a microcrystal (300 × 10 × 10 µm) on the RIKEN targeted protein beamline BL32XU and diffraction spots were obtained to 3.0 Å resolution. The native BVR crystal belonged to space group P212121, with unit-cell parameters a = 58.8, b = 88.4, c = 132.6 Å. Assuming that two molecules are present in the asymmetric unit, V
M (the Matthews coefficient) was calculated to be 2.37 Å3 Da−1 and the solvent content was estimated to be 48.1%. The structure of cyanobacterial BVR may provide insights into the mechanisms of its enzymatic and physiological functions.
bilirubin; biliverdin; microcrystals; microfocus beamline
Here we report a tracking X-ray microscopy (TrXM) as a novel methodology by using upper right lung apices alveoli in live intact mice. By enabling tracking of individual alveolar movements during respiration, TrXM identifies alveolar dynamics: individual alveoli in the upper lung apices show a small size increment as 4.9 ± 0.4% (mean ± s.e.m.) during respiration while their shapes look almost invariant. TrXM analysis in alveolar dynamics would be significant for better understanding of alveolar-based diseases, for instance, ventilator induced lung injury (VILI) in acute respiratory distress syndrome (ARDS).
Lung and gastric cancers are the first and second leading causes of death from cancer worldwide, and are especially prevalent in Eastern Asia. Relatively few reports are available in relation to the treatment and outcome of synchronous lung and gastric cancers, although there are increasing numbers of patients with these cancers. Efforts to develop more effective drugs for the treatment of synchronous cancers, without serious adverse effects, have been intensifying. Pemetrexed, a multi-targeted antifolate enzyme inhibitor, was approved by the United States Food and Drug Administration as a first-line chemotherapy for advanced non-squamous non-small cell lung cancer in 2007. Although clinical activity against several tumor types of adenocarcinoma, including gastric cancer, has been demonstrated, the efficacy of pemetrexed for gastric cancer remains to be fully evaluated.
We report a case involving a 62-year-old Japanese woman with synchronous locally-advanced poorly-differentiated lung adenocarcinoma and poorly-differentiated gastric adenocarcinoma, containing signet-ring cells distinguished by immunohistochemical profiles. She had been treated with carboplatin and pemetrexed as a first-line chemotherapy for lung cancer, and had achieved partial responses for both lung and gastric cancers. These responses led to a favorable 12-month progression-free survival after the initiation of chemotherapy, and the patient is still alive more than 33 months after diagnosis.
This case suggests a new chemotherapeutic regimen for patients with synchronous multiple primary cancers that have an adenocarcinoma background.
The process of wound healing involves complex interactions between circulating immune cells and local epithelial and endothelial cells. Studies in murine models indicate that cells of the innate immune system activated via their Toll-like receptors (TLR) can accelerate wound healing. This work examines whether immunostimulatory CpG oligodeoxynucleotides (ODN) designed to trigger human immune cells via TLR9 can promote the healing of excisional skin biopsies in rhesus macaques. Results indicate that ‘K’ type CpG ODN significantly accelerate wound closure in non-human primates (p < 0.05). Contributing to this outcome was a CpG-dependent increase in both the production of basic fibroblast growth factor and in keratinocyte migration. Of interest, IL-1a and TGFa normally present at sites of skin injury facilitated these effects. Current findings support the conclusion that the local administration of CpG ODN may provide an effective strategy for accelerating wound healing in humans.
CpG oligonucleotide; primate; TLR9; wound healing
To elucidate which RNA polymerase structural state a particular T. thermophilus Gre-family protein (Gfh1) associates with, the T. thermophilus RNAP elongation complex was cocrystallized with Gfh1.
RNA polymerase (RNAP) elongates RNA by iterative nucleotide-addition cycles (NAC). A specific structural state (or states) of RNAP may be the target of transcription elongation factors. Gfh1, a Thermus thermophilus Gre-family protein, inhibits NAC. To elucidate which RNAP structural state Gfh1 associates with, the T. thermophilus RNAP elongation complex (EC) was cocrystallized with Gfh1. Of the 70 DNA/RNA scaffolds tested, two (for EC1 and EC2) were successfully crystallized. In the presence of Gfh1, EC1 and EC2 yielded crystals belonging to space group P21 with similar unit-cell parameters (crystals 1 and 2, respectively). X-ray diffraction data sets were obtained at 3.6 and 3.8 Å resolution, respectively.
GreA; nucleotide-addition cycle; transcript cleavage
The bacterial PPM phosphatase RsbX from B. subtilis was expressed in E. coli, purified and crystallized. The crystal belonged to space group P1 and diffracted to 1.06 Å resolution.
RsbX from Bacillus subtilis is a manganese-dependent PPM phosphatase and negatively regulates the signal transduction of the general stress response by the dephosphorylation of RsbS and RsbR, which are activators of the alternative RNA polymerase σ factor SigB. In order to elucidate the structural–functional relationship of its Ser/Thr protein-phosphorylation mechanism, an X-ray crystallographic diffraction study of RsbX was performed. Recombinant RsbX was expressed in Escherichia coli, purified and crystallized. Crystals were obtained using the sitting-drop vapour-diffusion method and X-ray diffraction data were collected to 1.06 Å resolution with an R
merge of 8.1%. The crystals belonged to the triclinic space group P1, with unit-cell parameters a = 33.3, b = 41.7, c = 68.6 Å, α = 98.8, β = 90.0, γ = 108.4°.
PPM phosphatases; RsbX; general stress response; Bacillus subtilis
Wound healing is mediated through complex interactions between circulating immune cells and local epithelial and endothelial cells. Elements of the innate immune system are triggered when Toll like receptors (TLR) are stimulated by their cognate ligands, and previous studies suggest that such interactions can accelerate wound healing. This work examines the effect of treating excisional skin biopsies with immunostimulatory CpG oligodeoxynucleotides (ODN) that trigger via TLR9. Results indicate that CpG (but not control) ODN accelerate wound closure and reduce the total wound area exposed over time by >40% (p < 0.01). TLR9 KO mice, a strain unresponsive to the immunomodulatory effects of CpG stimulation, are unresponsive to ODN treatment and exhibit a general delay in healing when compared to wild type mice. CpG ODN administration promoted the influx of macrophages to the wound site and increased the production of vascular endothelial growth factor (VEGF), expediting neovascularization of the wound bed (p < 0.01 for both parameters). Stimulation via TLR9 thus represents a novel strategy to accelerate wound healing.
CpG oligonucleotide; wound healing; TLR9; VEGF
A blend of volatile organic compounds (VOCs) emitted from plants induced by herbivory enables the priming of defensive responses in neighboring plants. These effects may provide insights useful for pest control achieved with transgenic-plant-emitted volatiles. We therefore investigated, under both laboratory and greenhouse conditions, the priming of defense responses in plants (lima bean and corn) by exposing them to transgenic-plant-volatiles (VOCos) including (E)-β-ocimene, emitted from transgenic tobacco plants (NtOS2) that were constitutively overexpressing (E)-β-ocimene synthase. When lima bean plants that had previously been placed downwind of NtOS2 in an open-flow tunnel were infested by spider mites, they were more defensive to spider mites and more attractive to predatory mites, in comparison to the infested plants that had been placed downwind of wild-type tobacco plants. This was similarly observed when the NtOS2-downwind maize plants were infested with Mythimna separata larvae, resulting in reduced larval growth and greater attraction of parasitic wasps (Cotesia kariyai). In a greenhouse experiment, we also found that lima bean plants (VOCos-receiver plants) placed near NtOS2 were more attractive when damaged by spider mites, in comparison to the infested plants that had been placed near the wild-type plants. More intriguingly, VOCs emitted from infested VOCos-receiver plants affected their conspecific neighboring plants to prime indirect defenses in response to herbivory. Altogether, these data suggest that transgenic-plant-emitted volatiles can enhance the ability to prime indirect defenses via both plant-plant and plant-plant-plant communications.
We evaluated the prevalence and the types of infectious foci in oral as well as ear, nose, and throat diseases, and we examined incidence of renal involvement with active treatment for focal infection in children with Henoch-Schönlein Purpura. A total of 96 children who presented at Aichi Children's Health and Medical Center and were diagnosed as having HSP were evaluated for infectious foci in the ear, nose, throat, and oral cavities. Seventy-one of 96 children (74.0%) had some type of infectious lesion, such as sinusitis or tonsillitis, and the prevalence of sinusitis was the highest (51 cases, 53.7%). In 44 HSP patients without renal involvement at the first examination, the incidence of nephritis was lower (13.6%) than in previous reports (17–54%) due to our aggressive intervention for infectious foci.
The number of patients with non-HIV Pneumocystis pneumonia (PCP) is increasing with widespread immunosuppressive treatment. We investigated the clinical characteristics of non-HIV PCP and its association with microbiological genotypes.
Between January 2005 and March 2010, all patients in 2 university hospitals who had been diagnosed with PCP by PCR were enrolled in this study. Retrospective chart review of patients, microbiological genotypes, and association with 30-day mortality were examined.
Of the 82 adult patients investigated, 50 patients (61%) had inflammatory diseases, 17 (21%) had solid malignancies, 12 (15%) had hematological malignancies, and 6 (7%) had received transplantations. All patients received immunosuppressive agents or antitumor chemotherapeutic drugs. Plasma (1→3) β-D-glucan levels were elevated in 80% of patients, and were significantly reduced after treatment in both survivors and non-survivors. However, β-D-glucan increased in 18% of survivors and was normal in only 33% after treatment. Concomitant invasive pulmonary aspergillosis was detected in 5 patients. Fifty-six respiratory samples were stored for genotyping. A dihydropteroate synthase mutation associated with trimethoprim-sulfamethoxazole resistance was found in only 1 of the 53 patients. The most prevalent genotype of mitochondrial large-subunit rRNA was genotype 1, followed by genotype 4. The most prevalent genotype of internal transcribed spacers of the nuclear rRNA operon was Eb, followed by Eg and Bi. Thirty-day mortality was 24%, in which logistic regression analysis revealed association with serum albumin and mechanical ventilation, but no association with genotypes.
In non-HIV PCP, poorer general and respiratory conditions at diagnosis were independent predictors of mortality. β-D-glucan may not be useful for monitoring the response to treatment, and genotypes were not associated with mortality.
Leukotriene (LT) C4 and its metabolites, LTD4 and
LTE4, are involved in the pathobiology of bronchial asthma.
LTC4 synthase is the nuclear membrane-embedded enzyme responsible
for LTC4 biosynthesis, catalyzing the conjugation of two substrates
that have considerably different water solubility; that amphipathic
LTA4 as a derivative of arachidonic acid and a water-soluble
glutathione (GSH). A previous crystal structure revealed important details of
GSH binding and implied a GSH activating function for Arg-104. In addition,
Arg-31 was also proposed to participate in the catalysis based on the putative
LTA4 binding model. In this study enzymatic assay with mutant
enzymes demonstrates that Arg-104 is required for the binding and activation of
GSH and that Arg-31 is needed for catalysis probably by activating the epoxide
group of LTA4.
Crystal Structure; Eicosanoid-specific Enzymes; Enzyme Mechanisms; Enzyme Structure; Membrane Proteins; LTC4S; Leukotriene C4 Synthase
Crystals of the LOV1 domains of phototropin 1 and 2 from A. thaliana were obtained which diffracted X-rays to a resolution of at least 2.1 Å.
Phototropin is a blue-light receptor protein in plants that is responsible for phototropic responses, stomata opening and photo-induced relocation of chloroplasts. Higher plants such as Arabidopsis thaliana have two isoforms of phototropin: phototropin 1 and phototropin 2. Both isoforms comprise a tandem pair of blue-light-absorbing light–oxygen–voltage domains named LOV1 and LOV2 in the N-terminal half and a serine/threonine kinase domain in the C-terminal half. The LOV1 domain is thought to function as a dimerization site. In the present study, recombinant LOV1 domains of A. thaliana phototropin 1 and phototropin 2 were crystallized. The crystal of the LOV1 domain of phototropin 1 belonged to the orthorhombic space group P212121, with unit-cell parameters a = 61.2, b = 64.9, c = 70.8 Å, and diffracted X-rays to a resolution of 2.1 Å. The crystal of the LOV1 domain of phototropin 2 belonged to space group P21, with unit-cell parameters a = 32.5, b = 66.5, c = 56.7 Å, β = 92.4°, and diffracted X-rays to beyond 2.0 Å resolution. In both crystals, two LOV1 domains occupied the crystallographic asymmetric unit.
phototropins; LOV domains; blue-light receptors
Recently, we reported that a complex with an essential role in the degradation of Fructose-1,6-bisphosphatase in yeast is well conserved in mammalian cells; we named this mammalian complex C-terminal to the Lissencephaly type-1-like homology (CTLH) complex. Although the function of the CTLH complex remains unclear, here we used yeast two-hybrid screening to isolate Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) as a protein binding to a key component of CTLH complex, Armadillo repeat containing 8 (ARMc8) α. The association was confirmed by a yeast two-hybrid assay and a co-immunoprecipitation assay. The proline-rich domain of HRS was essential for the association. As demonstrated through immunofluorescence microscopy, ARMc8α co-localized with HRS. ARMc8α promoted the interaction of HRS with various ubiquitinated proteins through the ubiquitin-interacting motif. These findings suggest that HRS mediates protein endosomal trafficking partly through its interaction with ARMc8α.
ARMc8α; FBPase; monoubiquitination; HRS; UIM.
A shutterless continuous rotation method using an X-ray complementary metal-oxide semiconductor (CMOS) detector has been developed for high-speed, precise data collection in protein crystallography. The new method and detector were applied to the structure determination of three proteins by multi- and single-wavelength anomalous diffraction phasing and have thereby been proved to be applicable in protein crystallography.
A new shutterless continuous rotation method using an X-ray complementary metal-oxide semiconductor (CMOS) detector has been developed for high-speed, precise data collection in protein crystallography. The principle of operation and the basic performance of the X-ray CMOS detector (Hamamatsu Photonics KK C10158DK) have been shown to be appropriate to the shutterless continuous rotation method. The data quality of the continuous rotation method is comparable to that of the conventional oscillation method using a CCD detector and, furthermore, the combination with fine ϕ slicing improves the data accuracy without increasing the data-collection time. The new method is more sensitive to diffraction intensity because of the narrow dynamic range of the CMOS detector. However, the strong diffraction spots were found to be precisely measured by recording them on successive multiple images by selecting an adequate rotation step. The new method has been used to successfully determine three protein structures by multi- and single-wavelength anomalous diffraction phasing and has thereby been proved applicable in protein crystallography. The apparatus and method may become a powerful tool at synchrotron protein crystallography beamlines with important potential across a wide range of X-ray wavelengths.
protein crystallography; shutterless continuous rotation method; X-ray CMOS detectors; X-ray wavelength capabilities