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Isao Tanaka

Publications and source records attributed to Isao Tanaka.

At least 37 records · Page 2Linked to original sources
PubMed2006

Structure of the catalytic nucleotide-binding subunit A of A-type ATP synthase from Pyrococcus horikoshii reveals a novel domain related to the peripheral stalk.

H(+)-transporting ATP synthase is a multi-subunit enzyme involved in the production of ATP, which is an essential molecule for living organisms as a source of energy. Archaeal A-type ATPase (A-ATPase) is thought to act as a functional ATP synthase in archaea and is thought to have chimeric properties of F-ATPase and V-ATPase. Previous structural studies of F-ATPase indicated that the major nucleotide-binding subunits alpha and beta consist of three domains. The catalytic nucleotide-binding subunit A of V/A-ATPase contains an insertion of about 90 residues which is absent from the F(1)-ATPase beta subunit. Here, the first X-ray structure of the catalytic nucleotide-binding subunit A of an A(1)-ATPase is described, determined at 2.55 A resolution. A(1)-ATPase subunit A from Pyrococcus horikoshii consists of four domains. A novel domain, including part of the insertion, corresponds to the 'knob-like structure' observed in electron microscopy of A(1)-ATPase. Based on the structure, it is highly likely that this inserted domain is related to the peripheral stalk common to the A- and V-ATPases. The arrangement of this inserted domain suggests that this region plays an important role in A-ATPase as well as in V-ATPase.

Amino Acid Sequence↗
PubMed2006

LAFIRE: software for automating the refinement process of protein-structure analysis.

Manual intervention is usually required in the multiple rounds of refinement of protein crystal structures, including linking and/or extending the fragments of the initial model and rebuilding (fitting) ill-matched residues using computer-graphics software. Such manual modification is both time-consuming and requires a great deal of expertise in crystallography. Consequently, the refinement process becomes the bottleneck for high-throughput structure analysis. A program, Local correlation coefficient-based Automatic FItting for REfinement (LAFIRE), has been developed to achieve manual intervention-free refinement. This program was designed to perform the entire process of protein structural refinement automatically using the refinement programs CNS1.1 (CNS v.1.1) or REFMAC5. The automatic process begins from an initial model, which can be approximate, fragmentary or even only main-chain, and refines it to the final model including water molecules, controlled by monitoring the R(free) factor. More than 30 structures have now been refined successfully in a fully or semi-automatic manner within a few hours or days using LAFIRE.

Animals↗
PubMed2006

Crystal structure of protein Ph1481p in complex with protein Ph1877p of archaeal RNase P from Pyrococcus horikoshii OT3: implication of dimer formation of the holoenzyme.

Ribonuclease P (RNase P) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 consists of a catalytic RNA and five protein subunits. We previously determined crystal structures of four protein subunits. Ph1481p, an archaeal homologue for human hPop5, is the protein component of the P.horikoshii RNase P for which no structural information is available. Here we report the crystal structure of Ph1481p in complex with another protein subunit, Ph1877p, determined at 2.0 A resolution. Ph1481p consists of a five-stranded antiparallel beta-sheet and five helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. Ph1481p is, however, distinct from the typical RNP domain in that it has additional helices at the C terminus, which pack against one face of the beta-sheet. The presence of two complexes in the asymmetric unit, together with gel filtration chromatography indicates that the heterotetramer is stable in solution and represents a fundamental building block in the crystals. In the heterotetrameric structure (Ph1877p-(Ph1481p)(2)-Ph1877p), a homodimer of Ph1481p sits between two Ph1877p monomers. Ph1481p dimerizes through hydrogen bonding interaction from the loop between alpha1 and alpha2 helices, and each Ph1481p interacts with two Ph1877p molecules, where alpha2 and alpha3 in Ph1481p interact with alpha7 in one Ph1877p and alpha8 in the other Ph1877p molecule, respectively. Deletion of the alpha1-alpha2 loop in Ph1481p caused heterodimerization with Ph1877p, and abolished ability to homodimerize itself and heterotetramerize with Ph1877p. Furthermore, the reconstituted particle containing the deletion mutant Ph1481p (mPh1481p) exhibited significantly reduced nuclease activity. These results suggest the presence of the heterotetramer of Ph1481p and Ph1877p in P.horikoshii RNase P.

Amino Acid Sequence↗
PubMed2006

Crystallization and preliminary X-ray studies of the DNA-binding domain of hepatocyte nuclear factor-6alpha complexed with DNA.

Hepatocyte nuclear factor 6 (HNF-6)/OC-1, a part of liver-enriched transcription factor, controls pancreas and liver development and regulates expression of several hepatic genes. DNA-binding region of HNF-6alpha bound to a 14-mer DNA fragment has been crystallized by the hanging drop vapor diffusion method. The crystals belong to space group P2 with unit cell parameters of a = 73.0 A, b = 39.0 A, c = 106.5 A, beta = 107.6 degrees. X-ray diffraction data were collected to 2.0 A resolution.

Base Sequence↗
PubMed2005

Comparison of phasing methods for sulfur-SAD using in-house chromium radiation: case studies for standard proteins and a 69 kDa protein.

Phasing of the crystal structures of four standard proteins (lysozyme, trypsin, glucose isomerase and thaumatin) and a novel 69 kDa protein from Thermus thermophilus, TT0570, was performed using the single-wavelength anomalous diffraction of S atoms intrinsically present within the native protein molecules. To utilize the sulfur anomalous diffraction, the data sets were collected using the loopless data-collection method with chromium Kalpha X-rays of wavelength 2.29 A. Three phasing methods, MLPHARE, SHARP and OASIS-2004, were tested in combination with the DM or SOLOMON density-modification method. The results showed that the solvent contents are still an important factor for phasing with the S-SAD method, even when longer wavelength Cr Kalpha radiation is used. Of the three procedures, the improved direct phasing of OASIS-2004 with its implemented fragment feedback to the direct-method probability calculation gave the best results in determining the initial phases. For all five proteins, almost the entire models could be built automatically.

Aldose-Ketose Isomerases↗
PubMed2005

The CGL2612 protein from Corynebacterium glutamicum is a drug resistance-related transcriptional repressor: structural and functional analysis of a newly identified transcription factor from genomic DNA analysis.

The emergence of antibiotic-resistant bacteria often causes serious clinical problems. The TetR family is one of the major transcription factor families that regulate expression of genes involved in bacterial antimicrobial resistance systems. CGL2612 protein is a transcription factor newly identified by genomic DNA analysis on Corynebacterium glutamicum, which belongs to the mycolic acid-containing Actinomycetales, including the well known pathogens Corynebacterium diphtheriae and Mycobacterium tuberculosis. Crystal structure analysis showed that the CGL2612 protein exhibits significant structural similarity to the multidrug resistance (MDR)-related transcription factor QacR from Staphylococcus aureus, despite poor amino acid sequence similarity between these proteins. Binding DNA sequence analysis of CGL2612 protein using the systematic evolution of ligands by the exponential enrichment (systematic evolution of ligands by exponential enrichment, or SELEX) method revealed that this protein is a new member of the TetR family, which regulates expression of the immediately upstream gene, cgl2611, probably encoding a major facilitator superfamily permease. Subsequent functional analyses confirmed a function of the CGL2612 as a transcriptional repressor responsible for the antimicrobial resistance system in C. glutamicum. The strategy used in the present study is one of the most convenient and powerful methods to analyze functionally unknown transcription factors, and the results obtained here will contribute to our understanding of the drug resistance mechanism not only in C. glutamicum but also in the related bacteria, C. diphtheriae and M. tuberculosis.

Amino Acid Sequence↗
PubMed2005

Crystal structure of a ribonuclease P protein Ph1601p from Pyrococcus horikoshii OT3: an archaeal homologue of human nuclear ribonuclease P protein Rpp21.

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the removal of 5' leader sequences from tRNA precursors (pre-tRNA). The human protein Rpp21 is essential for human RNase P activity in tRNA processing in vitro. The crystal structure of Ph1601p from the hyperthermophilic archaeon Pyrococcus horikoshii OT3, the archaeal homologue of Rpp21, was determined using the multiple anomalous dispersion (MAD) method with the aid of anomalous scattering in zinc and selenium at 1.6 A resolution. Ph1601p comprises an N-terminal domain (residues 1-55), a central linker domain (residues 56-79), and a C-terminal domain (residues 80-120), forming an L-shaped structure. The N-terminal domain consists of two long alpha-helices, while the central and C-terminal domains fold in a zinc ribbon domain. The electrostatic potential representation indicates the presence of positively charged clusters along the L arms, suggesting a possible role in RNA binding. A single zinc ion binds the well-ordered binding site that consists of four Cys residues (Cys68, Cys71, Cys97, and Cys100) and appears to stabilize the relative positions of the N- and C-domains. Mutations of Cys68 and Cys71 or Cys97 and Cys100 to Ser destabilize the protein structure, which results in inactivation of the RNase P activity. In addition, site-directed mutagenesis suggests that Lys69 at the central loop and Arg86 and Arg105 at the zinc ribbon domain are strongly involved in the functional activity, while Arg22, Tyr44, Arg65, and Arg84 play a modest role in the activity.

Amino Acid Sequence↗
PubMed2005

Low p53 expression of acute myelocytic leukemia cells with t(8;21) chromosome abnormality: association with low p14(ARF) expression.

In this study, the mRNA expression of p14(ARF) in t(8;21)AML cells was found to be significantly lower than acute myelocytic leukemia (AML) cells without t(8;21) chromosome abnormality, which was concordant with previous observation by Linggi et al. that AML1-MTG8 represses the transcription of p14(ARF). Although p53 mRNA expression level of t(8;21)AML cells was not low, p53 protein expression was reduced in t(8;21)AML cells. Genotoxic damage by ionizing radiation did not induce p53 upregulation in t(8;21)AML cells. Since p14(ARF) has been demonstrated to inhibit p53 degradation by binding to MDM2, repression of p14(ARF) expression in t(8;21)AML may facilitate the degradation of p53 by MDM2. Low p14(ARF) in t(8;21)AML may also account for the absence of upregulation of p53 by ionizing radiation. Then, we have shown that p53 expression level was inversely correlated with S/G2/M population of cell cycle in AML cells. Most of the t(8;21)AML are considered to be in p53(low) S/G2/M(high). It is now widely known that formation of AML1-MTG8 by t(8;21) translocation is a very early event in leukemogenesis, and AML1-MTG8 alone might have limited proliferative potential. Then, secondary oncogenic events such as activated receptor tyrosine kinase (like c-kit mutation), is necessary to become full-blown leukemia. Low p53 protein expression and insufficient induction of p53 by genotoxic damage might increase the opportunity to obtain additional oncogenic events, since genome guard function of p53 does not work in t(8;21)AML cells.

Cell Cycle↗
PubMed2005

Molecular basis of alanine discrimination in editing site.

AlaX is the homologue of the class II alanyl-tRNA synthetase editing domain and has been shown to exhibit autonomous editing activity against mischarged tRNA(Ala). Here, we present the structures of AlaX from the archaeon Pyrococcus horikoshii in apo form, complexed with zinc, and with noncognate amino acid l-serine and zinc. Together with mutational analysis, we demonstrated that the conserved Thr-30 hydroxyl group located near the beta-methylene of the bound serine is responsible for the discrimination of noncognate serine from cognate alanine, based on their chemical natures. Furthermore, we confirmed that the conserved Gln-584 in alanyl-tRNA synthetase, which corresponds to Thr-30 of AlaX, is also critical for discrimination. These observations strongly suggested conservation of the chemical discrimination among trans- and cis-editing of tRNA(Ala).

Acylation↗
PubMed2005

Structure determination of a novel protein by sulfur SAD using chromium radiation in combination with a new crystal-mounting method.

A novel and easy crystal-mounting technique was developed for the sulfur SAD method using Cr Kalpha radiation (2.29 A). Using this technique, the cryo-buffer and cryoloop around the protein crystal can be removed before data collection in order to eliminate their X-ray absorption. The superiority and reproducibility of the data sets with this mounting technique were demonstrated using tetragonal hen egg-white lysozyme crystals. The structure of a novel protein, PH1109, from Pyrococcus horikoshii OT3 was solved using this technique. At the wavelength of Cr Kalpha radiation, the anomalous signal |DeltaF|/|F| of PH1109 is expected to be 1.72% as this protein of 144 residues includes four methionines and two cysteines. Sulfur SAD phasing was performed using SHELXD and SHELXE. In the case of the data set obtained using this novel crystal-mounting technique, 54.9% of all residues were built with side chains automatically by RESOLVE. On the other hand, only 16.0% were built with side chains for the data set collected using the standard cryoloop. These results indicated that this crystal-mounting technique was superior to the standard loop-mounting method for the measurement of small anomalous differences at longer wavelength and yielded better results in sulfur-substructure solution and initial phasing. The present study demonstrates that the sulfur SAD method with a chromium source becomes enhanced and more practical for macromolecular structure determination using the new crystal-mounting technique.

Absorption↗
PubMed2005

Electronic structure of lithium nickel oxides by electron energy loss spectroscopy.

The electronic structures of NiO, LiNiO2, and NiO2 are studied by the electron energy loss spectroscopy at Ni L(2,3), Ni M(2,3), and O K edges. The Ni L(2,3) edge spectra suggest that the formal charge of nickel is +2 in NiO, +3 with a low-spin state in LiNiO2, and +4 with a low-spin state in NiO2. This is well confirmed by first-principles calculations. The Ni M(2,3) edge spectra show similar chemical shifts to those of the Ni L(2,3) edge. Superposition of the Li K edge spectrum, however, hinders further analysis. Although the formal charge of oxygen is -2 in all the three phases, the O K edge spectra indicate a more remarkable difference in the electronic structure of the oxygen in NiO2 than that in either NiO or LiNiO2. The spectra suggest that lithium extraction from LiNiO2 reinforces the covalent bonding between the oxygen and nickel atoms and causes a notable reduction in electron density at the oxygen atoms.

Journal Article↗
PubMed2005

Crystal structure of archaeal toxin-antitoxin RelE-RelB complex with implications for toxin activity and antitoxin effects.

The Escherichia coli chromosome encodes toxin-antitoxin pairs. The toxin RelE cleaves mRNA positioned at the A-site in ribosomes, whereas the antitoxin RelB relieves the effect of RelE. The hyperthermophilic archaeon Pyrococcus horikoshii OT3 has the archaeal homologs aRelE and aRelB. Here we report the crystal structure of aRelE in complex with aRelB determined at a resolution of 2.3 A. aRelE folds into an alpha/beta structure, whereas aRelB lacks a distinct hydrophobic core and extensively wraps around the molecular surface of aRelE. Neither component shows structural homology to known ribonucleases or their inhibitors. Site-directed mutagenesis suggests that Arg85, in the C-terminal region, is strongly involved in the functional activity of aRelE, whereas Arg40, Leu48, Arg58 and Arg65 play a modest role in the toxin's activity.

Antitoxins↗
PubMed2005

Structure of the type I L-asparaginase from the hyperthermophilic archaeon Pyrococcus horikoshii at 2.16 angstroms resolution.

The crystal structure of the L-asparaginase from the hyperthermophilic archaeon Pyrococcus horikoshii (PhA) was determined by the multiwavelength anomalous diffraction (MAD) method and was refined to a resolution of 2.16 angstroms with a crystallographic R factor and free R factor of 21.1 and 25.3%, respectively. This is the first report of the three-dimensional structure of a type I L-asparaginase. These enyzmes are known as cytosolic L-asparaginases with lower affinities for substrate than the type II L-asparaginases. Although the overall fold of PhA was closely related to the structure of the well characterized type II L-asparaginase, structural differences were also detected. PhA forms a homodimer that corresponds to half the homotetramer of type II L-asparaginases. Structure comparison at the active site reveals that most catalytic residues are conserved except for two residues that recognize the amino group of the substrate. Additionally, a remarkable structural difference is found in the so-called 'active-site flexible loop'. In PhA this loop is stabilized by beta-hairpin formation and by elaborate interactions with the type-I-specific alpha-helical region derived from the other subunit forming the PhA dimer. The flexible loop of the type II enzyme is considered to serve as a mobile gate to the active site. Therefore, the loop stabilization observed in the PhA structure may cause limitation of the access of the substrate to the active site.

Amino Acid Sequence↗
PubMed2005

Crystallization and preliminary crystallographic analysis of the cellulose biosynthesis-related protein CMCax from Acetobacter xylinum.

The cellulose biosynthesis-related protein CMCax from Acetobacter xylinum was overexpressed in Escherichia coli, purified and crystallized. Single crystals of selenomethionine (SeMet) substituted CMCax were obtained by the hanging-drop vapour-diffusion method at 293 K, primarily using polyethylene glycol 4000 as a precipitant. The crystals belong to the primitive hexagonal space group P6(1) or P6(5), with unit-cell parameters a = b = 89.1, c = 94.2 A. The predicted Matthews coefficient (VM) value is 3.0 A3 Da(-1) for one CMCax monomer in the asymmetric unit. A single-wavelength anomalous dispersion (SAD) data set was collected to a resolution of 2.3 A using synchrotron radiation.

Bacterial Proteins↗
PubMed2005

Crystallization and preliminary X-ray analysis of alpha-xylosidase from Escherichia coli.

Glycoside hydrolases have been implicated in many biological processes. To date, they have been classified into 93 glycoside hydrolase (GH) families based on amino-acid sequence similarity. alpha-Xylosidase from Escherichia coli belongs to GH family 31 and catalyzes the release of alpha-xylose from the non-reducing terminal side of alpha-xyloside. Single crystals of alpha-xylosidase have been grown by vapour diffusion at 293 K from 10%(w/v) PEG 20K, 2%(v/v) 2-propanol, 2%(v/v) glycerol and 0.1 M 2-morpholinoethanesulfonic acid pH 5.5. These crystals belong to space group P2(1)2(1)2(1) and X-ray diffraction data were collected to a resolution of 2.75 A. Crystals of selenomethionyl-substituted alpha-xylosidase were also obtained, which diffracted to at least 3.0 A. Based on the value of VM, the asymmetric unit in these crystals was assumed to contain six molecules.

Escherichia coli↗