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T Tsukihara

Publications and source records attributed to T Tsukihara.

At least 19 recordsLinked to original sources
PubMed2001

Intermonomer interactions in dimer of bovine heart cytochrome c oxidase.

The X-ray structure of bovine heart cytochrome c oxidase solved for orthorhombic crystals showed a dimeric structure stabilized by four subunit-subunit contacts, namely, subunit Vb-subunit Vb on the matrix side, subunit I-subunit VIa, subunit VIa-subunit I in the transmembrane region and subunit VIb-subunit VIb on the intermembrane side. The same intermonomer contacts as in the orthorhombic crystals were observed in both hexagonal and tetragonal crystals, the X-ray structures of which were determined by the molecular-replacement method. These results suggest that the dimeric structure also exists under physiological conditions. These contacts, especially the subunit IVa-subunit I contact, in which the N-terminal portion of subunit IVa is placed on the surface of subunit I near the dioxygen-reduction site, indicate that the function of the bovine heart enzyme is likely to be controlled by perturbation of the monomer-monomer association.

Animals↗
PubMed2001

X-ray crystalline structures of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus, and its cys-free mutant.

In order to elucidate the mechanism of the thermostability of proteins from hyperthermophiles, X-ray crystalline structures of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus (PfPCP), and its mutant protein with Ser substituted at Cys142 and Cys188 were determined at 2.2 and 2.7 A resolution, respectively. The obtained structures were compared with those previously reported for pyrrolidone carboxyl peptidases from a hyperthermophilie, Thermococcus litoralis (TlPCP), and from a mesophile, Bacillus amyloliquefaciens (BaPCP). The PfPCP structure is a tetramer of four identical subunits similar to that of the TlPCP and BaPCP. The largest structural changes among the three PCPs were detected in the C-terminal protrusion, which interacts with that of another subunit. A comparison of the three structures indicated that the high stability of PfPCP is caused by increases in hydrophobic interactions and hydrogen bonds, the formation of an intersubunit ion-pair network, and improvement to an ideal conformation. On the basis of the structures of the three proteins, it can be concluded that PfPCP does not have any special factors responsible for its extremely high stability and that the conformational structure of PfPCP is superior in its combination of positive and negative stabilizing factors compared with BaPCP.

Amino Acid Sequence↗
PubMed2001

Purification and crystallization of Japanese pear S-RNase associated with gametophytic self-incompatibility.

S-RNase is a 25 kDa pistil-specific protein associated with gametophytic self-incompatibility. The S-RNase is secreted into the transmitting tissue of the pistil and is responsible for the discrimination of pollen S-alleles. Crystals of Japanese pear S(3)-RNase were obtained by the hanging-drop vapour-diffusion method. Preliminary X-ray data showed that the crystals diffract to a 1.5 A resolution and belong to the space group P2(1), with unit-cell parameters a = 45.4, b = 52.4, c = 47.4 A, alpha = gamma = 90, beta = 106.5 degrees.

Crystallization↗
PubMed2000

The adoption of a twisted structure of importin-beta is essential for the protein-protein interaction required for nuclear transport.

Importin-beta is a nuclear transport factor which mediates the nuclear import of various nuclear proteins. The N-terminal 1-449 residue fragment of mouse importin-beta (impbeta449) possesses the ability to bidirectionally translocate through the nuclear pore complex (NPC), and to bind RanGTP. The structure of the uncomplexed form of impbeta449 has been solved at a 2.6 A resolution by X-ray crystallography. It consists of ten copies of the tandemly arrayed HEAT repeat and exhibits conformational flexibility which is involved in protein-protein interaction for nuclear transport. The overall conformation of the HEAT repeats shows that a twisted motion produces a significantly varied superhelical architecture from the previously reported structure of RanGTP-bound importin-beta. These conformational changes appear to be the sum of small conformational changes throughout the polypeptide. Such a flexibility, which resides in the stacked HEAT repeats, is essential for interaction with RanGTP or with NPCs. Furthermore, it was found that impbeta449 has a structural similarity with another nuclear migrating protein, namely beta-catenin, which is composed of another type of helix-repeated structure of ARM repeat. Interestingly, the essential regions for NPC translocation for both importin-beta and beta-catenin are spatially well overlapped with one another. This strongly indicates the importance of helix stacking of the HEAT or ARM repeats for NPC-passage.

Amino Acid Sequence↗
PubMed2000

Solubilization of PNS myelin membrane proteins by detergents.

We have applied 25 different detergents to the PNS myelin membrane. The extracts produced were analyzed regarding the total amount of protein solubilized, and which myelin glycoproteins were solubilized. The degree of protein extraction was correlated with the critical micelle concentration. For some detergents the protein extraction ability depends clearly on critical micelle concentration. These detergents are more potent if critical micelle concentration is small. The other type of extraction is independent on the critical micelle concentration, but extractability of proteins is low. The best detergents for solubilization of myelin proteins are neutral of the alkyl chain length n =9-11, as follows: dodecyl-beta-D-maltopyranoside, decyl-beta-D-maltopyranoside, n-dodecylsulfobetaine. We expect that these detergents will also be suitable for crystallization of P0 and PASII/PMP22 glycoproteins.

Animals↗
PubMed2000

Crystal structure of N-carbamyl-D-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases.

BACKGROUND: N-carbamyl-D-amino acid amidohydrolase (DCase) catalyzes the hydrolysis of N-carbamyl-D-amino acids to the corresponding D-amino acids, which are useful intermediates in the preparation of beta-lactam antibiotics. To understand the catalytic mechanism of N-carbamyl-D-amino acid hydrolysis, the substrate specificity and thermostability of the enzyme, we have determined the structure of DCase from Agrobacterium sp. strain KNK712. RESULTS: The crystal structure of DCase has been determined to 1.7 A resolution. The enzyme forms a homotetramer and each monomer consists of a variant of the alpha + beta fold. The topology of the enzyme comprises a sandwich of parallel beta sheets surrounded by two layers of alpha helices, this topology has not been observed in other amidohydrolases such as the N-terminal nucleophile (Ntn) hydrolases. CONCLUSIONS: The catalytic center could be identified and consists of Glu46, Lys126 and Cys171. Cys171 was found to be the catalytic nucleophile, and its nucleophilic character appeared to be increased through general-base activation by Glu46. DCase shows only weak sequence similarity with a family of amidohydrolases, including beta-alanine synthase, aliphatic amidases and nitrilases, but might share highly conserved residues in a novel framework, which could provide a possible explanation for the catalytic mechanism for this family of enzymes.

Amidohydrolases↗
PubMed2000

Crystal structure of tobacco necrosis virus at 2.25 A resolution.

The crystal structure of tobacco necrosis virus (TNV) has been determined by real-space averaging with 5-fold non-crystallographic symmetry, and refined to R=25.3 % for diffraction data to 2.25 A resolution. A total of 180 subunits form a T=3 virus shell with a diameter of about 280 A and a small protrusion at the 5-fold axis. In 276 amino acid residues, the respective amino terminal 86, 87 and 56 residues of the A, B and C subunits are disordered. No density for the RNA was found. The subunits have a "jelly roll" beta-barrel structure, as have the structures of the subunits of other spherical viruses. The tertiary and quaternary structures of TNV are, in particular, similar to those of southern bean mosaic virus, although they are classified in different groups. Invisible residues 1 to 56 with a high level of basic residues are considered to be located inside the particle. Sequence comparison of the coat proteins of several TNV strains showed that the sequences of the disordered segment diverge considerably as compared with those of the ordered segment, consistent with a small tertiary structural constraint being imposed on the N-terminal segment. Basic residues are localized on the subunit interfaces or inner surface of the capsid. Positive charges of the basic residues facing the interior, as well as those of the N-terminal segment, may neutralize the negative charge of the RNA inside. Five calcium ions per icosahedral asymmetric unit are located at the subunit interfaces; three are close to the exterior surface, the other two away from it. The environments of the first three are similar, and those of the other two sites are similar. These calcium ions are assumed to be responsible for the stabilization/transition of the quaternary structure of the shell. Three peptide segments ordered only in the C subunits are clustered around each 3-fold (quasi-6-fold) axis forming a beta-annulus, and may lead to quasi-equivalent interactions for the organization of the T=3 shell.

Amino Acid Sequence↗
PubMed2000

X-ray structure of beta-carbonic anhydrase from the red alga, Porphyridium purpureum, reveals a novel catalytic site for CO(2) hydration.

The carbonic anhydrases (CAs) fall into three evolutionarily distinct families designated alpha-, beta-, and gamma-CAs based on their primary structure. beta-CAs are present in higher plants, algae, and prokaryotes, and are involved in inorganic carbon utilization. Here, we describe the novel x-ray structure of beta-CA from the red alga, Porphyridium purpureum, at 2.2-A resolution using intrinsic zinc multiwavelength anomalous diffraction phasing. The CA monomer is composed of two internally repeating structures, being folded as a pair of fundamentally equivalent motifs of an alpha/beta domain and three projecting alpha-helices. The motif is obviously distinct from that of either alpha- or gamma-CAs. This homodimeric CA appears like a tetramer with a pseudo 222 symmetry. The active site zinc is coordinated by a Cys-Asp-His-Cys tetrad that is strictly conserved among the beta-CAs. No water molecule is found in a zinc-liganding radius, indicating that the zinc-hydroxide mechanism in alpha-CAs, and possibly in gamma-CAs, is not directly applicable to the case in beta-CAs. Zinc coordination environments of the CAs provide an interesting example of the convergent evolution of distinct catalytic sites required for the same CO(2) hydration reaction.

Amino Acid Sequence↗
PubMed2000

X-ray structure and the reaction mechanism of bovine heart cytochrome c oxidase.

X-ray structure of bovine heart cytochrome c oxidase in the fully oxidized state shows a peroxide bridging between Fe2+ and Cu2+ in the O2 reduction site. The bond distances for Fe-O and Cu-O are 2.52 and 2.16 A, respectively. The structure is consistent with antiferromagnetic coupling between the two metals, which has long been known and to recent redox titration results [J. Biol. Chem. 274 (1999) 33403]. The trigonal planer coordination of Cu1+ in the O2 reduction site is consistent with the very weak interaction between Cu1+ and O2 bound at Fe2+ revealed by time-resolved resonance Raman investigations. One of the three histidine imidazoles coordinated to the Cu ion in the O2 reduction site fixes a tyrosine phenol group near the O2 reduction site with the direct covalent link between the two groups. The structure suggests that the phenol group is the site for donating protons to the bound O2. Redox-coupled conformational change in an extramembrane loop indicates that an aspartate (Asp51) in the loop apart from the O2 reduction site is the site for proton pumping.

Animals↗
PubMed2000

Roles of functional loops and the C-terminal segment of a single-stranded DNA binding protein elucidated by X-Ray structure analysis.

The single-stranded DNA (ssDNA) binding protein from Escherichia coli (EcoSSB) plays a central role in DNA replication, recombination and repair. The tertiary structure of EcoSSB was determined at 2.2 A resolution. This is rather higher resolution than previously reported. Crystals were grown from the homogeneous intact protein but the EcoSSB tetramer in the crystals contains truncated subunits lacking a part of the C-terminal. The structure determined includes biologically important flexible loops and C-terminal regions, and revealed the existence of concavities. These concavities include the residues important for ssDNA binding. An ssDNA can be fitted on the concavities and further stabilized through interactions with the loops forming flexible lids. It seems likely to play a central role in the binding of ssDNA.

Amino Acid Sequence↗
PubMed2000

New crystal forms and low resolution structure analysis of 20S proteasomes from bovine liver.

20S proteasomes from higher eukaryotes have immunological functions rather than those from archibacteria or yeast. To clarify the mechanism of the sorting and production of antigen-presenting peptides, it is important and worthwhile to determine the structure of mammalian proteasomes using a third generation synchrotron radiation source. Here we report new crystal forms of 20S proteasomes from bovine liver and preliminary structure analysis of them. The crystals belong to the same space group but have different cell dimensions. One crystal (form I) belongs to space group P2(1)2(1)2(1) with unit cell dimensions of a = 124.8, b =197.4, c =323.8 A, and diffracts to 3.0 A resolution. The other crystal (form II) belongs to the same space group with a =115.1, b =205.6, c =316. 0 A, and diffracts to 4.0 A resolution. The diffraction data for the form I crystal provided an interpretable electron density map for presenting the structural differences from yeast proteasomes.

Animals↗
PubMed2000

X-ray structure of azide-bound fully oxidized cytochrome c oxidase from bovine heart at 2.9 A resolution.

Two azide ions were identified, one between the Fe and Cu atoms in the O(2)-reduction site and the other at the transmembrane surface of the enzyme, in the crystal structure of the azide-bound form of bovine heart cytochrome c oxidase at 2.9 A resolution. Two geometries, a mu-1,3 type geometry between the Fe and Cu atoms and a terminal geometry on the Fe atom, are equally possible for an azide ion in the O(2)--reduction site. The other azide molecule was hydrogen bonded to an amide group of an asparagine and a hydroxyl group of tyrosine in a mu-1,1 type geometry. The antisymmetric infrared bands arising from these azide ions, which show essentially identical intensity [Yoshikawa & Caughey (1992), J. Biol. Chem. 267, 9757-9766], strongly suggest terminal binding of the azide to Fe. The electron density of all three imidazole ligands to Cu(B) was clearly seen in the electron-density map of the azide-bound form of bovine heart enzyme, in contrast to the crystal structure of the azide-bound form of the bacterial enzyme [Iwata et al. (1995), Nature (London), 376, 660-669], which lacks one of the three imidazole ligands to Cu(B).

Animals↗
PubMed1999

Quantitative reevaluation of the redox active sites of crystalline bovine heart cytochrome c oxidase.

Approximately 30% of the iron contained in a bovine heart cytochrome c oxidase preparation was removed by crystallization, giving a molecular extinction coefficient 1.25-1.4 times higher than those reported thus far. Six electron equivalents provided by dithionite were required for complete reduction of the crystalline cytochrome c oxidase preparation. The fully reduced enzyme was oxidized with 4 oxidation equivalents provided by molecular oxygen, giving an absorption spectrum slightly, but significantly, different from that of the original fully oxidized form. Four electron equivalents were required for complete reduction of the O(2)-oxidized enzyme. The O(2)-oxidized form, when exposed to excess amounts of O(2), was converted to the original oxidized form which required 6 electrons for complete reduction. A slow reduction of the O(2)-oxidized form without any external reductant added indicates the existence of internal electron donors for heme irons in the enzyme. These results suggest that the 2 extra oxidation equivalents in the original oxidized form, compared with the O(2)-oxidized form, are due to a bound peroxide produced by O(2) and electrons from the internal donors, consistently with a peroxide at the O(2) reduction site in the crystal structure of the enzyme (Yoshikawa, S., Shinzawa-Itoh, K. , Nakashima, R., Yaono, R., Yamashita, E., Inoue, N., Yao, M., Fei, M. J., Peters Libeu, C., Mizushima, T., Yamaguchi, H., Tomizaki, T., and Tsukihara, T. (1998) Science 280, 1723-1729).

Animals↗
PubMed1999

Structure analysis of bovine heart cytochrome c oxidase at 2.8 A resolution.

The crystal structure of bovine heart cytochrome c oxidase has been determined at 2.8 A resolution by the multiple isomorphous replacement (MIR) method with three heavy-atom derivatives. An asymmetric unit of the crystal has a molecular weight of 422 kDa. Eight heavy atoms as main sites of a CH3HgCl derivative were clearly located by solving the difference Patterson function. The electron density obtained by the MIR method was refined by density modification, consisting of solvent flattening, histogram matching and non-crystallographic symmetry averaging. The enzyme exhibits a dimeric structure in the crystal. Out of 3606 amino-acid residues in 26 subunits in the dimer, 3560 residues were located in the electron-density map. The structure was refined by X-PLOR. The final R factor and the free R factor were 0.199 and 0.252 at 2.8 A resolution, respectively. One monomer in the dimeric structure with a stronger packing interaction has a lower averaged temperature factor than the other, by 16 A2. The region +/-12 A from the centre of the transmembrane part is almost 100% alpha-helix, despite the glycine residue content being as high as 7.1% in the transmembrane region. The residues around haem a of animals have evolved away from those of bacteria in contrast with the residues of the haem a3. The hierarchy of the structural organization of the enzyme complex has been proposed on the basis of intersubunit interactions.

Animals↗
PubMed1999

A low-resolution structure of rice dwarf virus determined by ab initio phasing.

Rice dwarf virus crystals belong to space group I222 with cell parameters a = 770 (2), b = 795 (5), c = 814 (5) A and alpha = beta = gamma = 90 degrees. The unit cell of the crystal contains two viruses at the origin and body-centred positions. Using data synthesized from a rice dwarf virus model crystal in the space group I222, the possibility of ab initio phasing was thoroughly examined. The centric nature of the initial phases was unexpectedly broken by extensive iteration of the non-crystallographic symmetry averaging. The structure of rice dwarf virus was then solved with ab initio phasing up to 20 A resolution. The triangulation number determined by the present study is T = 13, which is different from the triangulation number, T = 9, previously determined by electron microscopy [Uyeda & Shikata (1982). Ann. Phytopathol. Soc. Jpn, 48, 295-300].

Capsid↗