Biomedical subjects

B Mikami

Publications and source records attributed to B Mikami.

At least 19 recordsLinked to original sources

PubMed2001

Essential contribution of germline-encoded lysine residues in Jgamma1.2 segment to the recognition of nonpeptide antigens by human gammadelta T cells.

Human gammadelta T cells display unique repertoires of Ag specificities largely imposed by selective usages of distinct Vgamma and Vdelta genes. Among them, Vgamma2/Vdelta2(+) T cells predominate in the circulation of healthy adults and respond to various microbial small molecular mass nonpeptide Ags. The present results indicate that the primary Vgamma2/Vdelta2(+) T cells stimulated with the distinct groups of nonpeptide Ags, including monoethyl pyrophosphate, isobutyl amine, and aminobisphosphonate, invariably exhibit Jgamma1.2 in the Vgamma2(+) TCR-gamma chains. Gene transfer studies revealed that most of the randomly cloned Vgamma2/Jgamma1.2(+) TCR-gamma genes bearing diverse Vgamma/Jgamma junctional sequences could confer the responsiveness to all these nonpeptide Ags, while none of the Vgamma2/Jgamma1.1(+) or Vgamma2/Jgamma1.3(+) TCR-gamma genes could do so. Furthermore, mutation of the lysine residues encoded by the Jgamma1.2 gene, which are unique in human Jgamma1.2 and absent in other human or mouse Jgamma segments, completely abrogated the responsiveness to all the nonpeptide Ags without affecting the response to anti-CD3 mAb. These results strongly suggested that the positively charged lysine residues in the TCR-gamma chain CDR3 region encoded by the germline Jgamma1.2 gene play a key role in the recognition of diverse small molecular mass nonpeptide Ags.

Amino Acid Sequence↗

PubMed2001

Super-channel in bacteria: function and structure of the macromolecule import system mediated by a pit-dependent ABC transporter.

In a soil isolate, Sphingomonas sp. A1, the transport of a macromolecule (alginate: 27 kDa) is mediated by a pit-dependent ATP-binding cassette (ABC) transporter. The transporter is different from other ABC transporters so far analyzed in that its function is dependent on a pit, a mouth-like organ formed on the cell surface only when cells are compelled to assimilate macromolecules, and in that it allows direct import of macromolecules into cells. The ABC transporter coupled with the pit, which functions as a funnel and/or concentrator of macromolecules to be imported, was designated the 'super-channel', and in this review, we discuss the three-dimensional structure and specific function of the 'super-channel' for macromolecule import found for the first time in a bacterium.

ATP-Binding Cassette Transporters↗

PubMed2001

Crystallization and preliminary X-ray analysis of NAD kinase from Mycobacterium tuberculosis H37Rv.

NAD kinase from Mycobacterium tuberculosis H37Rv utilizes ATP or inorganic polyphosphate [poly(P)] as a phosphoryl donor for the phosphorylation of NAD. The enzyme overexpressed in Escherichia coli was purified and crystallized by means of the hanging-drop vapour-diffusion method with polyethylene glycol 4000 as the precipitant. Preliminary X-ray analysis of the resultant crystals revealed they belonged to the monoclinic space group C2 and had unit-cell parameters a = 140.0, b = 69.3, c = 106.3 A, beta = 130.1 degrees. The molecular weight of the NAD kinase is 35 kDa; assuming that a crystal contains two subunits of the NAD kinase in an asymmetric unit, the solvent content V(sol) is 0.62. X-ray diffraction data to 2.99 A have been collected from the native crystal.

Crystallization↗

PubMed2001

Anion-mediated Fe3+ release mechanism in ovotransferrin C-lobe: a structurally identified SO4(2-) binding site and its implications for the kinetic pathway.

The differential properties of anion-mediated Fe(3+) release between the N- and C-lobes of transferrins have been a focus in transferrin biochemistry. The structural and kinetic characteristics for isolated lobe have, however, been documented with the N-lobe only. Here we demonstrate for the first time the quantitative Fe(3+) release kinetics and the anion-binding structure for the isolated C-lobe of ovotransferrin. In the presence of pyrophosphate, sulfate, and nitrilotriacetate anions, the C-lobe released Fe(3+) with a decelerated rate in a single exponential progress curve, and the observed first order rate constants displayed a hyperbolic profile as a function of the anion concentration. The profile was consistent with a newly derived single-pathway Fe(3+) release model in which the holo form is converted depending on the anion concentration into a "mixed ligand" intermediate that releases Fe(3+). The apo C-lobe was crystallized in ammonium sulfate solution, and the structure determined at 2.3 A resolution demonstrated the existence of a single bound SO(4)(2-) in the interdomain cleft, which interacts directly with Thr(461)-OG1, Tyr(431)-OH, and His(592)-NE2 and indirectly with Tyr(524)-OH. The latter three groups are Fe(3+)-coordinating ligands, strongly suggesting the facilitated Fe(3+) release upon the anion occupation at this site. The SO(4)(2-) binding structure supported the single-pathway kinetic model.

Anions↗

PubMed2001

Domain closure mechanism in transferrins: new viewpoints about the hinge structure and motion as deduced from high resolution crystal structures of ovotransferrin N-lobe.

The crystal structure of holo hen ovotransferrin N-lobe refined at 1.65 A resolution has been obtained. The final model gave an R-factor of 0.173 in the resolution range between 10.0 and 1.65 A. The comparison of the structure with previous high-resolution apo and Fe(3+)-loaded, domain-opened intermediate structures provides new viewpoints on the domain closure mechanism upon Fe(3+) uptake in ovotransferrin N-lobe. Overall, conformational transition follows the common mechanism that has been first demonstrated for lactoferrin N-lobe; the domains 1 and 2 rotate 49.7 degrees as rigid bodies with a translation of 2.1 A around a screw-axis that passes through the two interdomain beta-strands (89-94 and 244-249). It is generally believed that the two strands display a hinge-like motion. Here, the latter strand indeed displays an ideal hinge nature: the segments 244-246 and 248-249 behave as a part of the rigid body of domain 2 and that of domain 1, respectively, and a sharp bend upon the domain closure is largely accounted for by the changes in the torsion angles phi and psi of Val247. We find, however, that the mode of the conformational change in the first beta-strand is much more complex. Two of the five inter beta-strand hydrogen bonds undergo crucial exchanges: from Ser91-N...Val247-O and Thr89-O...Ala249-N in the open apo and intermediate structures into Tyr92-N...Val247-O and Thr90-O...Ala249-N in the closed holo structure. These exchanges, which may be triggered in the intermediate state by modulation in the topological relation between the Fe(3+)-ligated hinge residue Tyr92-OH and the anion anchor residues of helix 5, are accompanied by a large conformational change and extensive hydrogen bonding rearrangements in a long stretch of segment of Glu82 to Tyr92. Such structural transition would work as a driving force for the domain closure, which highlights a "door closer"-like role, in addition to the canonical-hinge role, for the interdomain polypeptide segment pair. As an alternative hinge that secures the correct domain motion by being placed on a significant distance from the beta-strand hinge, we point out the participation of the van der Waals contacts formed between domain 1 residue of Met331 and domain 2 residues of Trp125, Ile129 and Trp140.

Animals↗

PubMed2001

Crystallization and preliminary X-ray analysis of AlgS, a bacterial ATP-binding cassette (ABC) protein specific to macromolecule import.

Sphingomonas sp. A1 possesses a macromolecule (alginate; average molecular size 25 700 Da) uptake system mediated by a novel pit-dependent ABC transporter. In this system, AlgS (363 amino-acid residues; 40 kDa) functions as an ATPase and provides energy for the translocation of high molecular-weight alginate across the cytoplasmic membrane. Hexahistidine-tagged AlgS of Sphingomonas sp. A1 was overexpressed in Escherichia coli and crystallized by means of the hanging-drop vapour-diffusion method with ammonium dihydrogen monophosphate as the precipitant. Preliminary X-ray analysis of the resultant crystals was performed; they belonged to the monoclinic space group P2(1) and had unit-cell parameters a = 57.4, b = 92.7, c = 65.8 A, beta = 102.3 degrees. X-ray diffraction data to 3.2 A have been collected from the native crystal.

ATP-Binding Cassette Transporters↗

PubMed2001

Crystal structure of alginate lyase A1-III complexed with trisaccharide product at 2.0 A resolution.

The structure of A1-III from a Sphingomonas species A1 complexed with a trisaccharide product (4-deoxy-l-erythro-hex-4-enepyranosyluronate-mannuronate-mannuronic acid) was determined by X-ray crystallography at 2.0 A with an R-factor of 0.16. The final model of the complex form comprising 351 amino acid residues, 245 water molecules, one sulfate ion and one trisaccharide product exhibited a C(alpha) r.m.s.d. value of 0.154 A with the reported apo form of the enzyme. The trisaccharide was bound in the active cleft at subsites -3 approximately -1 from the non-reducing end by forming several hydrogen bonds and van der Waals interactions with protein atoms. The catalytic residue was estimated to be Tyr246, which existed between subsites -1 and +1 based on a mannuronic acid model oriented at subsite +1.

Binding Sites↗

PubMed2001

Crystal structure of soybean proglycinin A1aB1b homotrimer.

Soybean glycinin is a member of the 11 S globulin family. The crystal structure of proglycinin was determined by X-ray crystallography at 2.8 A resolution with an R-factor of 0.199 and a free R-factor of 0.250. A trimer molecule was found in an asymmetric unit of crystals. The trimer model contains three A1aB1b subunits and comprises 1128 amino acid residues and 34 water molecules. The constituent protomers of the homo-trimeric protein are arranged around a 3-fold symmetry axis with dimensions of 95 Ax95 Ax40 A. The protomer model is composed of five fragments which correspond roughly to conserved regions based on the sequence alignment of various 11 S globulins. The core of the protomer consists of two jelly-roll beta-barrels and two extended helix domains. This structure of proglycinin is similar to those of canavalin and phaseolin belonging to the 7 S globulin family, strongly supporting the hypothesis that both 7 S and 11 S globulins are derived from a common ancestor. The inter and intra-chain disulfide bonds conserved in the 11 S globulin family are clearly observed. It is found that the face with the inter-chain disulfide bond (IE face) contains more hydrophobic residues than that with the intra-chain disulfide bond. This suggests that a mature hexamer is formed by the interaction between the IE faces after processing.

Amino Acid Sequence↗

PubMed2001

Crystal structures of recombinant and native soybean beta-conglycinin beta homotrimers.

The crystal structures of recombinant and native beta homotrimers of soybean beta-conglycinin were determined by X-ray crystallography at 2.7 and 2.8 A resolutions, respectively. The crystals of the recombinant and native beta homotrimers belong to space group P21 with cell parameters a = 80.51 A, b = 63.48 A, c = 131.43 A, and beta = 90.01 degrees and with cell parameters a = 82.78 A, b = 69.47 A, c = 125.33 A and beta = 97.22 degrees, respectively. The beta monomers consist of amino-terminal and carboxyl-terminal modules that are very similar to each other and are related by a pseudo-dyad axis. Each module of the beta monomer is subdivided into a core and a loop domain. These structural features of both beta homotrimers are consistent with those of canavalin and phaseolin, which are similar vicilin class proteins. The superposition of the models of the native and recombinant beta monomers shows a root mean square deviation of 0.43-0.51 A for 343 common Calpha atoms within 2.0 A. This result indicates that the N-linked glycans do not influence the final structure of the beta homotrimer. Comparison of the models of beta-conglycinin, phaseolin and canavalin indicates that beta-conglycinin resembles canavalin rather than phaseolin, and that canavalin and phaseolin differ the most among them. The evolutional relationships are discussed.

Amino Acid Sequence↗

PubMed2001

Super-channel in bacteria: function and structure of a macromolecule import system mediated by a pit-dependent ABC transporter.

In a soil isolate, Sphingomonas sp. A1, the transport of a macromolecule (alginate: 27 kDa) is mediated by a pit-dependent ATP-binding cassette (ABC) transporter. The transporter is different from other ABC transporters so far analyzed in that its function is dependent on the pit, a mouth-like organ formed on the cell surface only when the cells are compelled to assimilate macromolecules, and in that it allows direct import of macromolecules into cells. The ABC transporter coupled with the pit, which functions as a funnel and/or concentrator of macromolecules to be imported, was designated as the "Super-channel", and in this review, we discuss the three-dimensional structure and specific function of the "Super-channel" for macromolecule import found for the first time in a bacterium.

ATP-Binding Cassette Transporters↗

PubMed2000

Crystal structure of N-acyl-D-glucosamine 2-epimerase from porcine kidney at 2.0 A resolution.

The X-ray crystallographic structure of N-acyl-d-glucosamine 2-epimerase (AGE) from porcine kidney, which has been identified to be a renin-binding protein (RnBP), was determined by the multiple isomorphous replacement method and refined at 2.0 A resolution with a final R-factor of 16.9 % for 15 to 2.0 A resolution data. The refined structure of AGE comprised 804 amino acid residues (one dimer) and 145 water molecules. The dimer of AGE had an asymmetric unit with approximate dimensions 46 Ax48 Ax96 A. The AGE monomer is composed of an alpha(6)/alpha(6)-barrel, the structure of which is found in glucoamylase and cellulase. One side of the AGE alpha(6)/alpha(6)-barrel structure comprises long loops containing five short beta-sheets, and contributes to the formation of a deep cleft shaped like a funnel. The putative active-site pocket and a possible binding site for the substrate N-acetyl-d-glucosamine (GlcNAc) were found in the cleft. The other side of the alpha(6)/alpha(6)-barrel comprises short loops and contributes to the dimer formation. At the dimer interface, which is composed of the short loops and alpha-helices of the subunits, five strong ion-pair interactions were observed, which play a major role in the dimer assembly. This completely ruled out the previously accepted hypothesis that the formation of the RnBP homodimer and RnBP-renin heterodimer requires the leucine zipper motif present in RnBP.

Animals↗

PubMed2000

C8/119S mutation of major mite allergen Derf-2 leads to degenerate secondary structure and molecular polymerization and induces potent and exclusive Th1 cell differentiation.

Hyposensitization therapy for atopic diseases has been conducted for decades but suffered from many problems including anaphylactic reactions. We previously developed a mutant protein of the major mite allergen Derf-2, C8/119S, which showed reduced binding to IgE. The C8/119S mutant was shown to exhibit more efficient hyposensitizing effect than Derf-2 in the animal model of allergic bronchial asthma. In the present study, we indicate that C8/119S exhibits markedly augmented immunogenicity for the proliferation of Derf-2-specific human T cells and T cell clones irrespective of the epitope specificity as compared with Derf-2. Furthermore, C8/119S has induced potent and almost exclusive differentiation of Th1 cells from the peripheral blood of atopic patients in vitro. Neither Ag dosage effect nor absence of B cell-mediated Ag presentation could fully account for these effects. C8/119S has been indicated to lose the characteristic beta-barrel structure as judged by circular dichroism spectroscopic analysis and to polymerize solubly in physiological condition. Heating of Derf-2 also caused less stable molecular aggregation, but it hardly affected the secondary structure and failed to induce such a polarity toward the Th1 cell differentiation. These results have indicated that the degenerate secondary structure of C8/119S leading to stable molecular polymerization is primarily responsible for the marked increase in T cell-immunogenicity and the induction of exclusive Th1 cell differentiation in atopic patients. It has been suggested strongly that the recombinant C8/119S protein can provide an effective Ag with the least risk of anaphylaxis for allergen immunotherapy against house dust mite in human.

Adjuvants, Immunologic↗

PubMed2000

Crystallization and preliminary X-ray crystallographic analysis of NADPH: azodicarbonyl/quinone oxidoreductase, a plant zeta-crystallin.

Arabidopsis thaliana P1 protein was crystallized using the hanging drop vapor-diffusion method in 0.1 M piperazine-1, 4-bis(2-ethanesulfonic acid) buffer, containing 14% polyethylene glycol 6000 and 0.2 M magnesium acetate at pH 6.5 and 20 degrees C. The crystals are orthorhombic and belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a=49.8, b=122.4 and c=149. 9 A. The diffraction data up to 2.9 A were collected by a multiwire area detector.

Alkanesulfonic Acids↗

PubMed2000

Crystal structure at 1.9 A resolution of the apoovotransferrin N-lobe bound by sulfate anions: implications for the domain opening and iron release mechanism.

Several lines of functional evidence have shown that anion binding to a nonsynergistic site is a prerequisite for the anion-mediated iron release mechanism of transferrins. We report here structural evidence of the location of sulfate anion binding sites of the ovotransferrin N-lobe via the 1.90 A resolution apo crystal structure. The crystals were grown in an ammonium sulfate solution and belonged to space group P6(3)22 with the following unit cell dimensions: a = b = 125.17 A and c = 87.26 A. The structural determination was performed by isomorphous replacement, using Pt and Au derivatives. The structure refinement gave an R-factor of 0.187 in the resolution range of 7.0-1.90 A for the final model. From the electron density map, the existence of four bound SO(4)(2)(-) anions was detected. Three of them that exhibited reasonably low B-factors were all located in the opened interdomain cleft (sites 1-3). In site 1, the bound anion directly interacts with an Fe(3+)-coordinating ligand; SO(4) O1 and SO(4) O3 form hydrogen bonds with His250 NE2. Oxygen atom O3 of the same sulfate anion makes a hydrogen bond with Ser91 OG in a hinge strand. The sulfate anion in site 2 partially occupies the synergistic anion binding sites; SO(4) O2 and SO(4) O3 are hydrogen bonded to Arg121 NE and NH2, respectively, both of which are consensus anchor groups for CO(3)(2)(-) anion in holotransferrins. The former oxygen atom of SO(4)(2)(-) is also hydrogen bonded to Ser122 N, which forms a hydrogen bond with Fe(3+)-coordinating ligand Asp60 OD2 in holotransferrins. Some of the SO(4)(2)(-) oxygen atoms in sites 1 and 2 interact indirectly through H(2)O molecules with functionally important protein groups, such as the other Fe(3+)-coordinating ligands, Tyr92 OH and Tyr191 OH, and a dilysine trigger group, Lys209 NZ. In site 3, SO(4) O1 and SO(4) O4 form hydrogen bonds with Ser192 OG and Tyr191 N, respectively, and SO(4) O2 forms hydrogen bonds with Ser192 N and Ser192 OG. These structural data are consistent with the view that the anion bindings to the interdomain cleft, especially to sites 1 and 2, play crucial roles in the domain opening and synergistic carbonate anion release in the iron release mechanism of the ovotransferrin N-lobe.

Animals↗

PubMed2000

Crystallization and preliminary X-ray crystallographic analysis of alginate lyase A1-II from Sphingomonas species A1.

Alginate lyase A1-II of Sphingomonas species A1 was purified and crystallized using the hanging drop vapor-diffusion method in 0.1 M Tris-HCl buffer containing 43% saturated ammonium sulfate, 8% polyethylene glycol 4000 and 0.2 M Li(2)SO(4) at pH 8.5 and 20 degrees C. The crystals are tetragonal and belong to the space group P4(3)2(1)2 or P4(1)2(1)2 with unit cell dimensions of a=b=144.07 and c=296.38 A. The diffraction data up to 2.8 A were collected by a synchrotron radiation source at SPring-8 in Japan.

Bacterial Proteins↗

PubMed2000

Overexpression in Escherichia coli, purification, and characterization of Sphingomonas sp. A1 alginate lyases.

A bacterium Sphingomonas sp. A1 produces three kinds of alginate lyases [A1-I (66 kDa), A1-II (25 kDa), and A1-III (40 kDa)] from a single precursor, through posttranslational processing. Overexpression systems for these alginate lyases were constructed in Escherichia coli cells by controlling of the lyase genes under T7 promoter and terminator. Expression levels of A1-I, A1-II, and A1-III in E. coli cells were 3.50, 3.04, and 2.13 kU/liter of culture, respectively, and were over 10-fold higher than those in Sphingomonas sp. A1 cells. Purified A1-I, A1-II, and A1-III from E. coli cells were monomeric enzymes with molecular masses of 63, 25, and 40 kDa, respectively. The depolymerization pattern of alginate with A1-I and A1-II indicated that both enzymes cleaved the glycosidic bond of the polymer endolytically and by beta-elimination reaction. A1-II preferred polyguluronate rather than polymannuronate and released tri- and tetrasaccharides, which have unsaturated uronyl residues at the nonreducing terminal, from alginate as the major final products. A1-I acted equally on both homopolymers and produced di- and trisaccharides as the final products.

Alginates↗

PubMed1999

Crystal structure of hen apo-ovotransferrin. Both lobes adopt an open conformation upon loss of iron.

The three-dimensional crystal structure of hen apo-ovotransferrin has been solved by molecular replacement and refined by simulated annealing and restrained least squares to a 3.0-A resolution. The final model, which comprises 5312 protein atoms (residues 1 to 686) and 28 carbohydrate atoms (from two monosaccharides attached to Asn(473)), gives an R-factor of 0.231 for the 11,989 observed reflections between 20.0- and 3.0-A resolution. In the structure, both empty iron binding clefts are in the open conformation, lending weight to the theory that Fe(3+) binding or release in transferrin proceeds via a mechanism that involves domain opening and closure. Upon opening, the domains rotate essentially as rigid bodies. The two domains of the N-lobe rotate away from one another by 53 degrees, whereas the C-lobe domains rotate away each another by 35 degrees. These rotations take place about an axis that passes through the two beta-strands, linking the domains. The domains of each lobe make different contacts with one another in the open and closed forms. These contacts form two interdomain interfaces on either side of the rotation axis, and domain opening or closing produces a see-saw motion between these two alternative close-packed interfaces. The interdomain disulfide bridge (Cys(478)-Cys(671)), found only in the C-lobe, may restrict domain opening but does not completely prevent it.

Amino Acid Sequence↗

PubMed1999

Crystal structure of alginate lyase A1-III from Sphingomonas species A1 at 1.78 A resolution.

The three-dimensional structure of alginate lyase A1-III (ALYIII) from a Sphingomonas species A1 was determined by X-ray crystallography. The enzyme was crystallized by the hanging-drop vapour-diffusion method in the presence of 49% ammonium sulfate at 20 degrees C. The crystals are monoclinic and belong to the space group C2 with unit cell dimensions of a=49.18 A, b=93.08 A, c=82.10 A and beta=104.12 degrees. There was one molecule of alginate lyase in the asymmetric unit of the crystal. The diffraction data up to 1. 71 A were collected with Rsymof 5.0%. The crystal structure of ALYIII was solved by the multiple isomorphous replacement method and refined at 1.78 A resolution using X-PLOR with a final R -factor of 18.0% for 10.0 to 1.78 A resolution data. The refined model of ALYIII contained 351 amino acid residues, 299 water molecules and two sulfate ions. The three-dimensional structure of ALYIII was abundant in helices and had a deep tunnel-like cleft in a novel (alpha6/alpha5)-barrel structure, which was similar to the (alpha6/alpha6)-barrel found in glucoamylase and cellulase. This structure presented the possibility that alginate molecules might penetrate into the cleft to interact with the catalytic site of ALYIII.

Alginates↗