Biomedical subjects

Atsushi Nakagawa

Publications and source records attributed to Atsushi Nakagawa.

At least 19 recordsLinked to original sources

PubMed2006

Crystal structure of mammalian alpha1,6-fucosyltransferase, FUT8.

Mammalian alpha1,6-fucosyltransferase (FUT8) catalyses the transfer of a fucose residue from a donor substrate, guanosine 5'-diphosphate-beta-L-fucose to the reducing terminal N-acetylglucosamine (GlcNAc) of the core structure of an asparagine-linked oligosaccharide. Alpha1,6-fucosylation, also referred to as core fucosylation, plays an essential role in various pathophysiological events. Our group reported that FUT8 null mice showed severe growth retardation and emphysema-like lung-destruction as a result of the dysfunction of epidermal growth factor and transforming growth factor-beta receptors. To elucidate the molecular basis of FUT8 with respect to pathophysiology, the crystal structure of human FUT8 was determined at 2.6 A resolution. The overall structure of FUT8 was found to consist of three domains: an N-terminal coiled-coil domain, a catalytic domain, and a C-terminal SH3 domain. The catalytic region appears to be similar to GT-B glycosyltransferases rather than GT-A. The C-terminal part of the catalytic domain of FUT8 includes a Rossmann fold with three regions that are conserved in alpha1,6-, alpha1,2-, and protein O-fucosyltransferases. The SH3 domain of FUT8 is similar to other SH3 domain-containing proteins, although the significance of this domain remains to be elucidated. The present findings of FUT8 suggest that the conserved residues in the three conserved regions participate in the Rossmann fold and act as the donor binding site, or in catalysis, thus playing key roles in the fucose-transferring reaction.

Acetylglucosamine↗

PubMed2006

Structural mechanism and photoprotective function of water-soluble chlorophyll-binding protein.

A water-soluble chlorophyll-binding protein (WSCP) is the single known instance of a putative chlorophyll (Chl) carrier in green plants. Recently the photoprotective function of WSCP has been demonstrated by EPR measurements; the light-induced singlet-oxygen formation of Chl in the WSCP tetramer is about four times lower than that of unbound Chl. This paper describes the crystal structure of the WSCP-Chl complex purified from leaves of Lepidium virginicum (Virginia pepperweed) to clarify the mechanism of its photoprotective function. The WSCP-Chl complex is a homotetramer comprising four protein chains of 180 amino acids and four Chl molecules. At the center of the complex one hydrophobic cavity is formed in which all of the four Chl molecules are tightly packed and isolated from bulk solvent. With reference to the novel Chl-binding mode, we propose that the photoprotection mechanism may be based on the inhibition of physical contact between the Chl molecules and molecular oxygen.

Binding Sites↗

PubMed2006

The first crystal structure of L-threonine dehydrogenase.

L-threonine dehydrogenase (TDH) is an enzyme that catalyzes the oxidation of L-threonine to 2-amino-3-ketobutyrate. We solved the first crystal structure of a medium chain L-threonine dehydrogenase from a hyperthermophilic archaeon, Pyrococcus horikoshii (PhTDH), by the single wavelength anomalous diffraction method using a selenomethionine-substituted enzyme. This recombinant PhTDH is a homo-tetramer in solution. Three monomers of PhTDHs were located in the crystallographic asymmetric unit, however, the crystal structure exhibits a homo-tetramer structure with crystallographic and non-crystallographic 222 symmetry in the cell. Despite the low level of sequence identity to a medium-chain NAD(H)-dependent alcohol dehydrogenase (ADH) and the different substrate specificity, the overall folds of the PhTDH monomer and tetramer are similar to those of the other ADH. Each subunit is composed of two domains: a nicotinamide cofactor (NAD(H))-binding domain and a catalytic domain. The NAD(H)-binding domain contains the alpha/beta Rossmann fold motif, characteristic of the NAD(H)-binding protein. One molecule of PhTDH contains one zinc ion playing a structural role. This metal ion exhibits coordination with four cysteine ligands and some of the ligands are conserved throughout the structural zinc-containing ADHs and TDHs. However, the catalytic zinc ion that is coordinated at the bottom of the cleft in the case of ADH was not observed in the crystal of PhTDH. There is a significant difference in the orientation of the catalytic domain relative to the coenzyme-binding domain that results in a larger interdomain cleft.

Alcohol Oxidoreductases↗

PubMed2006

Crystal structure of the DsbB-DsbA complex reveals a mechanism of disulfide bond generation.

Oxidation of cysteine pairs to disulfide requires cellular factors present in the bacterial periplasmic space. DsbB is an E. coli membrane protein that oxidizes DsbA, a periplasmic dithiol oxidase. To gain insight into disulfide bond formation, we determined the crystal structure of the DsbB-DsbA complex at 3.7 A resolution. The structure of DsbB revealed four transmembrane helices and one short horizontal helix juxtaposed with Cys130 in the mobile periplasmic loop. Whereas DsbB in the resting state contains a Cys104-Cys130 disulfide, Cys104 in the binary complex is engaged in the intermolecular disulfide bond and captured by the hydrophobic groove of DsbA, resulting in separation from Cys130. This cysteine relocation prevents the backward resolution of the complex and allows Cys130 to approach and activate the disulfide-generating reaction center composed of Cys41, Cys44, Arg48, and ubiquinone. We propose that DsbB is converted by its specific substrate, DsbA, to a superoxidizing enzyme, capable of oxidizing this extremely oxidizing oxidase.

Amino Acid Sequence↗

PubMed2006

Insulin responses to selective arterial calcium infusion under hyperinsulinemic euglycemic glucose clamps: case studies in adult nesidioblastosis and childhood insulinoma.

Selective arterial calcium stimulation and hepatic venous sampling (ASVS) for insulin secretion is used as a diagnostic procedure in patients with insulinomas or adult nesidioblastosis. In some of those patients, severe hypoglycemia requiring urgent glucose administration occurs during the procedure. Such glucose administration, however, may affect the results and damage the validity of the test. We report two cases of hyperinsulinemic hypoglycemia, in which ASVS tests were successfully performed under hyperinsulinemic euglycemic glucose clamps. A 40-year-old male with nesidioblastosis developed continual severe hypoglycemia several years after a Billroth II-Braun gastrectomy, and continuous glucose infusion could not be stopped even during ASVS tests. A 9-year-old girl with an insulinoma that showed atypical hypovascularity on imaging examinations had ASVS tests under a glucose clamp for safety. Hyperinsulinemic (approximately 100 microU/ml) euglycemic (approximately 90 mg/dl) clamps were achieved by an artificial endocrine pancreas. The insulin analogue lispro was utilized for clamps and endogenous insulin was measured with an assay that does not cross-react with the analogue. Diagnostically significant responses (more than twofold) of insulin secretion were observed under hyperinsulinemic clamps in both cases. The use of the hyperinsulinemic glucose clamp technique during the ASVS test should be considered for maintaining the safety of some hypoglycemic patients.

Adult↗

PubMed2006

Conformation of amyloid fibrils of beta2-microglobulin probed by tryptophan mutagenesis.

Beta2-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis, adopts an immunoglobulin domain fold in its native state. Although beta2-m has Trp residues at positions 60 and 95, both are located near the surface of the domain. Hence, beta2-m does not have a conserved Trp common to other immunoglobulin domains, which is buried in close proximity to the disulfide bond. To study the structure of amyloid fibrils in relation to their native fold, we prepared a series of Trp mutants. Trp60 and Trp95 were both replaced with Phe, and a single Trp was introduced at various positions. Among various mutants, W39-beta2-m, in which a Trp was introduced at the position corresponding to the conserved Trp, exhibited a remarkable quenching of fluorescence in the native state, as observed for other immunoglobulin domains. An x-ray structural analysis revealed that W39-beta2-m assumes the native fold with Trp39 located in the vicinity of the disulfide bond. Comparison of the fluorescence spectra of various mutants for the native and fibrillar forms indicated that, while the Trp residues introduced in the middle of the beta2-m sequence tend to be buried in the fibrils, those located in the C-terminal region are more exposed. In addition, the fluorescence spectra of fibrils prepared at pH 2.5 and 7.0 revealed a large difference in the fluorescence intensity for W60-beta2-m, implying a major structural difference between them.

Amino Acid Sequence↗

PubMed2006

Targeting AMAP1 and cortactin binding bearing an atypical src homology 3/proline interface for prevention of breast cancer invasion and metastasis.

Invasive potentials of carcinomas greatly contribute to their metastasis, which is a major threat in most cancers. We have recently shown that Arf6 plays a pivotal role in breast cancer invasive activities and identified AMAP1 as an effector of GTP-Arf6 in invasion. Expression of AMAP1 correlates well with invasive phenotypes of primary tumors of the human breast. We also have shown that AMAP1 functions by forming a trimeric protein complex with cortactin and paxillin. In this complex, AMAP1 binds to the src homology 3 (SH3) domain of cortactin via its proline-rich peptide, SKKRPPPPPPGHKRT. SH3 domains are known to bind generally to the proline-rich ligands with a one-to-one stoichiometry. We found that AMAP1/cortactin binding is very atypical in its stoichiometry and interface structure, in which one AMAP1 proline-rich peptide binds to two cortactin SH3 domains simultaneously. We made a cell-permeable peptide derived from the AMAP1 peptide, and we show that this peptide specifically blocks AMAP1/cortactin binding, but not other canonical SH3/proline bindings, and effectively inhibits breast cancer invasion and metastasis. Moreover, this peptide was found to block invasion of other types of cancers, such as glioblastomas and lung carcinomas. We also found that a small-molecule compound, UCS15A, which was previously judged as a weak inhibitor against canonical SH3/proline bindings, effectively inhibits AMAP1/cortactin binding and breast cancer invasion and metastasis. Together with fine structural analysis, we propose that the AMAP1/cortactin complex, which is not detected in normal mammary epithelial cells, is an excellent drug target for cancer therapeutics.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Crystallization and preliminary X-ray analysis of rat SHPS-1.

SHPS-1, a receptor-type transmembrane protein, is abundantly expressed in neural and myeloid tissues. The most amino-terminal immunoglobulin-like domain of SHPS-1 plays an important role in a variety of cell functions by binding its ligand CD47. Interaction between SHPS-1 and CD47 is thought to be involved in negative regulation of phagocytosis. The ligand-binding domain of rat SHPS-1 was purified and crystallized using the vapour-diffusion method with the solution-stirring technique. Preliminary X-ray diffraction data were collected from SHPS-1 crystals to 2.8 A resolution and reduced to primitive hexagonal space group P622. Unit-cell parameters are a = b = 100.5, c = 101.3 A.

Animals↗

PubMed2006

Orexin-A is composed of a highly conserved C-terminal and a specific, hydrophilic N-terminal region, revealing the structural basis of specific recognition by the orexin-1 receptor.

Orexins-A and B, also called hypocretins-1 and 2, respectively, are neuropeptides that regulate feeding and sleep-wakefulness by binding to two orphan G protein-coupled receptors named orexin-1 (OX(1)R) and orexin-2 (OX(2)R). The sequences and functions of orexins-A and B are similar to each other, but the high sequence homology (68%) is limited in their C-terminal half regions (residues 15-33). The sequence of the N-terminal half region of orexin-A (residues 1-14), containing two disulfide bonds, is very different from that of orexin-B. The structure of orexin-A was determined using two-dimensional homonuclear and (15)N and (13)C natural abundance heteronuclear NMR experiments. Orexin-A had a compact conformation in the N-terminal half region, which contained a short helix (III:Cys6-Gln9) and was fixed by the two disulfide bonds, and a helix-turn-helix conformation (I:Leu16-Ala23 and II:Asn25-Thr32) in the remaining C-terminal half region. The C-terminal half region had both hydrophobic and hydrophilic residues, which existed on separate surfaces to provide an amphipathic character in helices I and II. The nine residues on the hydrophobic surface are also well conserved in orexin-B, and it was reported that the substitution of each of them with alanine resulted in a significant drop in the functional potency at the receptors. Therefore, we suggest that they form the surface responsible for the main hydrophobic interaction with the receptors. On the other hand, the residues on the hydrophilic surface, together with the hydrophilic residues in the N-terminal half region that form a cluster, are known to make only small contributions to the binding to the receptors through similar alanine-scan experiments. However, since our structure of orexin-A showed that large conformational and electrostatical differences between orexins-A and B were rather concentrated in the N-terminal half regions, we suggest that the region of orexin-A is important for the preference for orexin-A of OX(1)R.

Amino Acid Sequence↗

PubMed2006

The intracellular region of ClC-3 chloride channel is in a partially folded state and a monomer.

In contrast to bacterial ClC chloride channels, all eukaryotic ClC chloride channels have a conserved long intracellular region that makes up of the carboxyl terminus of the protein and is necessary for channel functions as a channel gate. Little is known, however, about the molecular structure of the intracellular region of ClC chloride channels so far. Here, for the first time, we have expressed and purified the intracellular region of the rat ClC-3 chloride channel (C-ClC-3) as a water-soluble protein under physiological conditions, and investigated its structural characteristics and assembly behavior by means of circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC), size exclusion chromatography and analytical ultracentrifugation. The far-UV CD spectra of C-ClC-3 in the native state and in the presence of urea clearly show that the protein has a significantly folded secondary structure consisting of alpha-helices and beta-sheets, while the near-UV CD spectra and DSC experiments indicate the protein is deficient in well-defined tertiary packing. Its Stokes radius is larger than its expected size as a folded globular protein, as determined on size exclusion chromatography. Furthermore, the DisEMBL program, a useful computational tool for the prediction of disordered/unstructured regions within a protein sequence, predicts that the protein is in a partially folded state. Based on these results, we conclude that C-ClC-3 is partially folded. On the other hand, both size exclusion chromatography and sedimentation equilibrium analysis show that C-ClC-3 exists as a monomer in solution, not a dimer like the whole ClC-3 molecule.

Amino Acid Sequence↗

PubMed2006

Improvement on production of (R)-4-chloro-3-hydroxybutyrate and (S)-3-hydroxy-gamma-butyrolactone with recombinant Escherichia coli cells.

(R)-4-Chloro-3-hydroxybutyrate (CHB) and (S)-3-hydroxy-gamma-butyrolactone (HL) are used for the synthesis of biologically and pharmacologically important compounds. Enterobacter sp. DS-S-75 was found to have the unique activity to convert (S)-CHB in the racemate to (S)-HL through asymmetric dechlorination, hydrolysis, and lactonization. As a result, the remaining (R)-CHB and formed (S)-HL could be obtained in a one-pot reaction. We purified the CHB degrading enzyme which catalyzing these reactions and isolated the coding gene from the strain DS-S-75 in order to improve the productivity of these compounds using the transformant. Interestingly, the purified enzyme showed not only dechlorinating, but also hydrolyzing activities on CHB and the similar carboxylic esters, it was then designated CHB hydrolase, and appears to be a novel enzyme. The gene had 1101 bp encoding 367 amino acids including a signal peptide composed of 25 residues. The deduced amino acid sequence contained a conserved region generally found in esterases and lipases, but did not have significant similarity. When asymmetric degradation of racemic methyl CHB (CHBM) was performed using a culture broth of Escherichia coli DH5alpha transformed with the isolated gene, the reaction time was shortened 20-fold over that of the strain DS-S-75, and the maximum concentration of the substrate could be increased from 8% to 15% (w/v). Moreover, both of the obtained residual (R)-CHBM and the formed (S)-HL had high optical purities (>99% e.e.).

4-Butyrolactone↗

PubMed2005

Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia.

T-protein, a component of the glycine cleavage system, catalyzes the formation of ammonia and 5,10-methylenetetrahydrofolate from the aminomethyl moiety of glycine attached to the lipoate cofactor of H-protein. Several mutations in the human T-protein gene cause non-ketotic hyperglycinemia. To gain insights into the effect of disease-causing mutations and the catalytic mechanism at the molecular level, crystal structures of human T-protein in free form and that bound to 5-methyltetrahydrofolate (5-CH3-H4folate) have been determined at 2.0 A and 2.6 A resolution, respectively. The overall structure consists of three domains arranged in a cloverleaf-like structure with the central cavity, where 5-CH3-H4folate is bound in a kinked shape with the pteridine group deeply buried into the hydrophobic pocket and the glutamyl group pointed to the C-terminal side surface. Most of the disease-related residues cluster around the cavity, forming extensive hydrogen bonding networks. These hydrogen bonding networks are employed in holding not only the folate-binding space but also the positions and the orientations of alpha-helix G and the following loop in the middle region, which seems to play a pivotal role in the T-protein catalysis. Structural and mutational analyses demonstrated that Arg292 interacts through water molecules with the folate polyglutamate tail, and that the invariant Asp101, located close to the N10 group of 5-CH3-H4folate, might play a key role in the initiation of the catalysis by increasing the nucleophilic character of the N10 atom of the folate substrate for the nucleophilic attack on the aminomethyl lipoate intermediate. A clever mechanism of recruiting the aminomethyl lipoate arm to the reaction site seems to function as a way of avoiding the release of toxic formaldehyde.

Amino Acid Sequence↗

PubMed2005

Crystal structure of lipoate-protein ligase A from Escherichia coli. Determination of the lipoic acid-binding site.

Lipoate-protein ligase A (LplA) catalyzes the formation of lipoyl-AMP from lipoate and ATP and then transfers the lipoyl moiety to a specific lysine residue on the acyltransferase subunit of alpha-ketoacid dehydrogenase complexes and on H-protein of the glycine cleavage system. The lypoyllysine arm plays a pivotal role in the complexes by shuttling the reaction intermediate and reducing equivalents between the active sites of the components of the complexes. We have determined the X-ray crystal structures of Escherichia coli LplA alone and in a complex with lipoic acid at 2.4 and 2.9 angstroms resolution, respectively. The structure of LplA consists of a large N-terminal domain and a small C-terminal domain. The structure identifies the substrate binding pocket at the interface between the two domains. Lipoic acid is bound in a hydrophobic cavity in the N-terminal domain through hydrophobic interactions and a weak hydrogen bond between carboxyl group of lipoic acid and the Ser-72 or Arg-140 residue of LplA. No large conformational change was observed in the main chain structure upon the binding of lipoic acid.

Amino Acid Sequence↗

PubMed2005

Ab initio crystal structure determination of spherical viruses that exhibit a centrosymmetric location in the unit cell.

Ab initio phasing by non-crystallographic symmetry averaging coupled with solvent flattening has previously been used to determine the structure of canine parvovirus. As CPV particles were located at general positions, initial phases were generated with a spherical shell deviating from the centre of symmetry. In many virus crystals, the viral particles are located at positions with a centre of symmetry in the unit cell. Thus, the initial phases calculated with a spherical shell model have a centre of symmetry. The inherent difficulty in structural determination was breaking the centre of symmetry in the initial phase. The centric nature of the initial phases of the rice dwarf virus crystal, however, was successfully broken at low resolution by iteration of the density-modification method described by Tsao et al. (1992). In this study, ab initio phasing was tested for seven viruses ranging from 82 to 344 A in radius. Although the crystal structures of the initial spherical shell models for each virus had a centre of symmetry, the centric natures of the initial phases were successfully broken by non-crystallographic symmetry averaging coupled with solvent flattening at a low resolution; these phases were then successfully extended to high-resolution measurements. A novel procedure of ab initio phasing for spherical virus crystals is proposed.

Crystallography↗

PubMed2005

Crystallization and preliminary X-ray diffraction analysis of a chitin-binding domain of hyperthermophilic chitinase from Pyrococcus furiosus.

The crystallization and preliminary X-ray diffraction analysis of the chitin-binding domain of chitinase from a hyperthermophilic archaeon, Pyrococcus furiosus, are reported. The recombinant protein was prepared using an Escherichia coli overexpression system and was crystallized by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected to 1.70 A resolution. The crystal belonged to space group P4(3)2(1)2 or P4(1)2(1)2. The unit-cell parameters were determined to be a = b = 48.8, c = 85.0 A.

Chitin↗

PubMed2005

Crystallization and preliminary X-ray analysis of hyperthermophilic L-threonine dehydrogenase from the archaeon Pyrococcus horikoshii.

Recombinant L-threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus horikoshii was prepared using an Escherichia coli expression system. The hyperthermostable L-threonine dehydrogenase consists of 348 amino acids with a molecular weight of 37.7 kDa. The enzyme was crystallized by the hanging-drop vapour-diffusion method at 277 K and preliminary X-ray crystallographic analysis was carried out. Diffraction data were collected to 2.20 A resolution under cryogenic conditions. P. horikoshii L-threonine dehydrogenase crystals belong to space group I4(1)22, with unit-cell parameters a = b = 143.84, c = 304.13 A. The presence of three subunits of the enzyme per asymmetric unit was estimated to give a Matthews coefficient (VM) of 3.5 A3 Da(-1) and a solvent content of 64.7%(v/v).

Alcohol Oxidoreductases↗

PubMed2005

Transcapsidation and the conserved interactions of two major structural proteins of a pair of phytoreoviruses confirm the mechanism of assembly of the outer capsid layer.

The strongly conserved amino acid sequences of the P8 outer capsid proteins of Rice dwarf virus (RDV) and Rice gall dwarf virus (RGDV) and the distribution of electrostatic potential on the proteins at the interfaces between structural proteins suggested the possibility that P8-trimers of RGDV might bind to the 3-fold symmetrical axes of RDV core particles, with vertical interaction between heterologous P3 and P8 proteins and lateral binding of homologous P8 proteins, thereby allowing formation of the double-layered capsids that are characteristic of viruses that belong to the family Reoviridae. We proved this hypothesis using chimeric virus-like particles composed of the P3 core capsid protein of RDV and the P8 outer capsid protein of RGDV, which were co-expressed in a baculovirus expression system. This is the first report on the molecular biological proof of the mechanism of the assembly of the double-layered capsids with disparate icosahedral lattices.

Amino Acid Sequence↗

PubMed2005

The crystal structures of semi-synthetic aequorins.

The photoprotein aequorin emits light by an intramolecular reaction in the presence of a trace amount of Ca(2+). Semi-synthetic aequorins, produced by replacing the coelenterazine moiety in aequorin with the analogues of coelenterazine, show widely different sensitivities to Ca(2+). To understand the structural basis of the Ca(2+)-sensitivity, we determined the crystal structures of four semi-synthetic aequorins (cp-, i-, br- and n-aequorins) at resolutions of 1.6-1.8 A. In general, the protein structures of these semi-synthetic aequorins are almost identical to native aequorin. Of the four EF-hand domains in the molecule, EF-hand II does not bind Ca(2+), and the loop of EF-hand IV is clearly deformed. It is most likely that the binding of Ca(2+) with EF-hands I and III triggers luminescence. Although little difference was found in the overall structures of aequorins investigated, some significant differences were found in the interactions between the substituents of coelenterazine moiety and the amino acid residues in the binding pocket. The coelenterazine moieties in i-, br-, and n-aequorins have bulky 2-substitutions, which can interfere with the conformational changes of protein structure that follow the binding of Ca(2+) to aequorin. In cp-aequorin, the cyclopentylmethyl group that substitutes for the original 8-benzyl group does not interact hydrophobically with the protein part, giving the coelenterazine moiety more conformational freedom to promote the light-emitting reaction. The differences of various semi-synthetic aequorins in Ca(2+)-sensitivity and reaction rate are explained by the capability of the involved groups and structures to undergo conformational changes in response to the Ca(2+)-binding.

Aequorin↗