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Hiroshi Hirota

Publications and source records attributed to Hiroshi Hirota.

At least 19 recordsLinked to original sources
PubMed2006

Cation-pi interaction in the polyolefin cyclization cascade uncovered by incorporating unnatural amino acids into the catalytic sites of squalene cyclase.

It has been assumed that the pi-electrons of aromatic residues in the catalytic sites of triterpene cyclases stabilize the cationic intermediates formed during the polycyclization cascade of squalene or oxidosqualene, but no definitive experimental evidence has been given. To validate this cation-pi interaction, natural and unnatural aromatic amino acids were site-specifically incorporated into squalene-hopene cyclase (SHC) from Alicyclobacillus acidocaldarius and the kinetic data of the mutants were compared with that of the wild-type SHC. The catalytic sites of Phe365 and Phe605 were substituted with O-methyltyrosine, tyrosine, and tryptophan, which have higher cation-pi binding energies than phenylalanine. These replacements actually increased the SHC activity at low temperature, but decreased the activity at high temperature, as compared with the wild-type SHC. This decreased activity is due to the disorganization of the protein architecture caused by the introduction of the amino acids more bulky than phenylalanine. Then, mono-, di-, and trifluorophenylalanines were incorporated at positions 365 and 605; these amino acids reduce cation-pi binding energies but have van der Waals radii similar to that of phenylalanine. The activities of the SHC variants with fluorophenylalanines were found to be inversely proportional to the number of the fluorine atoms on the aromatic ring and clearly correlated with the cation-pi binding energies of the ring moiety. No serious structural alteration was observed for these variants even at high temperature. These results unambiguously show that the pi-electron density of residues 365 and 605 has a crucial role for the efficient polycyclization reaction by SHC. This is the first report to demonstrate experimentally the involvement of cation-pi interaction in triterpene biosynthesis.

Alanine↗
PubMed2006

An alpha2-macroglobulin-like protein is the cue to gregarious settlement of the barnacle Balanus amphitrite.

Many benthic marine invertebrates, like barnacles, have a planktonic larval stage whose primary purpose is dispersal. How these species colonize suitable substrata is fundamental to understanding their evolution, population biology, and wider community dynamics. Unlike larval dispersal, settlement occurs on a relatively small spatial scale and involves larval behavior in response to physical and chemical characteristics of the substratum. Biogenic chemical cues have been implicated in this process. Their identification, however, has proven challenging, no more so than for the chemical basis of barnacle gregariousness, which was first described >50 years ago. We now report that a biological cue to gregarious settlement, the settlement-inducing protein complex (SIPC), of the major fouling barnacle Balanus amphitrite is a previously undescribed glycoprotein. The SIPC shares a 30% sequence homology with the thioester-containing family of proteins that includes the alpha(2)-macroglobulins. The cDNA (5.2 kb) of the SIPC encodes a protein precursor comprising 1,547 aa with a 17-residue signal peptide region. A number of structural characteristics and the absence of a thioester bond in the SIPC suggest that this molecule is a previously undescribed protein that may have evolved by duplication from an ancestral alpha(2)-macroglobulin gene. Although the SIPC is regarded as an adult cue that is recognized by the cyprid at settlement, it is also expressed in the juvenile and in larvae, where it may function in larva-larva settlement interactions.

Amino Acid Sequence↗
PubMed2006

Solution structure of the antifreeze-like domain of human sialic acid synthase.

The structure of the C-terminal antifreeze-like (AFL) domain of human sialic acid synthase was determined by NMR spectroscopy. The structure comprises one alpha- and two single-turn 3(10)-helices and two beta-strands, and is similar to those of the type III antifreeze proteins. Evolutionary trace analyses of the type III antifreeze protein family suggested that the class-specific residues in the human and bacterial AFL domains are important for their substrate binding, while the class-specific residues of the fish antifreeze proteins are gathered on the ice-binding surface.

Amino Acid Sequence↗
PubMed2006

Asteropine A, a sialidase-inhibiting conotoxin-like peptide from the marine sponge Asteropus simplex.

Marine sponges contain structurally intriguing and biologically active peptides of nonribosomal peptide synthase origin, often containing amino acids with novel structures. Here we report the discovery of asteropine A (APA), a cystine knot to be isolated from marine sponges. The solution structure of APA as determined by NMR belongs to the four-loop class of cystine knots similar to those of some conotoxins and spider toxins. However, the highly negatively charged surface of APA is uncommon among other cystine knots. APA competitively inhibits bacterial sialidases, but not a viral sialidase. APA was inactive against all other enzymes tested and did not have any apparent antitumor activity. Our data suggest that APA and other knotting peptides may be important leads for antibacterial and even antiviral drug development.

Amino Acid Sequence↗
PubMed2005

Axinelloside A, an unprecedented highly sulfated lipopolysaccharide inhibiting telomerase, from the marine sponge, Axinella infundibula.

Axinelloside A was isolated from the lipophilic extract of the Japanese marine sponge Axinella infundibula as a strong human telomerase inhibitor (IC(50) 2.0 microg/mL). It has the molecular weight of 4780.4 as the monoisotopic mass of the 19 sodium salt. The chemical structure was elucidated mainly by spectroscopic methods (2D NMR and MS). Axinelloside A consists of twelve sugars, e.g., a scyllo-inositol, a D-arabinose, 5 D-galactoses, and 5 L-fucoses, together with an (R)-3-hydroxy-octadecanoic acid, 3 (E)-2-hexadecenoic acids, and 19 sulfates.

Animals↗
PubMed2005

On-probe sample preparation without washes for matrix-assisted laser desorption/ionization mass spectrometry using an anion exchange medium.

When the mass spectra of biological samples (proteins, peptides, and so on) are obtained routinely by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), a serious problem is the reduction of the ionization efficiency by impurities, such as buffer salts and detergents. We focused our attention on devising a method to maintain the ionization efficiency of protein samples, even in the presence of sodium dodecyl sulfate (SDS), without any extra purification step. Although no protein ion peaks are observed in the presence of 2.5% SDS with the usual methods, the addition of a granular anion exchange silica gel to the matrix solution allowed the protein ion peaks to be obtained with an excellent signal-to-noise ratio. Together with other supporting experiments, we suggest that the positively charged surface (the basic environment derived from the anion exchange groups) and the roughness of the particles were important for good ionization in the presence of a high SDS concentration. For a very uneven surface, the SDS might be absorbed into the particle interiors during the process of cocrystallization with the matrix and analytes, which is known as the molecular sieve effect, and the SDS concentration in the surface crystalline film might be reduced. As a result, we developed an on-probe sample preparation method without washes for MALDI, using a strong anion exchange silica gel. This method is applicable even in the presence of 2.5% SDS, and is not only very simple but also inexpensive, because it can be used with the standard MALDI target plates.

Anions↗
PubMed2005

Solution structure of Vanabin2, a vanadium(IV)-binding protein from the vanadium-rich ascidian Ascidia sydneiensis samea.

Ascidians belonging to the suborder Phlebobranchia are known to accumulate high levels of a transition metal, vanadium, in their blood cells, called vanadocytes, although the mechanism for this biological phenomenon remains unclear. Recently, we identified vanadium(IV)-binding proteins, designated as Vanabins, from vanadium-accumulating ascidians. Here, we report the first 3D structure of Vanabin2 from an ascidian, Ascidia sydneiensis samea, in an aqueous solution. The structure revealed a novel bow-shaped conformation, with four alpha-helices connected by nine disulfide bonds. There are no structural homologues reported so far. The 15N heteronuclear single-quantum coherence (HSQC) perturbation experiments of Vanabin2 indicated that vanadyl cations, which are exclusively localized on the same face of the molecule, are coordinated by amine nitrogens derived from amino acid residues such as lysines, arginines, and histidines, as suggested by the electron paramagnetic resonance (EPR) results. The present NMR studies provide information that will contribute toward elucidating the mechanism of vanadium accumulation in ascidians.

Amino Acid Sequence↗
PubMed2005

Crystal structure of a novel polyisoprenoid-binding protein from Thermus thermophilus HB8.

The isoprenoid quinones exist widely among prokaryotes and eukaryotes. They play essential roles in respiratory electron transport and in controlling oxidative stress and gene regulation. In the isoprenoid quinone biosynthetic pathway, polyprenyl pyrophosphates are used as isoprenoid side-chain precursors. Here we report the crystal structure of a novel polyprenyl pyrophosphate binding protein, TT1927b, from Thermus thermophilus HB8, complexed with its ligand. This protein belongs to the YceI-like family in the Pfam database, and its sequence homologs are present in a broad range of bacteria and archaea. The structure consists of an extended, eight-stranded, antiparallel beta-barrel. In the hydrophobic pore of the barrel, the protein binds the polyisoprenoid chain by hydrophobic interactions. Its overall structure resembles the lipocalin fold, but there is no sequence homology between TT1927b and the lipocalin family of proteins.

Amino Acid Sequence↗
PubMed2005

Solution structure of the PWWP domain of the hepatoma-derived growth factor family.

Among the many PWWP-containing proteins, the largest group of homologous proteins is related to hepatoma-derived growth factor (HDGF). Within a well-conserved region at the extreme N-terminus, HDGF and five HDGF-related proteins (HRPs) always have a PWWP domain, which is a module found in many chromatin-associated proteins. In this study, we determined the solution structure of the PWWP domain of HDGF-related protein-3 (HRP-3) by NMR spectroscopy. The structure consists of a five-stranded beta-barrel with a PWWP-specific long loop connecting beta2 and beta3 (PR-loop), followed by a helical region including two alpha-helices. Its structure was found to have a characteristic solvent-exposed hydrophobic cavity, which is composed of an abundance of aromatic residues in the beta1/beta2 loop (beta-beta arch) and the beta3/beta4 loop. A similar ligand binding cavity occurs at the corresponding position in the Tudor, chromo, and MBT domains, which have structural and probable evolutionary relationships with PWWP domains. These findings suggest that the PWWP domains of the HDGF family bind to some component of chromatin via the cavity.

Amino Acid Motifs↗
PubMed2005

13-Deoxytedanolide, a marine sponge-derived antitumor macrolide, binds to the 60S large ribosomal subunit.

13-Deoxytedanolide is a potent antitumor macrolide isolated from the marine sponge Mycale adhaerens. In spite of its remarkable activity, the mode of action of 13-deoxytedanolide has not been elucidated. [11-3H]-(11S)-13-Deoxydihydrotedanolide derived from the macrolide was used for identifying the target molecule from the yeast cell lysate. Fractionation of the binding protein revealed that the labeled 13-deoxytedanolide derivative strongly bound to the 80S ribosome as well as to the 60S large subunit, but not to the 40S small subunit. In agreement with this observation, 13-deoxytedanolide efficiently inhibited the polypeptide elongation. Interestingly, competition studies demonstrated that 13-deoxytedanolide shared the binding site on the 60S large subunit with pederin and its marine-derived analogues. These results indicate that 13-deoxytedanolide is a potent protein synthesis inhibitor and is the first macrolide to inhibit the eukaryotic ribosome.

Animals↗
PubMed2005

Structure-activity relationship study on 13-deoxytedanolide, a highly antitumor macrolide from the marine sponge Mycale adhaerens.

To obtain information of structure-activity relationships (SARs) of 13-deoxytedanolide, its chemical transformation has been carried out, targeting on such functional groups as an epoxide, hydroxyls, ketones, and olefins. A total of 10 derivatives have been prepared and their cytotoxicity against P388 murine leukemia cells and inhibitory activity of polypeptide elongation in yeast cell lysate provided some important SARs; the southern hemisphere comprises the pharmacophore, while the epoxide-bearing side chain is essential for the activity.

Animals↗
PubMed2005

Himeic acid A: a new ubiquitin-activating enzyme inhibitor isolated from a marine-derived fungus, Aspergillus sp.

A new ubiquitin-activating enzyme (E1) inhibitor, himeic acid A, was isolated from a culture of marine-derived fungus, Aspergillus sp. The structure was determined by spectroscopic analysis. The formation of an E1-ubiquitin (Ub) intermediate was 65% inhibited by himeic acid A at the concentration of 50 microM, while two new related compounds, himeic acids B and C, showed little inhibitory activity even at 100 microM.

Aspergillus↗
PubMed2005

A structure-based strategy for discovery of small ligands binding to functionally unknown proteins: combination of in silico screening and surface plasmon resonance measurements.

In the postgenomic era, many researchers and organizations have been engaged in structural and functional analyses of proteins. As a part of these efforts, searching for small organic compounds that bind specifically to target proteins is quite important. In this study, we have developed a rational strategy for ligand discovery based on the three-dimensional structures of target proteins, which were elucidated by X-ray crystallography and nuclear magnetic resonance spectroscopy. The strategy has three features: (i) rapid selection of candidate compounds by in silico screening, (ii) automated preparation of sample solutions with robotics, and (iii) reliable evaluation of the candidates with surface plasmon resonance. Applying the strategy to a protein, At2g24940 from Arabidopsis thaliana, we discovered four small ligands out of a commercially available library of about 150 000 compounds. Although these compounds had only weak affinities to the target protein, with dissociation constants ranging from 68 to 120 microM, they apparently possess common structural features. They would be leads for the development of specific inhibitors/drugs for At2g24940, and provide important clues toward elucidation of the protein function.

Arabidopsis Proteins↗
PubMed2005

NMR analysis of the Mg2+-binding properties of aequorin, a Ca2+-binding photoprotein.

Aequorin, which is a calcium-sensitive photoprotein and a member of the EF-hand superfamily, binds to Mg2+ under physiological conditions, which modulates its light emission. The Mg2+ binding site and its stabilizing influence were examined by NMR spectroscopy. The binding of Mg2+ to aequorin prevented the molecule from aggregating and stabilized it in the monomeric form. To determine the structural differences between Mg2+-bound and free aequorin, we have performed backbone NMR assignments of aequorin in the Mg2+-free state. Mg2+ binding induces conformational changes that are localized in the EF-hand loops. The chemical shift difference data indicated that there are two Mg2+-binding sites, EF-hands I and III. The Mg2+ titration experiment revealed that EF-hand III binds to Mg2+ with higher affinity than EF-hand I, and that only EF-hand III seems to be occupied by Mg2+ under physiological conditions.

Aequorin↗
PubMed2004

Solution structure of the SEA domain from the murine homologue of ovarian cancer antigen CA125 (MUC16).

Human CA125, encoded by the MUC16 gene, is an ovarian cancer antigen widely used for a serum assay. Its extracellular region consists of tandem repeats of SEA domains. In this study we determined the three-dimensional structure of the SEA domain from the murine MUC16 homologue using multidimensional NMR spectroscopy. The domain forms a unique alpha/beta sandwich fold composed of two alpha helices and four antiparallel beta strands and has a characteristic turn named the TY-turn between alpha1 and alpha2. The internal mobility of the main chain is low throughout the domain. The residues that form the hydrophobic core and the TY-turn are fully conserved in all SEA domain sequences, indicating that the fold is common in the family. Interestingly, no other residues are conserved throughout the family. Thus, the sequence alignment of the SEA domain family was refined on the basis of the three-dimensional structure, which allowed us to classify the SEA domains into several subfamilies. The residues on the surface differ between these subfamilies, suggesting that each subfamily has a different function. In the MUC16 SEA domains, the conserved surface residues, Asn-10, Thr-12, Arg-63, Asp-75, Asp-112, Ser-115, and Phe-117, are clustered on the beta sheet surface, which may be functionally important. The putative epitope (residues 58-77) for anti-MUC16 antibodies is located around the beta2 and beta3 strands. On the other hand the tissue tumor marker MUC1 has a SEA domain belonging to another subfamily, and its GSVVV motif for proteolytic cleavage is located in the short loop connecting beta2 and beta3.

Amino Acid Sequence↗
PubMed2004

Solution structure of a BolA-like protein from Mus musculus.

The BolA-like proteins are widely conserved from prokaryotes to eukaryotes. The BolA-like proteins seem to be involved in cell proliferation or cell-cycle regulation, but the molecular function is still unknown. Here we determined the structure of a mouse BolA-like protein. The overall topology is alphabetabetaalphaalphabetaalpha, in which beta(1) and beta(2) are antiparallel, and beta(3) is parallel to beta(2). This fold is similar to the class II KH fold, except for the absence of the GXXG loop, which is well conserved in the KH fold. The conserved residues in the BolA-like proteins are assembled on the one side of the protein.

Amino Acid Sequence↗