<jats:title>Abstract</jats:title><jats:p><jats:italic>Watasenia scintillans</jats:italic>, a sparkling enope squid, has bioluminescence organs to illuminate its body with its own luciferase activity. To clarify the molecular mechanism underlying its scintillation, we analysed high-throughput sequencing data acquired previously and obtained draft genome sequences accomplished with comparative genomic data among the cephalopods. The genome mapped by transcriptome data showed that (1) RNA editing contributed to transcriptome variation of lineage specific genes, such as <jats:italic>W. scintillans</jats:italic> luciferase, and (2) two types of luciferase enzymes were characterized with reasonable 3D models docked to a luciferin molecule. We report two different types of luciferase in one organism and possibly related to variety of colour types in the <jats:italic>W. scintillans</jats:italic> fluorescent organs.</jats:p>

© 2020 Kagaya et al. Otter clam farming in Vietnam has recently encountered difficulties due to swollen-siphon disease. Here, we report the metagenome sequences of microorganisms extracted from the siphon tissue of infected otter clams. The data comprised bacterial and viral sequences which likely include those derived from the disease-causing agent.

<title>Abstract</title>Potential inhibitors of a target biomolecule, NAD-dependent deacetylase Sirtuin 1, were identified by a contest-based approach, in which participants were asked to propose a prioritized list of 400 compounds from a designated compound library containing 2.5 million compounds using <italic>in silico</italic> methods and scoring. Our aim was to identify target enzyme inhibitors and to benchmark computer-aided drug discovery methods under the same experimental conditions. Collecting compound lists derived from various methods is advantageous for aggregating compounds with structurally diversified properties compared with the use of a single method. The inhibitory action on Sirtuin 1 of approximately half of the proposed compounds was experimentally accessed. Ultimately, seven structurally diverse compounds were identified.

Caspase-3-related DEVDase activity is initiated upon apoptosis in unfertilized starfish eggs. In this study, we cloned a starfish procaspase-3 corresponding to mammalian effector caspase containing a CARD that is similar to the amino terminal CARD of mammalian capsase-9, and we named it procaspase-3/9. Recombinant procaspase-3/9 expressed at 15 °C was cleaved to form active caspase-3/9 which has DEVDase activity. Microinjection of the active caspase-3/9 into starfish oocytes/eggs induced apoptosis. An antibody against the recombinant protein recognized endogenous procaspase-3/9 in starfish oocytes, which was cleaved upon apoptosis in aged unfertilized eggs. These results indicate that caspase-3/9 is an effector caspase in starfish. To verify the mechanism of caspase-3/9 activation, we cloned starfish Apaf-1 containing a CARD, a NOD, and 11 WD40 repeat regions, and we named it sfApaf-1. Recombinant sfApaf-1 CARD interacts with recombinant caspase-3/9 CARD and with endogenous procaspase-3/9 in cell-free preparations made from starfish oocytes, causing the formation of active caspase-3/9. When the cell-free preparation without mitochondria was incubated with inactive recombinant procaspase-3/9 expressed at 37 °C, DEVDase activity increased and apoptosome-like complexes were formed in the high molecular weight fractions containing both sfApaf-1 and cleaved caspase-3/9. These results suggest that sfApaf-1 activation is not dependent on cytochrome c.

The neocortex exhibits a six-layered structure that is formed by radial migration of excitatory neurons, for which the multipolar-to-bipolar transition of immature migrating multipolar neurons is required. Here, we report that subplate neurons, one of the first neuron types born in the neocortex, manage the multipolar-to-bipolar transition of migrating neurons. By histochemical, imaging, and microarray analyses on the mouse embryonic cortex, we found that subplate neurons extend neurites toward the ventricular side of the subplate and form transient glutamatergic synapses on the multipolar neurons just below the subplate. NMDAR (N-methyl-D-aspartate receptor)-mediated synaptic transmission from subplate neurons to multipolar neurons induces themultipolar-to-bipolar transition, leading to a change inmigration mode from slow multipolar migration to faster radial glial-guided locomotion. Our data suggested that transient synapses formed on early immature neurons regulate radialmigration.

Background: More than 7000 papers related to "protein refolding" have been published to date, with approximately 300 reports each year during the last decade. Whilst some of these papers provide experimental protocols for protein refolding, a survey in the structural life science communities showed a necessity for a comprehensive database for refolding techniques. We therefore have developed a new resource "REFOLDdb" that collects refolding techniques into a single, searchable repository to help researchers develop refolding protocols for proteins of interest.
Results: We based our resource on the existing REFOLD database, which has not been updated since 2009. We redesigned the data format to be more concise, allowing consistent representations among data entries compared with the original REFOLD database. The remodeled data architecture enhances the search efficiency and improves the sustainability of the database. After an exhaustive literature search we added experimental refolding protocols from reports published 2009 to early 2017. In addition to this new data, we fully converted and integrated existing REFOLD data into our new resource. REFOLDdb contains 1877 entries as of March 17th, 2017, and is freely available at http://p4d-info.nig.ac.jp/refolddb/.
Conclusion: REFOLDdb is a unique database for the life sciences research community, providing annotated information for designing new refolding protocols and customizing existing methodologies. We envisage that this resource will find wide utility across broad disciplines that rely on the production of pure, active, recombinant proteins. Furthermore, the database also provides a useful overview of the recent trends and statistics in refolding technology development.

Protein is the major component of the organism. A concave (pocket) on a protein surface is known to be thebest target for a drug to react. We previously presented astudy on distance analysis between pockets and amino acidresidue. We firstly identified pockets on the protein surfaceand then calculated distances between atoms of an amino acidresidue and the deepest points or the outer loops of the pockets. We extracted proteins which at least one of the pockets areclose to arbitrary pairs of amino acid residues, calculated theratios of druggable proteins, and visualized the distributionof the ratios as a colored matrix. We suggested from thevisualization results that particular pairs of amino acid residuesmay affect the druggability of the proteins in our previousstudy. This paper presents an extension of our study to explorethe relevance between druggability of proteins and distancesbetween a set of amino acid residues and protein surfacepockets. Our technique treats the pockets as 20-dimensionalvectors consisting of distances to each of amino acid residues, and applies GeodesicSOM with the set of the vectors. Sphericalmaps generated by GeodesicSOM are used to visualizationof distribution of the pockets in the 20-dimensional vectorspace, and estimation of druggability of proteins with the 20-dimensional vectors of the pockets.

The sparkling enope squid, Watasenia scintillans, is a deep-sea mollusk inhabiting the western part of the Pacific Ocean. It has the peculiar ability to illuminate its body without the involvement of other organisms. In this study, we extracted the brain DNA from a single squid female caught in the Japan Sea and determined the complete genome sequence of its mitochondrial DNA using the Illumina sequencing platform. The circular sequence is 20,089 bp in length. Using the next-generation sequencing data, we also estimated the mean copy number of mitochondria per cell in the brain to be 108 by comparig the depths of the read data in the nuclear and mitochondrial genomes. The haploid genome size was calculated to be 4.78 Gb. Six heteroplasmy sites were also identified, together with their allele frequencies, in this individual. Our methodology is shown to be useful in mitochondrion-related studies.

Life science research now heavily relies on all sorts of databases for genome sequences, transcription, protein three-dimensional (3D) structures, protein–protein interactions, phenotypes and so forth. The knowledge accumulated by all the omics research is so vast that a computer-aided search of data is now a prerequisite for starting a new study. In addition, a combinatory search throughout these databases has a chance to extract new ideas and new hypotheses that can be examined by wet-lab experiments. By virtually integrating the related databases on the Internet, we have built a new web application that facilitates life science researchers for retrieving experts’ knowledge stored in the databases and for building a new hypothesis of the research target. This web application, named VaProS, puts stress on the interconnection between the functional information of genome sequences and protein 3D structures, such as structural effect of the gene mutation. In this manuscript, we present the notion of VaProS, the databases and tools that can be accessed without any knowledge of database locations and data formats, and the power of search exemplified in quest of the molecular mechanisms of lysosomal storage disease. VaProS can be freely accessed at http://p4d-info.nig.ac.jp/vapros/.

Lampreys are eel-like jawless fishes evolutionarily positioned between invertebrates and vertebrates, and have been used as model organisms to explore vertebrate evolution. In this study we determined the complete genome sequence of the mitochondrial DNA of the Japanese river lamprey, Lethenteron japonicum, using next-generation sequencers. The sequence was 16,272 bp in length. The gene content and order were identical to those of the sea lamprey, Petromyzon marinus, which has been the reference among lamprey species. However, the sequence similarity was less than 90%, suggesting the need for the whole-genome sequencing of L. japonicum.

Eye-field transcription factors (EFTFs) are a set of genes that compose a regulatory network for eye development in animals, which are highly conserved among various animal phyla. To investigate the processes of conservation and diversification of the transcription factors for eye development, we examined the structural changes in the EFTF proteins by estimating the ancestral sequences with the available genome information. Among the different types of EFTFs, we selected otx2, tbx3, rx1, pax6, six3/6, Ihx2 and nr2e1 because they are highly conserved in bilaterian animals. We searched the genome sequences of representative animal phyla for EFTF protein sequences. With deduced ancestral sequences and three-dimensional structures of EFTFs, we traced the evolutionary changes in amino add residues and found that the DNA-binding domains were always more conserved than other regions, and that the other regions showed distinct evolutionary rates. The EFTF rx1, which resides at the pivotal part of the EFTF network, had a faster evolutionary rate than the others. These results indicated that the evolutionary rates of each protein in the EFTF network, which were expected to be consistent with each other to maintain the interactions in the network, were not constant among or within the factors, but rather, varied to a significant extent. (C) 2014 Published by Elsevier B.V.

Steroid hormone is extensively used for transmitting variety of biological signals in organisms. Natural steroid hormone is synthesized from cholesterol in adrenal cortex and in sexual gland in vertebrates. Appropriately dosed synthetic steroid hormones can be used for medication. Despite their positive effects as medicine, they sometimes cause significant side effects due to their wide range of actions, and the studies for discovering the mechanisms of side effects were carried out aiming to reduce the side effects. The fundamental cause of the side effects seems to be interactions between the steroid and a non-target protein. To understand the possible range of interaction of steroid molecule, we gathered all the three-dimensional structures of protein-steroid complex determined by X-ray crystallography, compared the atomic environments of the steroid-binding sites in proteins and classified the pattern of steroid binding. Protein Data Bank contained 871 structures of steroid-protein complexes in 382 entries. For this study, we selected 832 steroid binding proteins. Using a newly developed method to describe the atomic environments of these steroid molecules and their function, we were able to separate the environments into six patterns. This classification had a potential to predict the function of function-unknown proteins with a co-crystallized steroid molecule. We speculated that the proteins grouped into the same pattern of nuclear receptors were the candidates of non-targeted proteins causing a side effect by a therapeutic prescription of steroid hormone. (C) 2015 Elsevier Inc. All rights reserved.

Tafazzin is a mitochondrial phospholipid transacylase, and its mutations cause Barth syndrome (BTHS). Human tafazzin gene produces four distinct alternatively spliced transcripts. To understand the molecular mechanisms of tafazzin deficiency, we performed an atomic resolution analysis of the influence of the BTHS mutations and of alternative splicing on the structure and function of tafazzin. From the three-dimensional (3D) homology modeling of tafazzin, we identified candidate amino acid residues that contribute to cardiolipin binding and to mitochondrial membrane associations that facilitate acyl-transfer reactions. Primate specific exon 5, which is alternatively spliced, is predicted to correspond to an intrinsically unstructured region in the protein. We proposed that this region should change the substrate-binding affinity and/or contribute to primate-specific molecular interactions. Exon 7, another alternatively spliced exon, encodes a region forming a part of the putative substrate-binding cleft, suggesting that the gene products lacking exon 7 will lose their substrate-binding ability. We demonstrate a clear localization of the BTHS mutations at residues responsible for membrane association, substrate binding, and the conformational stability of tafazzin. These findings provide new insights into the function of defective tafazzin and the pathogenesis of BTHS at the level of protein 3D structure and the evolution of alternatively spliced exons in primates.

The biochemical analysis of Phytolacca americana DOPA dioxygenases (PaDOD1 and PaDOD2) was carried out. The recombinant protein of PaDOD1 catalyzed the conversion of DOPA to betalamic acid, whereas DOD activity was not detected in PaDOD2 in vitro. While the reported motif conserved in DODs from betalain-producing plants was found in PaDOD1, a single amino acid residue alteration was detected in PaDOD2. A mutated PaDOD1 protein with a change of 177 Asn to Gly showed reduced specific activity compared with PaDOD1, while DOPA dioxygenase activity was not observed for a mutated PaDOD2 protein which had its conserved motif replaced with that of PaDOD1. A three-dimensional (3D) structural model of PaDOD1 and PaDOD2 showed that the conserved motif in DODs was located in the N-terminal side of a loop, which was found close to the putative active site. The difference in stability of the loop may affect the enzymatic activity of PaDOD2.

The androgen receptor (AR) plays important roles in multiple physiological functions, including differentiation, growth, and maintenance of male reproductive organs, and also has effects on hair and skin. In this paper, we report the synthesis of nonsteroidal AR antagonists having a 4-benzyl-1-(2H)-phthalazinone skeleton. Among the synthesized compounds, 11c with two ortho-substituents on the phenyl group potently inhibited SC-3 cell proliferation (IC50: 0.18 mu M) and showed high wt AR-binding affinity (IC50: 10.9 mu M), comparable to that of hydroxyflutamide (3). Compound 11c also inhibited proliferation of LNCaP cells containing T877A-mutated AR. Docking study of 11c with the AR ligand-binding domain indicated that the benzyl group is important for the antagonism. These phthalazinone derivatives may be useful for investigating potential clinical applications of AR antagonists. (C) 2015 Elsevier Masson SAS. All rights reserved.

Flagellar dyneins are essential microtubule motors in eukaryotes, as they drive the beating motions of cilia and flagella. Unlike myosin and kinesin motors, the track binding mechanism of dyneins and the regulation between the strong and weak binding states remain obscure. Here we report the solution structure of the microtubule-binding domain of flagellar dynein-c/DHC9 (dynein-c MTBD). The structure reveals a similar overall helix-rich fold to that of the MTBD of cytoplasmic dynein (cytoplasmic MTBD), but dynein-c MTBD has an additional flap, consisting of an antiparallel b sheet. The flap is positively charged and highly flexible. Despite the structural similarity to cytoplasmic MTBD, dynein-c MTBD shows only a small change in the microtubule- binding affinity depending on the registry change of coiled coil-sliding, whereby lacks the apparent strong binding state. The surface charge distribution of dynein-c MTBD also differs from that of cytoplasmic MTBD, which suggests a difference in the microtubule-binding mechanism.

Protein is the major component of the organism. It has a unique three-dimensional structure determined by its amino acid sequence. A concave (pocket) on the surface of a protein is known to be the best target for a drug to react. We started analyzing how' drug ability' of proteins related to the location of amino acids in a pocket. For as tarter of the study, this paper presents a visualization tool for distance analysis between pockets and the amino acid residue. Provided that a protein surface is described by a triangular mesh, this tool first identifies pockets on the protein surface, specifies the deepest point and outer loops of the pocket, and calculates distances between atoms of an amino acid residue and the deepest point or the outer loops of the pocket. The tool then visualizes the statistics of the distance calculation results by poly line charts and the distribution by scatter plots. This paper proposes a biological interpretation of the visualization results.

Previous studies have reported that the developmental processes of vertebrate eyes are controlled by four Pax-6 splicing variants, each modulating different downstream genes, whereas those of insect eyes are controlled by duplicated Pax-6 genes. Cephalopods belong to the Protostomes but possess a camera-type eye similar to those in vertebrates. We examined Pax-6 variations in the squid and found five types of Pax-6 splicing variants but no duplication of the Pax-6 gene. In the five splicing variants, the splicing patterns were produced by the combination of two additional exons to the ortholog and one jettisoned exon containing most of the Homeobox domain (HD). These five variants show spatio-temporal patterns of gene expression during development in the squid. Our study suggests that cephalopods acquired Pax-6 splicing variants independent of those in vertebrates and that these variants were similarly utilized in the development of the squid eye.

Metabolic enzymes utilize the cofactor flavin adenine dinucleotide (FAD) to catalyze essential biochemical reactions. Because these enzymes have been implicated in disease pathways, it will be necessary to target them via FAD-based structural analogues that can either activate/inhibit the enzymatic activity. To achieve this, it is important to explore the conformational space of FAD in the enzyme-bound and free states. Herein, we analyze X-ray crystallographic data of the enzyme-bound FAD conformations and sample conformations of the molecule in explicit water by molecular dynamics (MD) simulations. Enzyme-bound FAD conformations segregate into five distinct groups based on dihedral angle principal component analysis (PCA). A notable feature in the bound FADs is that the adenine base and isoalloxazine ring are oppositely oriented relative to the pyrophosphate axis characterized by near trans hypothetical dihedral angle ?V values. Not surprisingly, MD simulations in water show final compact but not perfectly stacked ring structures in FAD. Simulation data did not reveal noticeable changes in overall conformational dynamics of the dinucleotide in reduced and oxidized forms and in the presence and/or absence of ions. During unfoldingfolding dynamics, the riboflavin moiety is more flexible than the adenosine monophosphate group in the molecule. Conversely, the isoalloxazine ring is more stable than the variable adenine base. The pyrophosphate group depicts an unusually highly organized fluctuation illustrated by its dihedral angle distribution. Conformations sampled from enzymes and MD are quantified. The extent to which the protein shifts the distribution from the unbound state is discussed in terms of prevalent FAD shapes and dihedral angle population.

Flagellar dyneins are essential microtubule motors in eukaryotes, as they drive the beating motions of cilia and flagella. Unlike myosin and kinesin motors, the track binding mechanism of dyneins and the regulation between the strong and weak binding states remain obscure. Here we report the solution structure of the microtubule-binding domain of flagellar dynein-c/DHC9 (dynein-c MTBD). The structure reveals a similar overall helix-rich fold to that of the MTBD of cytoplasmic dynein (cytoplasmic MTBD), but dynein-c MTBD has an additional flap, consisting of an antiparallel beta sheet. The flap is positively charged and highly flexible. Despite the structural similarity to cytoplasmic MTBD, dynein-c MTBD shows only a small change in the microtubule-binding affinity depending on the registry change of coiled coil-sliding, whereby lacks the apparent strong binding state. The surface charge distribution of dynein-c MTBD also differs from that of cytoplasmic MTBD, which suggests a difference in the microtubule-binding mechanism.

Adenosine triphosphate (ATP) is a versatile molecule used mainly for energy and a phosphate source. The hydrolysis of γ phosphate initiates the reactions and these reactions almost always start when ATP binds to protein. Therefore, there should be a mechanism to prevent spontaneous hydrolysis reaction and a mechanism to lead ATP to a pure energy source or to a phosphate source. To address these questions, we extensively analyzed the effect of protein to ATP conformation based on the sampling of the ATP solution conformations obtained from molecular dynamics simulation and the sampling of ATP structures bound to protein found in a protein structure database. The comparison revealed mainly the following three points
1) The ribose ring in ATP molecule, which puckers in many ways in solution, tends to assume either C2′ exo or C2′ endo when it binds to protein. 2) The adenine ring in ATP molecule, which takes open-book motion with the two ring structures, has two distinct structures when ATP binds to protein. 3) The glycosyl-bond and the bond between phosphate and the ribose have unique torsion angles, when ATP binds to protein. The combination of torsion angles found in protein-bound forms is under-represented in ATP molecule in water. These findings suggest that ATP-binding protein exerts forces on ATP molecule to assume a conformation that is rarely found in solution, and that this conformation change should be a trigger for the reactions on ATP molecule. © 2013 The Biophysical Society of Japan.

Lysosomal β-galactosidase (β-Gal) deficiency causes a group of disorders that include neuronopathic GM1 gangliosidosis and non-neuronopathic Morquio B disease. We have previously proposed the use of small molecule ligands of β-Gal as pharmacological chaperones (PCs) for the treatment of GM1 gangliosidosis brain pathology. Although it is still under development, PC therapy has yielded promising preclinical results in several lysosomal diseases. In this study, we evaluated the effect of bicyclic 1-deoxygalactonojirimycin (DGJ) derivative of the sp2-iminosugar type, namely 5N,6S-(N′-butyliminomethylidene)-6-thio-1- deoxygalactonojirimycin (6S-NBI-DGJ), as a novel PC for human mutant β-Gal. In vitro, 6S-NBI-DGJ had the ability to inhibit the activity of human β-Gal in a competitive manner and was able to protect this enzyme from heat-induced degradation. Computational analysis supported that the rigid glycone bicyclic core of 6S-NBI-DGJ binds to the active site of the enzyme, with the aglycone N′-butyl substituent, in a precise E-orientation, located at a hydrophobic region nearby. Chaperone potential profiling indicated significant increases of enzyme activity in 24 of 88 β-Gal mutants, including four common mutations. Finally, oral administration of 6S-NBI-DGJ ameliorated the brain pathology of GM1 gangliosidosis model mice. These results suggest that 6S-NBI-DGJ is a novel PC that may be effective on a broad range of β-Gal mutants. Copyright © 2013, The American Society of Gene &amp
Cell Therapy.

Background: Lynch syndrome is a hereditary cancer predisposition syndrome caused by a mutation in one of the DNA mismatch repair (MMR) genes. About 24% of the mutations identified in Lynch syndrome are missense substitutions and the frequency of missense variants in MSH6 is the highest amongst these MMR genes. Because of this high frequency, the genetic testing was not effectively used in MSH6 so far. We, therefore, developed CoDP (Combination of the Different Properties), a bioinformatics tool to predict the impact of missense variants in MSH6.
Methods: We integrated the prediction results of three methods, namely MAPP, PolyPhen-2 and SIFT. Two other structural properties, namely solvent accessibility and the change in the number of heavy atoms of amino acids in the MSH6 protein, were further combined explicitly. MSH6 germline missense variants classified by their associated clinical and molecular data were used to fit the parameters for the logistic regression model and to assess the prediction. The performance of CoDP was compared with those of other conventional tools, namely MAPP, SIFT, PolyPhen-2 and PON-MMR.
Results: A total of 294 germline missense variants were collected from the variant databases and literature. Of them, 34 variants were available for the parameter training and the prediction performance test. We integrated the prediction results of MAPP, PolyPhen-2 and SIFT, and two other structural properties, namely solvent accessibility and the change in the number of heavy atoms of amino acids in the MSH6 protein, were further combined explicitly. Variants data classified by their associated clinical and molecular data were used to fit the parameters for the logistic regression model and to assess the prediction. The values of the positive predictive value (PPV), the negative predictive value (NPV), sensitivity, specificity and accuracy of the tools were compared on the whole data set. PPV of CoDP was 93.3% (14/15), NPV was 94.7% (18/19), specificity was 94.7% (18/19), sensitivity was 93.3% (14/15) and accuracy was 94.1% (32/34). Area under the curve of CoDP was 0.954, that of MAPP for MSH6 was 0.919, of SIFT was 0.864 and of PolyPhen-2 HumVar was 0.819. The power to distinguish between pathogenic and non-pathogenic variants of these methods was tested by Wilcoxon rank sum test (p &lt; 8.9 x 10(-6) for CoDP, p &lt; 3.3 x 10(-5) for MAPP, p &lt; 3.1 x 10(-4) for SIFT and p &lt; 1.2 x 10(-3) for PolyPhen-2 HumVar), and CoDP was shown to outperform other conventional methods.
Conclusion: In this paper, we provide a human curated data set for MSH6 missense variants, and CoDP, the prediction tool, which achieved better accuracy for predicting the impact of missense variants in MSH6 than any other known tools. CoDP is available at http://cib.cf.ocha.ac.jp/CoDP/.

Background: ORC binds to replication origins, but human ORC does not exhibit apparent sequence-specificity. Results: G-quadruplex (G4)-preferable RNA or single-stranded DNA competes for DNA binding of ORC. Conclusion: HumanORCbinds preferentially toRNAand single-strandedDNAthat form G4, and the certain domain in ORC1 is involved in this binding. Significance: This ability may correlate with the G4-formable motif in human replication origins. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

The MSH6 gene is one of the mismatch repair genes involved in Lynch syndrome and its mutations account for 10-20% of Lynch syndrome. Although previous studies suggested that the difference of the geographical region affects the clinical phenotype of Lynch syndrome, there has been no report on the detailed features of Japanese Lynch syndrome patients carrying an MSH6 mutation. The aim of the present study was to investigate the clinical and molecular features of MSH6 mutation carriers in Japan. Surgically resected 1720 colorectal carcinoma specimens were screened by microsatellite instability (MSI) testing and the MSI-high cases were subjected to a germline mutation analysis of the mismatch repair genes MLH1, MSH2 and MSH6. We investigated the clinical and molecular features of the MSH6 variants, such as the family cancer history, pathological findings, immunohistochemistry, methylation status of the MLH1 promoter and BRAF mutation in the colorectal tumor. Furthermore, the impact of the missense variants on MSH6 protein was predicted by using in silico tools. We identified nine novel pathogenic mutations and eight unclassified missense variants. Among the eight missense variants, three were suspected pathogenic by in silico analysis. We also found that most colorectal cancers in the MSH6 mutation carrier were diagnosed after the age of 50 and were localized distally. Furthermore, the mean age at diagnosis of endometrial cancer in Japanese MSH6 mutation carriers (49.2 years) was earlier than previous reports from Western countries (56.5 years). These results may improve the surveillance program for Japanese MSH6 mutation carriers.

Protein is the major component of the organism. A hole (pocket) on the surface of a protein is known to be the best target for a drug to react. We started analyzing how 'drug ability' of proteins related to the locations amino acids in a pocket. This poster presents a visualization tool for a distance distribution analysis between two types of amino acids in a pocket and proposes the biological interpretation of the visualization results. © 2013 IEEE.

Competitive inhibitors of either α-galactosidase (α-Gal) or β-galactosidase (β-Gal) with high affinity and selectivity have been accessed by exploiting aglycone interactions with conformationally locked sp 2-iminosugars. Selected compounds were profiled as potent pharmacological chaperones for mutant lysosomal α- and β-Gal associated with Fabry disease and GM 1 gangliosidosis. © 2012 The Royal Society of Chemistry.

In comparative modeling, the quality of amino acid sequence alignment still constitutes a major bottleneck in the generation of high quality models of protein three-dimensional (3D) structures. Substantial efforts have been made to improve alignment quality by revising the substitution matrix, introducing multiple sequences, replacing dynamic programming with hidden Markov models, and incorporating 3D structure information. Improvements in the gap penalty have not been a major focus, however, following the development of the affine gap penalty and of the secondary structure dependent gap penalty. We revisited the correlation between protein 3D structure and gap location in a large protein 3D structure data set, and found that the frequency of gap locations approximated to an exponential function of the solvent accessibility of the inserted residues. The nonlinearity of the gap frequency as a function of accessibility corresponded well to the relationship between residue mutation pattern and residue accessibility. By introducing this relationship into the gap penalty calculation for pairwise alignment between template and target amino acid sequences, we were able to obtain a sequence alignment much closer to the structural alignment. The quality of the alignments was substantially improved on a pair of sequences with identity in the "twi-light zone&apos;&apos; between 20 and 40%. The relocation of gaps by our new method made a significant improvement in comparative modeling, exemplified here by the Bacillus subtilis yitF protein. The method was implemented in a computer program, ALAdeGAP (ALignment with Accessibility dependent GAp Penalty), which is available at http://cib.cf.ocha.ac.jp/target_protein/. Proteins 2011; 79:1868-1877. (C) 2011 Wiley-Liss, Inc.

Motivation: Structural features at protein-protein interfaces can be studied to understand protein-protein interactions. It was noticed that in a dataset of 45 multimeric proteins the interface could either be described as. at against. at or protruding/interwound. In the latter, residues within one chain were surrounded by those in other chains, whereas in the former they were not.
Results: A simple method was developed that could distinguish between these two types with results that matched those made by a human annotator. Applying this automatic method to a large dataset of 888 structures, chains at interfaces were categorized as non-surrounded or surrounded. It was found that the surrounded set had a significantly lower folding tendency using a sequence based measure, than the non-surrounded set. This suggests that before complexation, surrounded chains are relatively unstable and may be involved in &apos;fly-casting&apos;. This is supported by the finding that terminal regions are overrepresented in the surrounded set.

Transcripts from mitochondrial and chloroplast DNA of land plants often undergo cytidine to uridine conversion-type RNA editing events. RESOPS is a newly built database that specializes in displaying RNA editing sites of land plant organelles on protein three-dimensional (3D) structures to help elucidate the mechanisms of RNA editing for gene expression regulation. RESOPS contains the following information: unedited and edited cDNA sequences with notes for the target nucleotides of RNA editing, conceptual translation from the edited cDNA sequence in pseudo-UniProt format, a list of proteins under the influence of RNA editing, multiple amino acid sequence alignments of edited proteins, the location of amino acid residues coded by codons under the influence of RNA editing in protein 3D structures and the statistics of biased distributions of the edited residues with respect to protein structures. Most of the data processing procedures are automated; hence, it is easy to keep abreast of updated genome and protein 3D structural data. In the RESOPS database, we clarified that the locations of residues switched by RNA editing are significantly biased to protein structural cores. The integration of different types of data in the database also help advance the understanding of RNA editing mechanisms. RESOPS is accessible at http://cib.cf.ocha.ac.jp/RNAEDITING/.

We have developed coliSNP, a database server (http://yayoi.kansai.jaea.go.jp/colisnp) that maps non-synonymous single nucleotide polymorphisms (nsSNPs) on the three-dimensional (3D) structure of proteins. Once a week, the SNP data from the dbSNP database and the protein structure data from the Protein Data Bank (PDB) are downloaded, and the correspondence of the two data sets is automatically tabulated in the coliSNP database. Given an amino acid sequence, protein name or PDB ID, the server will immediately provide known nsSNP information, including the amino acid mutation caused by the nsSNP, the solvent accessibility, the secondary structure and the flanking residues of the mutated residue in a single page. The position of the nsSNP within the amino acid sequence and on the 3D structure of the protein can also be observed. The database provides key information with which to judge whether an observed nsSNP critically affects protein function and/or stability. As far as we know, this is the only web-based nsSNP database that automatically compiles SNP and protein information in a concise manner.

DNA photolyase recognizes ultraviolet-damaged DNA and breaks improperly formed covalent bonds within the cyclobutane pyrimidine dimer by a light-activated electron transfer reaction between the flavin adenine dinucleotide, the electron donor, and cyclobutane pyrimidine dinner, the electron acceptor. Theoretical analysis of the electron-tunneling pathways of the DNA photolyase derived from Anacystis nidulans can reveal the active role of the protein environment in the electron transfer reaction. Here, we report the unexpectedly important role of the single methionine residue, Met-353, where busy trafficking of electron-tunneling currents is observed. The amino acid conservation pattern of Met-353 in the homologous sequences perfectly correlates with experimentally verified annotation as photolyases. The bioinformatics sequence analysis also suggests that the residue plays a pivotal role in biological function. Consistent findings from different disciplines of computational biology strongly suggest the pivotal role of Met-353 in the biological function of DNA photolyase.

Here we report the new features and improvements in our latest release of the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/), a comprehensive annotation resource for human genes and transcripts. H-InvDB, originally developed as an integrated database of the human transcriptome based on extensive annotation of large sets of full-length cDNA (FLcDNA) clones, now provides annotation for 120 558 human mRNAs extracted from the International Nucleotide Sequence Databases (INSD), in addition to 54 978 human FLcDNAs, in the latest release H-InvDB_4.6. We mapped those human transcripts onto the human genome sequences (NCBI build 36.1) and determined 34 699 human gene clusters, which could define 34 057 (98.1%) protein-coding and 642 (1.9%) non-protein-coding loci; 858 (2.5%) transcribed loci overlapped with predicted pseudogenes. For all these transcripts and genes, we provide comprehensive annotation including gene structures, gene functions, alternative splicing variants, functional non-protein-coding RNAs, functional domains, predicted sub cellular localizations, metabolic pathways, predictions of protein 3D structure, mapping of SNPs and microsatellite repeat motifs, co-localization with orphan diseases, gene expression profiles, orthologous genes, proteinprotein interactions (PPI) and annotation for gene families. The current H-InvDB annotation resources consist of two main views: Transcript view and Locus view and eight sub-databases: the DiseaseInfo Viewer, H-ANGEL, the Clustering Viewer, G-integra, the TOPO Viewer, Evola, the PPI view and the Gene family/group.

Integrin, a membrane protein with a huge extracellular domain, participates in cell-cell and cell-extracellular-matrix interactions for metazoan. A group of integrins is known to perform a large-scale structural change when the protein is activated, but the activation mechanism and generality of the conformational change remain to be elucidated. We performed normal-mode analysis of the elastic network model on integrin alpha(V)beta(3) ectodomain in the bent form and identified key residues that influenced molecular motions. Iterative normal-mode calculations demonstrated that the specific nonbonded interactions involving the key residues work as a snap to keep integrin in the bent form. The importance of the key residues for the conformational change was further verified by mutation experiments, in which integrin alpha(IIb)beta(3) was used. The conservation pattern of amino acid residues among the integrin family showed that the characteristic pattern of residues seen around these key residues is found in the limited groups of integrin beta-chains. This conservation pattern suggests that the molecular mechanism of the conformational change relying on the interactions found in integrin alpha(V)beta(3) is unique to the limited types of integrins.

Background: In plant organelles, specific messenger RNAs (mRNAs) are subjected to conversion editing, a process that often converts the first or second nucleotide of a codon and hence the encoded amino acid. No systematic patterns in converted sites were found on mRNAs, and the converted sites rarely encoded residues located at the active sites of proteins. The role and origin of RNA editing in plant organelles remain to be elucidated.
Results: Here we study the relationship between amino acid residues encoded by edited codons and the structural characteristics of these residues within proteins, e. g., in protein-protein interfaces, elements of secondary structure, or protein structural cores. We find that the residues encoded by edited codons are significantly biased toward involvement in helices and protein structural cores. RNA editing can convert codons for hydrophilic to hydrophobic amino acids. Hence, only the edited form of an mRNA can be translated into a polypeptide with helix-preferring and core-forming residues at the appropriate positions, which is often required for a protein to form a functional three-dimensional (3D) structure.
Conclusion: We have performed a novel analysis of the location of residues affected by RNA editing in proteins in plant organelles. This study documents that RNA editing sites are often found in positions important for 3D structure formation. Without RNA editing, protein folding will not occur properly, thus affecting gene expression. We suggest that RNA editing may have conferring evolutionary advantage by acting as a mechanism to reduce susceptibility to DNA damage by allowing the increase in GC content in DNA while maintaining RNA codons essential to encode residues required for protein folding and activity.

RNA editing in land plant organelles is a process primarily involving the conversion of cytidine to uridine in pre-mRNAs. The process is required for gene expression in plant organelles, because this conversion alters the encoded amino acid residues and improves the sequence identity to homologous proteins. A recent study uncovered that proteins encoded in the nuclear genome are essential for editing site recognition in chloroplasts; the mechanisms by which this recognition occurs remain unclear. To understand these mechanisms, we determined the genomic and cDNA sequences of moss Takakia lepidozioides chloroplast genes, then computationally analyzed the sequences within -30 to +10 nucleotides of RNA editing sites (neighbor sequences) likely to be recognized by trans-factors. As the T lepidozioides chloroplast has many RNA editing sites, the analysis of these sequences provides a unique opportunity to perform statistical analyses of chloroplast RNA editing sites. We divided the 302 obtained neighbor sequences into eight groups based on sequence similarity to identify group-specific patterns. The patterns were then applied to predict novel RNA editing sites in T. lepidozioides transcripts; similar to 60% of these predicted sites are true editing sites. The success of this prediction algorithm suggests that the obtained patterns are indicative of key sites recognized by trans-factors around editing sites of T. lepidozioides chloroplast genes.

A vast amount of DNA sequence data, protein three-dimensional (3D) structure data, and RNA expression data have been produced by the efforts of genome sequencing, structural genomics, and omics projects, and we are at the stage where comprehensive views of cell activity and molecular mechanisms of life can be deduced. But in reality, we are inundated with massive amounts of data and are still in the process of finding ways to fully utilize the data. In this report, I would like to present our observations on the growth of protein 3D structure data and our effort to deduce the functions from the 3D structures. We found that the 3D structure of quite a high proportion of proteins derived from genome sequences can be now predicted and methods to predict the functions from 3D structures are in high demand. The methods we have developed can be used to predict some functions, namely RNA and ligand interfaces, based on those 3D structures and DNA sequences with relatively high accuracy. The methods enable predictions that are accurate enough to help with deducing the atomic structures of the complexes.

The emerging grid computing technologies enable bioinformatics scientists to conduct their researches in a virtual laboratory, in which they share public databases, computational tools as well as their analysis workflows. However, the development of grid applications is still a nightmare for general bioinformatics scientists, due to the lack of grid programming environments, standards and high-level services. Here, we present a system, which we named Bioinformatics: Ask Any Questions (BAAQ), to automate this development procedure as much as possible. BAAQ allows scientists to store and manage remote biological data and programs, to build analysis workflows that integrate these resources seam-lessly, and to discover knowledge from available resources. This paper addresses two issues in building grid applications in bioinformatics: how to smoothly compose an analysis workflow using heterogeneous resources and how to efficiently discover and re-use available resources in the grid community. Correspondingly an intelligent grid programming environment and an active solution recommendation service are proposed. Finally, we present a case study applying BAAQ to a bioinformatics problem.

Genome sequencing and structural genomics projects are both proceeded to gain a new perspective of life, that is global views on mechanisms of life with comprehensive and unbiased fashion. We now have genome sequences of human and other species, and are going to have a three-dimensional structure of whole proteins. Those massive pieces of information can only be deciphered with collaboration of computational biology. In this paper, we will discuss the amount of data we have at the moment and one of the new views on mechanisms of cellular function regulation obtained based on the computational analyses of those data.

We have cloned, sequenced, and characterized cDNA of actins from five ciliate species of three different classes of the phylum Ciliophora: Karyorelictea (Loxodes striatus), Heterotrichea (Blepharisma japonicum, Blepharisma musculus), and Litostomatea (Didinium nasutum, Dileptus margaritifer). Loxodes striatus uses UGA as the stop codon and has numerous in-frame UAA and UAG, which are translated into glutamine. The other four species use UAA as the stop codon and have no in-frame UAG nor UGA. The putative amino acid sequences of the newly determined actin genes were found to be highly divergent as expected from previous findings of other ciliate actins. These sequences were also highly divergent from other ciliate actins, indicating that actin genes are highly diverse even within the phylum Ciliophora. Phylogenetic analysis showed high evolutionary rate of ciliate actins. Our results suggest that the evolutionary rate was accelerated because of the differences in molecular interactions.

The genomes of more than 100 species have been sequenced, and the biological functions of encoded proteins are now actively being researched. Protein function is based on interactions between proteins and other molecules. One approach to assuming protein function based on genomic sequence is to predict interactions between an encoded protein and other molecules. As a data source for such predictions, knowledge regarding known protein-small molecule interactions needs to be compiled. We have, therefore, surveyed interactions between proteins and other molecules in Protein Data Bank (PDB), the protein three-dimensional (3D) structure database. Among 20,685 entries in PDB (April, 2003), 4,189 types of small molecules were found to interact with proteins. Biologically relevant small molecules most often found in PDB were metal ions, such as calcium, zinc, and magnesium. Sugars and nucleotides were the next most common. These molecules are known to act as cofactors for enzymes and/or stabilizers of proteins. In each case of interactions between a protein and small molecule, we found preferred amino acid residues at the interaction sites. These preferences can be the basis for predicting protein function from genomic sequence and protein 3D structures. The data pertaining to these small molecules were collected in a database named Het-PDB Navi., which is freely available at http:// daisy.nagahama-i-bio.ac.jp/golab/hetpdbnavi.html and linked to the official PDB home page.

The catalytic domain of xylanases belonging to glycoside hydrolase family 10 ( GH10) can be divided into 22 modules (M1 to M22; Sato, Y., Niimura, Y., Yura, K., and Go, M. ( 1999) Gene (Amst.) 238, 93 - 101). Inspection of the crystal structure of a GH10 xylanase from Streptomyces olivaceoviridis E-86 (SoXyn10A) revealed that the catalytic domain of GH10 xylanases can be dissected into two parts, an N-terminal larger region and C-terminal smaller region, by the substrate binding cleft, corresponding to the module border between M14 and M15. It has been suggested that the topology of the substrate binding clefts of GH10 xylanases are not conserved (Charnock, S. J., Spurway, T. D., Xie, H., Beylot, M. H., Virden, R., Warren, R. A. J., Hazlewood, G. P., and Gilbert, H. J. ( 1998) J. Biol. Chem. 273, 32187 - 32199). To facilitate a greater understanding of the structure-function relationship of the substrate binding cleft of GH10 xylanases, a chimeric xylanase between SoXyn10A and Xyn10A from Cellulomonas fimi (CfXyn10A) was constructed, and the topology of the hybrid substrate binding cleft established. At the three-dimensional level, SoXyn10A and CfXyn10A appear to possess 5 subsites, with the amino acid residues comprising subsites -3 to +1 being well conserved, although the +2 subsites are quite different. Biochemical analyses of the chimeric enzyme along with SoXyn10A and CfXyn10A indicated that differences in the structure of subsite +2 influence bond cleavage frequencies and the catalytic efficiency of xylooligosaccharide hydrolysis. The hybrid enzyme constructed in this study displays fascinating biochemistry, with an interesting combination of properties from the parent enzymes, resulting in a low production of xylose.

The human genome sequence defines our inherent biological potential
the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB
http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology.

To facilitate an understanding of structure-function relationships, chimeric xylanases were constructed by module shuffling between the catalytic domains of the FXYN from Streptomyces olivaceoviridis E-86 and the Cex from Cellulomonas fimi. In the family F/10 xylanases, the modules M4 and M5 relate to substrate binding so that modules M4 and M5 of the FXYN were replaced with those of the Cex and the chimeric enzymes denoted FCF-C4, FCF-C5 and FCF-C4,5 were constructed. The kcat value of FCF-C5 for p-nitrophenyl-β-D-cellobioside was similar to that of the FXYN (2.2 s-1)
however, the kcat value of FCF-C4 for p-nitrophenyl-β-D-cellobioside was significantly higher (7.0 s-1). The loss of the hydrogen bond between E46 and S22 or the presence of the I49W mutation would be expected to change the position of Q88, which plays a pivotal role in discriminating between glucose and xylose, resulting in the increased kcat value observed for FCF-C4 acting on p-nitrophenyl-β-D-cellobioside since module M4 directly interacts with Q88. To investigate the synergistic effects of the different modules, module M10 of the FCF-C4 chimera was replaced with that of the Cex. The effects of replacement of module M4 and M10 were almost additive with regard to the Km and kcat values.

Eukaryotic genes encoding proteins are split by introns. Introns do not have information of protein amino acid sequences and they are spliced out during maturation of mRNA. Only exons are translated into proteins. Exon shuffling mediated by introns has been hypothesized as a molecular mechanism to shuffle protein segments, creating new functional proteins during evolution. We have shown that intron positions are correlated with module boundaries. A module is a small compact structural unit of a globular protein. As expected from the module-intron correlation, shuffling of a phosphate-binding module among DNA manipulating proteins was observed. Based on a compact conformation of a module, we expected mechanical stability of a single module. Molecular dynamics studies indeed show that a single module of barnase has mechanical stability. Such stability of a single module, together with the correlation between module boundaries and intron positions, implies that modules were combined into globular domains through exon shuffling during genome evolution. To investigate a role of module in protein folding, we designed a mini-protein by deleting one module from barnase and we could show conformational stability of the mini-barnase. The module engineering in barnase implies an independent behavior of the local region corresponding to a module on protein conformation. Hypothetical role of modules in folding process is also discussed.

Although the amino acid homology in the catalytic domain of FXYN xylanase from Streptomyces olivaceoviridis E-86 and Cex xylanase from Cellulomonas fimi is only 50%, an active chimeric enzyme was obtained by replacing module 10 in FXYN with module 10 from Cex, In the family F/10 xylanases, module 10 is an important region as it includes an acid/base catalyst and a substrate binding residue. In FXYN, module 10 consists of 15 amino acid residues, while in Cex it consists of 14 amino acid residues. The K-m and k(cat) values of the chimeric xylanase FCF-C10 for PKP-xylobioside (PNP-X-2) were 10-fold less than those for FXYN, CD spectral data indicated that the structure of the chimeric enzyme was similar to that of FXYN, Based on the comparison of the amino acid sequences of FXYN and Cex in module 10, we constructed four mutants of FXYN, When D133 or S135 of FXYN,vas deleted, the kinetic properties were not changed from those of FXYN. By deletion of both D133 and S135, the K-m value for PNP-X-2 decreased from the 2.0 mM of FXYN to 0.6 mM and the k(cat) value decreased from the 20 s(-1) of FXYN to 8.7 s(-1). Insertion of Q140 into the doubly deleted mutant further reduced the K-m value to 0.3 mM and the k(cat) value to 3.8 s(-1). These values are close to those for the chimeric enzyme FCF-C10, These results indicate that module 10 itself is able to accommodate changes in the sequence position of amino acids which are critical for enzyme function, Since changes of the spatial position of these amino acids would be expected to result in enzyme inactivation, module 10 must have some flexibility in its tertiary structure. The structure of module 10 itself also affects the substrate specificity of the enzyme. (C) 1999 Federation of European Biochemical Societies.

Helix–turn–helix motif is one of the common motifs observed in DNA-binding proteins. The motif interacts with DNA double helix and recognizes specific base sequences. It is assumed that the helix–turn–helix motif appears only once in seven prokaryotic transcriptional repressors of which 3-D structures have been determined by X-ray crystallographk studies. These prokaryotic repressors consist of several a-helices connected with turns. We report here that these repressors are decomposable into helix–turn–helix modules and their connectors. A module is defined as a compact structural unit with consecutive amino acid residues in a globular protein. Each of the helix- turn -helix motifs in the seven proteins corresponds approximately to a single helix-turn—helix module consisting of approximately 13 amino acids. Identification of modules of seven prokaryotic repressors and comparisons of then tertiary structures led to the conclusion that three of these DNA-binding proteins contain more than one helix–turn–helix module with a structure similar to the helix-turn-heUx motif. The difference in module organization of these DNA-binding proteins paves the way for further classification of the DNA-binding proteins with the helix- turn -helix motif. The structural repertoire of these transcriptional regulators was increased through different utilizations in the number of helix–turn–helix and other modules. The difference in DNA base recognition ability in these helix–turn–helix modules is ascribed to a difference in size of a side chain at the fifth residue from Gly, on the turn. © 1993, Oxford University Press.

人体の主成分である蛋白質は，アミノ酸が多数連結して複雑に折りたたまれることで，固有の立体構造を形成する．薬は蛋白質表面上の窪み （ポケットと呼ぶ) に入り込みやすいことが知られている．そこで我々は，薬との親和性と，アミノ酸のポケット内で位置関係がどのように関係しているか分析している．その一環として本報告では，ポケットとアミノ酸の距離分布分析のための可視化ツールを提案する．本手法では，蛋白質表面形状から抽出された各ポケットに対して最深部を求め，アミノ酸を構成する各原子との距離を算出し，その分布を可視化する．本報告では可視化結果から得られた傾向を示し，その生物的解釈の可能性について推察する．Protein is the major component of the organism. It has a unique three-dimensional structure determined by its amino acid sequence. A hole (pocket) on the surface of a protein is known to be the best target for a drug to react. We have started analyzing how "druggability" of proteins relates to the locations of amino acids in the pocket. For starter of the study, this paper presents a visualization tool for distance distribution analysis between two types of amino acids in the protein pocket. Providing protein surfaces by triangular meshes, this tool first identifies a pocket from the protein surface, specifies the deepest point in the pocket, and calculates distances between atoms of an amino acids and the deepest point of the pocket. The tool then visualizes the distribution of the distances by scatterplots. This paper presents a biological interpretation of the visualization results.

人体の主成分である蛋白質は,アミノ酸が多数連結して複雑に折りたたまれることで,固有の立体構造を形成する.薬は蛋白質表面上の窪み(ポケットと呼ぶ)に入り込みやすいことが知られている.そこで我々は,薬との親和性と,アミノ酸のポケット内で位置関係がどのように関係しているか分析している.その一環として本報告では,ポケットとアミノ酸の距離分布分析のための可視化ツールを提案する.本手法では,蛋白質表面形状から抽出された各ポケットに対して最深部を求め,アミノ酸を構成する各原子との距離を算出し,その分布を可視化する.本報告では可視化結果から得られた傾向を示し,その生物学的解釈の可能性について推察する.

We have been developing FAMSBASE, a protein homology-modeling database of whole ORFs predicted from genome sequences. The latest update of FAMSBASE ( http://daisy.nagahama-i-bio.ac.jp/Famsbase/ ), which is based on the protein three-dimensional (3D) structures released by November 2003, contains modeled 3D structures for 368,724 open reading frames (ORFs) derived from genomes of 276 species, namely 17 archaebacterial, 130 eubacterial, 18 eukaryotic and 111 phage genomes. Those 276 genomes are predicted to have 734,193 ORFs in total and the current FAMSBASE contains protein 3D structure of approximately 50% of the ORF products. However, cases that a modeled 3D structure covers the whole part of an ORF product are rare. When portion of an ORF with 3D structure is compared in three kingdoms of life, in archaebacteria and eubacteria, approximately 60% of the ORFs have modeled 3D structures covering almost the entire amino acid sequences, however, the percentage falls to about 30% in eukaryotes. When annual differences in the number of ORFs with modeled 3D structure are calculated, the fraction of modeled 3D structures of soluble protein for archaebacteria is increased by 5%, and that for eubacteria by 7% in the last 3 years. Assuming that this rate would be maintained and that determination of 3D structures for predicted disordered regions is unattainable, whole soluble protein model structures of prokaryotes without the putative disordered regions will be in hand within 15 years. For eukaryotic proteins, they will be in hand within 25 years. The 3D structures we will have at those times are not the 3D structure of the entire proteins encoded in single ORFs, but the 3D structures of separate structural domains. Measuring or predicting spatial arrangements of structural domains in an ORF will then be a coming issue of structural genomics. © 2006 Springer Science+Business Media B.V.

Protein-RNA interactions play essential roles in a number of regulatory mechanisms for gene expression such as RNA splicing, transport, translation and post-transcriptional control. As the number of available protein-RNA complex 3D structures has increased, it is now possible to statistically examine protein-RNA interactions based on 3D structures. We performed computational analyses of 86 representative protein-RNA complexes retrieved from the Protein Data Bank. Interface residue propensity, a measure of the relative importance of different amino acid residues in the RNA interface, was calculated for each amino acid residue type (residue singlet interface propensity). In addition to the residue singlet propensity, we introduce a new residue-based propensity, which gives a measure of residue pairing preferences in the RNA interface of a protein (residue doublet interface propensity). The residue doublet interface propensity contains much more information than the sum of two singlet propensities alone. The prediction of the RNA interface using the two types of propensities plus a position-specific multiple sequence profile can achieve a specificity of about 80%. The prediction method was then applied to the 3D structure of two mRNA export factors, TAP (Mex67) and UAP56 (Sub2). The prediction enables us to point out candidate RNA interfaces, part of which are consistent with previous experimental studies and may contribute to elucidation of atomic mechanisms of mRNA export.

Alternative splicing is a molecular mechanism that produces multiple proteins from a single gene, and is thought to produce variety in proteins translated from a limited number of genes. Here we analyzed how alternative splicing produced variety in protein structure and function, by using human full-length cDNAs on the assumption that all of the alternatively spliced mRNAs were translated to proteins. We found that the length of alternatively spliced amino acid sequences, in most cases, fell into a size shorter than that of average protein domain. We evaluated comprehensively the presumptive three-dimensional structures of the alternatively spliced products to assess the impact of alternative splicing on gene function. We found that more than half of the products encoded proteins which were involved in signal transduction, transcription and translation, and more than half of alternatively spliced regions comprised interaction sites between proteins and their binding partners, including substrates, DNA/RNA, and other proteins. Intriguingly, 67% of the alternatively spliced isoforms showed significant alterations to regions of the protein structural core, which likely resulted in large conformational change. Based on those findings, we speculate that there are a large number of cases that alternative splicing modulates protein networks through significant alteration in protein conformation. (c) 2006 Published by Elsevier B.V.

We report the first genome-wide identification and characterization of alternative splicing in human gene transcripts based on analysis of the full-length cDNAs. Applying both manual and computational analyses for 56 419 completely sequenced and precisely annotated full-length cDNAs selected for the H-Invitational human transcriptome annotation meetings, we identified 6877 alternative splicing genes with 18 297 different alternative splicing variants. A total of 37 670 exons were involved in these alternative splicing events. The encoded protein sequences were affected in 6005 of the 6877 genes. Notably, alternative splicing affected protein motifs in 3015 genes, subcellular localizations in 2982 genes and transmembrane domains in 1348 genes. We also identified interesting patterns of alternative splicing, in which two distinct genes seemed to be bridged, nested or having overlapping protein coding sequences (CDSs) of different reading frames (multiple CDS). In these cases, completely unrelated proteins are encoded by a single locus. Genome-wide annotations of alternative splicing, relying on full-length cDNAs, should lay firm groundwork for exploring in detail the diversification of protein function, which is mediated by the fast expanding universe of alternative splicing variants.

The genetic code of nuclear genes in some ciliates was found to differ from that of other organisms in the assignment of UGA, UAG, and UAA codons, which are normally assigned as stop codons. In some ciliate species, the universal stop codons UAA and UAG instead encode glutamine. In some other ciliates, the universal stop codon UGA appears to be translated as cysteine or tryptophan. Eukaryotic release factor 1 (eRF1) is a key protein, in stop codon recognition, thus, the protein is believed to play an important role in the stop codon reassignment in ciliates. We have cloned, sequenced, and analyzed the cDNA of eRF1 from four ciliate species of three different classes: Karyorelictea (Loxodes striatus), Heterotrichea (Blepharisma musculus), and Litostomatea (Didinium nasutum, Dileptus margaritifer). Phylogenetic analysis of these eRF1s supports the hypothesis that the genetic code in ciliates has deviated independently several times from the universal genetic code, and that different ciliate eRF1s may have undergone different processes to change the codon specificity. Using computational methods, we have also suggested areas on the surface of eRF1s that are important for stop codon recognition in ciliate eRF1s. (c) 2004 Elsevier B.V. All rights reserved.

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for nonprotein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology.

We have cloned, sequenced, and characterized cDNA of actins from five ciliate species of three different classes of the phylum Ciliophora: Karyorelictea (Loxodes striatus), Heterotrichea (Blepharisma japonicum, Blepharisma musculus), and Litostomatea (Didinium nasutum, Dileptus margaritifer). Loxodes striatus uses UGA as the stop codon and has numerous in-frame UAA and UAG, which are translated into glutamine. The other four species use UAA as the stop codon and have no in-frame UAG nor UGA. The putative amino acid sequences of the newly determined actin genes were found to be highly divergent as expected from previous findings of other ciliate actins. These sequences were also highly divergent from other ciliate actins, indicating that actin genes are highly diverse even within the phylum Ciliophora. Phylogenetic analysis showed high evolutionary rate of ciliate actins. Our results suggest that the evolutionary rate was accelerated because of the differences in molecular interactions.

2004.2予定

The genomes of more than 100 species have been sequenced, and the biological functions of encoded proteins are now actively being researched. Protein function is based on interactions between proteins and other molecules. One approach to assuming protein function based on genomic sequence is to predict interactions between an encoded protein and other molecules. As a data source for such predictions, knowledge regarding known protein-small molecule interactions needs to be compiled. We have, therefore, surveyed interactions between proteins and other molecules in Protein Data Bank (PDB), the protein three-dimensional (3D) structure database. Among 20,685 entries in PDB (April, 2003), 4,189 types of small molecules were found to interact with proteins. Biologically relevant small molecules most often found in PDB were metal ions, such as calcium, zinc, and magnesium. Sugars and nucleotides were the next most common. These molecules are known to act as cofactors for enzymes and/or stabilizers of proteins. In each case of interactions between a protein and small molecule, we found preferred amino acid residues at the interaction sites. These preferences can be the basis for predicting protein function from genomic sequence and protein 3D structures. The data pertaining to these small molecules were collected in a database named Het-PDB Navi., which is freely available at http:// daisy.nagahama-i-bio.ac.jp/golab/hetpdbnavi.html and linked to the official PDB home page.

We have cloned, sequenced, and characterized cDNA of actins from five ciliate species of three different classes of the phylum Ciliophora: Karyorelictea (Loxodes striatus), Heterotrichea (Blepharisma japonicum, Blepharisma musculus), and Litostomatea (Didinium nasutum, Dileptus margaritifer). Loxodes striatus uses UGA as the stop codon and has numerous in-frame UAA and UAG, which are translated into glutamine. The other four species use UAA as the stop codon and have no in-frame UAG nor UGA. The putative amino acid sequences of the newly determined actin genes were found to be highly divergent as expected from previous findings of other ciliate actins. These sequences were also highly divergent from other ciliate actins, indicating that actin genes are highly diverse even within the phylum Ciliophora. Phylogenetic analysis showed high evolutionary rate of ciliate actins. Our results suggest that the evolutionary rate was accelerated because of the differences in molecular interactions.

The genomes of more than 100 species have been sequenced, and the biological functions of encoded proteins are now actively being researched. Protein function is based on interactions between proteins and other molecules. One approach to assuming protein function based on genomic sequence is to predict interactions between an encoded protein and other molecules. As a data source for such predictions, knowledge regarding known protein-small molecule interactions needs to be compiled. We have, therefore, surveyed interactions between proteins and other molecules in Protein Data Bank (PDB), the protein three-dimensional (3D) structure database. Among 20,685 entries in PDB (April, 2003), 4,189 types of small molecules were found to interact with proteins. Biologically relevant small molecules most often found in PDB were metal ions, such as calcium, zinc, and magnesium. Sugars and nucleotides were the next most common. These molecules are known to act as cofactors for enzymes and/or stabilizers of proteins. In each case of interactions between a protein and small molecule, we found preferred amino acid residues at the interaction sites. These preferences can be the basis for predicting protein function from genomic sequence and protein 3D structures. The data pertaining to these small molecules were collected in a database named Het-PDB Navi., which is freely available at http:// daisy.nagahama-i-bio.ac.jp/golab/hetpdbnavi.html and linked to the official PDB home page.

An analysis of the genome sequence database revealed novel types of two-domain multi-copper oxidases. The two-domain proteins have the conspicuous combination of blue-copper and inter-domain trinuclear copper binding residues, which is common in ceruloplasmin and ascorbate oxidase but not in nitrite reductase, and therefore are considered to retain the characteristics of the plausible ancestral form of ceruloplasmin and ascorbate oxidase. A possible evolutionary relationship of these proteins is proposed. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Enlarged FAMSBASE is a relational database of comparative protein structure models for the whole genome of 41 species, presented in the GTOP database. The models are calculated by Full Automatic Modeling System ( FAMS). Enlarged FAMSBASE provides a wide range of query keys, such as name of ORF ( open reading frame), ORF keywords, Protein Data Bank ( PDB) ID, PDB heterogen atoms and sequence similarity. Heterogen atoms in PDB include cofactors, ligands and other factors that interact with proteins, and are a good starting point for analyzing interactions between proteins and other molecules. The data may also work as a template for drug design. The present number of ORFs with protein 3D models in FAMSBASE is 183 805, and the database includes an average of three models for each ORF. FAMSBASE is available at http: / / famsbase. bio. nagoya- u. ac. jp/ famsbase/

Enlarged FAMSBASE is a relational database of comparative protein structure models for the whole genome of 41 species, presented in the GTOP database. The models are calculated by Full Automatic Modeling System ( FAMS). Enlarged FAMSBASE provides a wide range of query keys, such as name of ORF ( open reading frame), ORF keywords, Protein Data Bank ( PDB) ID, PDB heterogen atoms and sequence similarity. Heterogen atoms in PDB include cofactors, ligands and other factors that interact with proteins, and are a good starting point for analyzing interactions between proteins and other molecules. The data may also work as a template for drug design. The present number of ORFs with protein 3D models in FAMSBASE is 183 805, and the database includes an average of three models for each ORF. FAMSBASE is available at http: / / famsbase. bio. nagoya- u. ac. jp/ famsbase/

An analysis of the genome sequence database revealed novel types of two-domain multi-copper oxidases. The two-domain proteins have the conspicuous combination of blue-copper and inter-domain trinuclear copper binding residues, which is common in ceruloplasmin and ascorbate oxidase but not in nitrite reductase, and therefore are considered to retain the characteristics of the plausible ancestral form of ceruloplasmin and ascorbate oxidase. A possible evolutionary relationship of these proteins is proposed. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Yoshihara, S., Geng, X.X., Okamoto, S., Yura, K., Murata, T., Go M., Ohmori, M., and ikeuchi, M.: Mutational analysis of genes involved in pilus structure, motility and transformation competency in the unicelluular motile cyanobacterium Synechocystis sp. PCC6803

To reveal structure-function relationships of family F/10 glycanases, an in vitro molecular level shuffling experiment was conducted to accumulate useful amino acid residues from two homologous F/10 xylanases, FXYN of Streptomyces olivaceoviridis E-86 and XylA of Thermomonospora alba ULJB1, into a single chimeric xylanase. The parent genes were shuffled by crossovers at selected module borders using self-priming Polymerase Chain Reaction (PCR)s. The shuffled constructs, designated as FXYN-M3/4-XylA, FXYN-M9/10-XylA, and FXYN-M14/15-XylA were cloned and their nucleotide sequences were confirmed. Two chimera, FXYN-M3/4-XylA and FXYN-M14/15-XylA, demonstrated activity against RBB-xylan and were over-expressed as His-tag fusion proteins under control of T5 promoter of pQE60. The homogeneously pure chimeric proteins, FXYN-M3/4-XylA and FXYN-M14/15-XylA showed improved thermal and pH profiles compared to those of one of the parents, FXYN. This was apparently due to the influence of amino acids inherited from thermophilic XylA. Measured K-m and kcat values were closer to those of the other parent, XylA. Interestingly, a significant level of heat tolerance up to 60 degreesC, was recorded for FXYN-M3/4-XylA in comparison to only 40 degreesC for FXYN-M14/15-XylA though their temperature optima did not correlates with their thermal stability. These results indicated that the amino acid residues of the larger T. alba XylA DNA fragment present in FXYN-M3/4-XylA were responsible for inducing its thermal stability. (C) 2001 Elsevier Science Inc. All rights reserved.

The relevance of pilus-related genes to motility, pilus structure on the cell surface and competency of natural transformation was studied by gene disruption analysis in the unicellular motile cyanobacterium Synechocystis sp. PCC 6803. The genes disrupted in this study were chosen as related to the pil genes for biogenesis of the type IV pill in a Gram-negative bacterium Pseudomonas aeruginosa, It was found that motility of Synechocystis cells was lost in the mutants of slr0063, slr1274, slr1275, slr1276, slr1277 and sll1694 together with a simultaneous loss of the thick pill on the cell surface. Competency of the natural transformation was lost in the mutants listed above and slr0197-disruptant. The gene slr0197 was previously predicted as a competence gene by a search with sequence-independent DNA-binding structure [Yura et al. (1999) DNA Res. 6: 75]. It was suggested that both DNA uptake for natural transformation and motility are mediated by a specific type IV-like pilus structure, while a putative DNA-binding protein encoded by slr0197 is additionally required for the DNA uptake. Based on the homology with the pil genes in P. aeruginosa, slr0063, slr1274, slr1275, slr1276, slr1277 and sll1694 were designated pilB1, pilM, pilN, pilO, pilQ and pilA1, respectively. The gene slr0197 was designated comA.

To reveal structure-function relationships of family F/10 glycanases, an in vitro molecular level shuffling experiment was conducted to accumulate useful amino acid residues from two homologous F/10 xylanases, FXYN of Streptomyces olivaceoviridis E-86 and XylA of Thermomonospora alba ULJB1, into a single chimeric xylanase. The parent genes were shuffled by crossovers at selected module borders using self-priming Polymerase Chain Reaction (PCR)s. The shuffled constructs, designated as FXYN-M3/4-XylA, FXYN-M9/10-XylA, and FXYN-M14/15-XylA were cloned and their nucleotide sequences were confirmed. Two chimera, FXYN-M3/4-XylA and FXYN-M14/15-XylA, demonstrated activity against RBB-xylan and were over-expressed as His-tag fusion proteins under control of T5 promoter of pQE60. The homogeneously pure chimeric proteins, FXYN-M3/4-XylA and FXYN-M14/15-XylA showed improved thermal and pH profiles compared to those of one of the parents, FXYN. This was apparently due to the influence of amino acids inherited from thermophilic XylA. Measured K-m and kcat values were closer to those of the other parent, XylA. Interestingly, a significant level of heat tolerance up to 60 degreesC, was recorded for FXYN-M3/4-XylA in comparison to only 40 degreesC for FXYN-M14/15-XylA though their temperature optima did not correlates with their thermal stability. These results indicated that the amino acid residues of the larger T. alba XylA DNA fragment present in FXYN-M3/4-XylA were responsible for inducing its thermal stability. (C) 2001 Elsevier Science Inc. All rights reserved.

Phycobiliproteins are basic building blocks of phycobilisomes, a supra-molecular assembly for the light-capturing function of photosynthesis in cyanobacteria and red algae. One functional form of phycobiliproteins is a trimeric form consisting of three identical units having C-3 symmetry, with each unit composed of two kinds of subunits, the alpha-subunit and beta-subunit. These subunits have similar chain folds and can be divided into either globin-like or X-Y helices domains. We studied the significance of this two-domain structure for their assembled structures and biological function (light-absorption) using a normal mode analysis to investigate dynamic aspects of their three-dimensional structures. We used C-phycocyanin (C-PC) as an example, and focused on the interactions between the two domains. The normal mode analysis was carried out for the following two cases: 1) the whole subunit, including the two domains; and 2) the globin-like domain alone. By comparing the dynamic properties, such as correlative movements between residues and the fluctuations of individual residues, we found that the X-Y helices domain plays an important role not only in the C-3 symmetry assemblies of the subunits in phycobiliproteins, but also in stabilizing the light absorption property by suppressing the fluctuation of the specific Asp residues near the chromophore. Interestingly, the conformation of the X-Y helices domain corresponds to that of a module in pyruvate phosphate dikinase (PPDK). The module in PPDK is involved in the interactions of two domains, just as the X-Y helices domain is involved in the interactions of two subunits. Finally, we discuss the mechanical construction of the C-PC subunits based on the normal mode analysis.

Kaneko, S., Iwamatsu, S., Kuno, A., Fujimoto, Z., Sato, Y., Yura, K., Go, M., Mizuno, H., Taira, K., Hasegawa, T., Kusakabe, I., Hayashi, K.: Module shuffling of a family F/10 xylanase: replacement of modules M4 and M5 of the FXYN of Streptomyces olivaceviridis E-86 with those of the Cex of Cellulomonas fimi.

Phycobiliproteins are basic building blocks of phycobilisomes, a supra-molecular assembly for the light-capturing function of photosynthesis in cyanobacteria and red algae. One functional form of phycobiliproteins is a trimeric form consisting of three identical units having C-3 symmetry, with each unit composed of two kinds of subunits, the alpha-subunit and beta-subunit. These subunits have similar chain folds and can be divided into either globin-like or X-Y helices domains. We studied the significance of this two-domain structure for their assembled structures and biological function (light-absorption) using a normal mode analysis to investigate dynamic aspects of their three-dimensional structures. We used C-phycocyanin (C-PC) as an example, and focused on the interactions between the two domains. The normal mode analysis was carried out for the following two cases: 1) the whole subunit, including the two domains; and 2) the globin-like domain alone. By comparing the dynamic properties, such as correlative movements between residues and the fluctuations of individual residues, we found that the X-Y helices domain plays an important role not only in the C-3 symmetry assemblies of the subunits in phycobiliproteins, but also in stabilizing the light absorption property by suppressing the fluctuation of the specific Asp residues near the chromophore. Interestingly, the conformation of the X-Y helices domain corresponds to that of a module in pyruvate phosphate dikinase (PPDK). The module in PPDK is involved in the interactions of two domains, just as the X-Y helices domain is involved in the interactions of two subunits. Finally, we discuss the mechanical construction of the C-PC subunits based on the normal mode analysis.

Kaneko, S., Iwamatsu, S., Kuno, A., Fujimoto, Z., Sato, Y., Yura, K., Go, M., Mizuno, H., Taira, K., Hasegawa, T., Kusakabe, I., Hayashi, K.: Module shuffling of a family F/10 xylanase: replacement of modules M4 and M5 of the FXYN of Streptomyces olivaceviridis E-86 with those of the Cex of Cellulomonas fimi.

Xylanases are classified into two families, numbered F/10 and G/11 according to the similarity of amino acid sequences of their catalytic domain (Henrissat, B., Bairoch, A., 1993. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 293, 781-788). Three-dimensional structure of the catalytic domain of the family F/10 xylanase was reported (White, A., Withers, S.G., Gilkes, N.R., Rose, D.R., 1994. Crystal structure of the catalytic domain of the beta-1,4-glycanase Cex from Cellulomonas fimi. Biochemistry 33, 12546-12552). The domain was decomposed into 22 modules by centripetal profiles (Gb, M., Nosaka, M., 1987. Protein architecture and the origin of introns. Cold Spring Harbor Symp. Quant. Biol. 52, 915-924; Noguti, T., Sakakibara, H., Gb, M., 1993. Localization of hydrogen-bonds within modules in barnase. Proteins 16, 357-363). A module is a contiguous polypeptide segment of amino acid residues having a compact conformation within a globular domain. Collected 31 intron sites of the family F/10 xylanase genes from fungus were found to be correlated to module boundaries with considerable statistical force (p values &lt;0.001). The relationship between the intron locations and protein structures provides supporting evidence for the ancient origin of introns, because such a relationship cannot be expected by random insertion of introns into eukaryotic genes, but it rather suggests pre-existence of introns in the ancestral genes of prokaryotes and eukaryotes. A phylogenetic tree of the fungal and bacterial xylanase sequences made two clusters; one includes both the bacterial and fungal genes, but the other consists of only fungal genes. The mixed cluster of bacterial genes without introns and the fungal genes with introns further supports the ancient origin of introns. Comparison of the conserved base sequences of introns indicates that sliding of a splice site occurred in Aspergillus kawachii gene by one base from the ancestral position. Substrate-binding sites of xylanase are localized on eight modules, and introns are found at both termini of six out of these functional modules. This result suggests that introns might play a functional role in shuffling the exons encoding the substrate-binding modules. (C) 1999 Elsevier Science B.V. All rights reserved.

Motifs for sequence specific-protein-DNA interactions, such as helix-turn-helix, zinc finger and leucine zipper, are now better understood as a result of extensive studies of three-dimensional (3D) structures of transcription factors. On the other hand, little attention has been paid to motifs for sequence nonspecific binding, namely DNA-phosphate binding. To address the question whether different transcription factors and DNA manipulation enzymes, that is enzymes that work on DNA, share a similar mode of phosphate binding, we sun eyed interactions between DNA and protein module, a structural unit of a globular protein. We analyzed the modular organization of DNA polymerase beta and found that residues making contact with DNA phosphates were localized to five modules. Structural comparison of these phosphate-binding modules against others in transcription factors and DNA manipulation enzymes revealed that DNA polymerase beta. the oct-1 POU domain, 434 Cro and the Are repressor have a phosphate-binding module with 3D structures similar to one another, This newly detected module, the phosphate-binding helix-turn-helix (pbHTH) module, named for its function and 3D structure, interacts with DNA by (i) making hydrogen bonds between a DNA phosphodiester oxygen and an amino hydrogen of the main chain located at the N-terminus of a C-terminal alpha-helix. and (ii) making electrostatic interactions between DNA phosphates and side chains of lysine or arginine. Finding structurally and functionally similar phosphate-binding units in different transcription factors and DNA manipulation enzymes suggests that shuffling of modules is not limited to the DNA base-recognition motif. Phosphate-binding modules are apparently also shuffled in DNA-binding proteins.

Genetic transformation is widely utilized in molecular biology as a tool for gene cloning in Escherichia coli and for gene mapping in Bacillus subtilis. Several strains of eubacteria can naturally take up exogenous DNA and integrate the DNA into their own genomes. Molecular details of natural transformation, however, remained to be elucidated. The complete genome of a cyanobacterium, Synechocystis sp. PCC6803, has been sequenced. This bacterium has been used to examine functions of a particular gene. The genome is considered to carry information on natural transformable characteristics of Synechocystis. The first step in genetic transformation is the uptake of exogenous DNA. Proteins with non-specific DNA binding features are required, because specificity in the exogenous DNA has not been demonstrated. Such proteins have modules interacting with the phosphate backbone of DNA, including helix-turn-helix modules. Using a consensus pattern of the phosphate-binding helix-turn-helix module, we searched through the genome data of Synechocystis for genes or open reading frame (ORF) products with the pattern in primary structures. We found that an ORF, slr0197, has the pattern in duplicate at the C-terminal region. We also found that the ORF product has a hydrophobic segment at the N-terminal region, which is followed by two internal repeats of the endonuclease domain. Based on these observations, we propose a model for the initial stage of genetic transformation. This is apparently the first report on molecular mechanisms of natural transformation.

Kaneko, S., Kuno, A., Fujimoto, Z., Shimizu, D., Machida, S., Sato, Y., Yura, K., Go, M.,Mizuno, H., Taira, K., Kusakabe, I. and Hayashi, K.: An investigation of the nature and function of module 10 in a family F/10 xylanase FXYN of Streptomyces olivaceoviridis E-86 by module shuffling with the Cex of Cellulomonas fimi and by site-directed mutagenesis.

Genetic transformation is widely utilized in molecular biology as a tool for gene cloning in Escherichia coli and for gene mapping in Bacillus subtilis. Several strains of eubacteria can naturally take up exogenous DNA and integrate the DNA into their own genomes. Molecular details of natural transformation, however, remained to be elucidated. The complete genome of a cyanobacterium, Synechocystis sp. PCC6803, has been sequenced. This bacterium has been used to examine functions of a particular gene. The genome is considered to carry information on natural transformable characteristics of Synechocystis. The first step in genetic transformation is the uptake of exogenous DNA. Proteins with non-specific DNA binding features are required, because specificity in the exogenous DNA has not been demonstrated. Such proteins have modules interacting with the phosphate backbone of DNA, including helix-turn-helix modules. Using a consensus pattern of the phosphate-binding helix-turn-helix module, we searched through the genome data of Synechocystis for genes or open reading frame (ORF) products with the pattern in primary structures. We found that an ORF, slr0197, has the pattern in duplicate at the C-terminal region. We also found that the ORF product has a hydrophobic segment at the N-terminal region, which is followed by two internal repeats of the endonuclease domain. Based on these observations, we propose a model for the initial stage of genetic transformation. This is apparently the first report on molecular mechanisms of natural transformation.

Kaneko, S., Kuno, A., Fujimoto, Z., Shimizu, D., Machida, S., Sato, Y., Yura, K., Go, M.,Mizuno, H., Taira, K., Kusakabe, I. and Hayashi, K.: An investigation of the nature and function of module 10 in a family F/10 xylanase FXYN of Streptomyces olivaceoviridis E-86 by module shuffling with the Cex of Cellulomonas fimi and by site-directed mutagenesis.<br />

Xylanases are classified into two families, numbered F/10 and G/11 according to the similarity of amino acid sequences of their catalytic domain (Henrissat, B., Bairoch, A., 1993. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 293, 781-788). Three-dimensional structure of the catalytic domain of the family F/10 xylanase was reported (White, A., Withers, S.G., Gilkes, N.R., Rose, D.R., 1994. Crystal structure of the catalytic domain of the beta-1,4-glycanase Cex from Cellulomonas fimi. Biochemistry 33, 12546-12552). The domain was decomposed into 22 modules by centripetal profiles (Gb, M., Nosaka, M., 1987. Protein architecture and the origin of introns. Cold Spring Harbor Symp. Quant. Biol. 52, 915-924; Noguti, T., Sakakibara, H., Gb, M., 1993. Localization of hydrogen-bonds within modules in barnase. Proteins 16, 357-363). A module is a contiguous polypeptide segment of amino acid residues having a compact conformation within a globular domain. Collected 31 intron sites of the family F/10 xylanase genes from fungus were found to be correlated to module boundaries with considerable statistical force (p values &lt;0.001). The relationship between the intron locations and protein structures provides supporting evidence for the ancient origin of introns, because such a relationship cannot be expected by random insertion of introns into eukaryotic genes, but it rather suggests pre-existence of introns in the ancestral genes of prokaryotes and eukaryotes. A phylogenetic tree of the fungal and bacterial xylanase sequences made two clusters; one includes both the bacterial and fungal genes, but the other consists of only fungal genes. The mixed cluster of bacterial genes without introns and the fungal genes with introns further supports the ancient origin of introns. Comparison of the conserved base sequences of introns indicates that sliding of a splice site occurred in Aspergillus kawachii gene by one base from the ancestral position. Substrate-binding sites of xylanase are localized on eight modules, and introns are found at both termini of six out of these functional modules. This result suggests that introns might play a functional role in shuffling the exons encoding the substrate-binding modules. (C) 1999 Elsevier Science B.V. All rights reserved.

Motifs for sequence specific-protein-DNA interactions, such as helix-turn-helix, zinc finger and leucine zipper, are now better understood as a result of extensive studies of three-dimensional (3D) structures of transcription factors. On the other hand, little attention has been paid to motifs for sequence nonspecific binding, namely DNA-phosphate binding. To address the question whether different transcription factors and DNA manipulation enzymes, that is enzymes that work on DNA, share a similar mode of phosphate binding, we sun eyed interactions between DNA and protein module, a structural unit of a globular protein. We analyzed the modular organization of DNA polymerase beta and found that residues making contact with DNA phosphates were localized to five modules. Structural comparison of these phosphate-binding modules against others in transcription factors and DNA manipulation enzymes revealed that DNA polymerase beta. the oct-1 POU domain, 434 Cro and the Are repressor have a phosphate-binding module with 3D structures similar to one another, This newly detected module, the phosphate-binding helix-turn-helix (pbHTH) module, named for its function and 3D structure, interacts with DNA by (i) making hydrogen bonds between a DNA phosphodiester oxygen and an amino hydrogen of the main chain located at the N-terminus of a C-terminal alpha-helix. and (ii) making electrostatic interactions between DNA phosphates and side chains of lysine or arginine. Finding structurally and functionally similar phosphate-binding units in different transcription factors and DNA manipulation enzymes suggests that shuffling of modules is not limited to the DNA base-recognition motif. Phosphate-binding modules are apparently also shuffled in DNA-binding proteins.

The human CCG1 gene, encoding CCG1/TAF(II)250/p250, was isolated by complementing tsBN462, a mutant BHK21 cell line that shows cell-cycle arrest at high temperature. Using the cDNA as a probe, the locations of exon-intron junctions were determined in the genomic DNA: Thirty-eight exons ranging from 68 to 219 bp in size were found. All the exon-intron junctions followed the GT-AG rule. Using a newly developed method, we performed a module analysis of the CCG1 protein. The functional domain previously predicted in CCG1 was further confirmed to be encoded in a single predicted module that is the minimal functional unit in the protein. The boundaries of the predicted modules show a close correlation to the intron/exon junction of CCG1. The entire gene, at least 110 kb long has been recovered in a YAC, which provides a route to the further study of module function.

The human CCG1 gene, encoding CCG1/TAF(II)250/p250, was isolated by complementing tsBN462, a mutant BHK21 cell line that shows cell-cycle arrest at high temperature. Using the cDNA as a probe, the locations of exon-intron junctions were determined in the genomic DNA: Thirty-eight exons ranging from 68 to 219 bp in size were found. All the exon-intron junctions followed the GT-AG rule. Using a newly developed method, we performed a module analysis of the CCG1 protein. The functional domain previously predicted in CCG1 was further confirmed to be encoded in a single predicted module that is the minimal functional unit in the protein. The boundaries of the predicted modules show a close correlation to the intron/exon junction of CCG1. The entire gene, at least 110 kb long has been recovered in a YAC, which provides a route to the further study of module function.

The human CCG1 gene, encoding CCG1/TAF(II)250/p250, was isolated by complementing tsBN462, a mutant BHK21 cell line that shows cell-cycle arrest at high temperature. Using the cDNA as a probe, the locations of exon-intron junctions were determined in the genomic DNA: Thirty-eight exons ranging from 68 to 219 bp in size were found. All the exon-intron junctions followed the GT-AG rule. Using a newly developed method, we performed a module analysis of the CCG1 protein. The functional domain previously predicted in CCG1 was further confirmed to be encoded in a single predicted module that is the minimal functional unit in the protein. The boundaries of the predicted modules show a close correlation to the intron/exon junction of CCG1. The entire gene, at least 110 kb long has been recovered in a YAC, which provides a route to the further study of module function.

Cytochrome P450 cam (CYP101) of Pseudomonas putida PpG1 in which Arg112 is substituted by Cys was isolated by in vitro random mutagenesis of the camC gene DNA coding for P450cam. The absorption spectra of the purified mutant enzyme were similar to those of the wild type enzyme, but its substrate-dependent NADH oxidation activity in the presence of putidaredoxin (Pd) and putidaredoxin reductase (PdR) was extremely low. The rate constant of electron transfer from reduced Pd to the heme of the mutant P450cam, measured on an anaerobic stopped flow apparatus, was 1/400 of that of the wild type enzyme and the dissociation constant of the mutant P450cam for oxidized Pd was several fold higher than that of the wild type enzyme. A considerable decrease in mid-point potential of the mutant enzyme was also noted. We conclude that Arg112, which is located on the surface of the P450cam molecule and hydrogen-bonded to one of the heme propionate chains, plays an essential role in the electron transfer from Pd.

Cytochrome P450 cam (CYP101) of Pseudomonas putida PpG1 in which Arg112 is substituted by Cys was isolated by in vitro random mutagenesis of the camC gene DNA coding for P450cam. The absorption spectra of the purified mutant enzyme were similar to those of the wild type enzyme, but its substrate-dependent NADH oxidation activity in the presence of putidaredoxin (Pd) and putidaredoxin reductase (PdR) was extremely low. The rate constant of electron transfer from reduced Pd to the heme of the mutant P450cam, measured on an anaerobic stopped flow apparatus, was 1/400 of that of the wild type enzyme and the dissociation constant of the mutant P450cam for oxidized Pd was several fold higher than that of the wild type enzyme. A considerable decrease in mid-point potential of the mutant enzyme was also noted. We conclude that Arg112, which is located on the surface of the P450cam molecule and hydrogen-bonded to one of the heme propionate chains, plays an essential role in the electron transfer from Pd.

Helix-turn-helix motif is one of the common motifs observed in DNA-binding proteins. The motif interacts with DNA double helix and recognizes specific base sequences. It is assumed that the helix - turn - helix motif appears only once in seven prokaryotic transcriptional repressors of which 3-D structures have been determined by X-ray crystallographic studies. These prokaryotic repressors consist of several alpha-helices connected with turns. We report here that these repressors are decomposable into helix - turn - helix modules and their connectors. A module is defined as a compact structural unit with consecutive amino acid residues in a globular protein. Each of the helix - turn - helix motifs in the seven proteins corresponds approximately to a single helix-turn-helix module consisting of approximately 13 amino acids. Identification of modules of seven prokaryotic repressors and comparisons of their tertiary structures led to the conclusion that three of these DNA-binding proteins contain more than one helix - turn - helix module with a structure similar to the helix-turn-helix motif. The difference in module organization of these DNA-binding proteins paves the way for further classification of the DNA-binding proteins with the helix-turn-helix motif. The structural repertoire of these transcriptional regulators was increased through different utilizations in the number of helix - turn - helix and other modules. The difference in DNA base recognition ability in these helix - turn - helix modules is ascribed to a difference in size of a side chain at the fifth residue from Gly, on the turn.

Helix-turn-helix motif is one of the common motifs observed in DNA-binding proteins. The motif interacts with DNA double helix and recognizes specific base sequences. It is assumed that the helix - turn - helix motif appears only once in seven prokaryotic transcriptional repressors of which 3-D structures have been determined by X-ray crystallographic studies. These prokaryotic repressors consist of several alpha-helices connected with turns. We report here that these repressors are decomposable into helix - turn - helix modules and their connectors. A module is defined as a compact structural unit with consecutive amino acid residues in a globular protein. Each of the helix - turn - helix motifs in the seven proteins corresponds approximately to a single helix-turn-helix module consisting of approximately 13 amino acids. Identification of modules of seven prokaryotic repressors and comparisons of their tertiary structures led to the conclusion that three of these DNA-binding proteins contain more than one helix - turn - helix module with a structure similar to the helix-turn-helix motif. The difference in module organization of these DNA-binding proteins paves the way for further classification of the DNA-binding proteins with the helix-turn-helix motif. The structural repertoire of these transcriptional regulators was increased through different utilizations in the number of helix - turn - helix and other modules. The difference in DNA base recognition ability in these helix - turn - helix modules is ascribed to a difference in size of a side chain at the fifth residue from Gly, on the turn.

Helix-turn-helix motif is one of the common motifs observed in DNA-binding proteins. The motif interacts with DNA double helix and recognizes specific base sequences. It is assumed that the helix - turn - helix motif appears only once in seven prokaryotic transcriptional repressors of which 3-D structures have been determined by X-ray crystallographic studies. These prokaryotic repressors consist of several alpha-helices connected with turns. We report here that these repressors are decomposable into helix - turn - helix modules and their connectors. A module is defined as a compact structural unit with consecutive amino acid residues in a globular protein. Each of the helix - turn - helix motifs in the seven proteins corresponds approximately to a single helix-turn-helix module consisting of approximately 13 amino acids. Identification of modules of seven prokaryotic repressors and comparisons of their tertiary structures led to the conclusion that three of these DNA-binding proteins contain more than one helix - turn - helix module with a structure similar to the helix-turn-helix motif. The difference in module organization of these DNA-binding proteins paves the way for further classification of the DNA-binding proteins with the helix-turn-helix motif. The structural repertoire of these transcriptional regulators was increased through different utilizations in the number of helix - turn - helix and other modules. The difference in DNA base recognition ability in these helix - turn - helix modules is ascribed to a difference in size of a side chain at the fifth residue from Gly, on the turn.

Koga, H., Sagara, Y., Yaoi, T., Tsujimura, M., Nakamura, K., Sekimizu, K., Makino, R., Shimada, H., Ishimura, Y., Yura, K., Go, M., Ikeguchi, M., and Horiuchi, T.: Essential role of the Arg112 residue of cytochrome P450cam for election transfer from reduced putidaredoxin.