ViralORFeome: an integrated database to generate a versatile collection of viral ORFs.

Large collections of protein-encoding open reading frames (ORFs) established in a versatile recombination-based cloning system have been instrumental to study protein functions in high-throughput assays. Such 'ORFeome' resources have been developed for several organisms but in virology, plasmid collections covering a significant fraction of the virosphere are still needed. In this perspective, we present ViralORFeome 1.0 (http://www.viralorfeome.com), an open-access database and management system that provides an integrated set of bioinformatic tools to clone viral ORFs in the Gateway(R) system. ViralORFeome provides a convenient interface to navigate through virus genome sequences, to design ORF-specific cloning primers, to validate the sequence of generated constructs and to browse established collections of virus ORFs. Most importantly, ViralORFeome has been designed to manage all possible variants or mutants of a given ORF so that the cloning procedure can be applied to any emerging virus strain. A subset of plasmid constructs generated with ViralORFeome platform has been tested with success for heterologous protein expression in different expression systems at proteome scale. ViralORFeome should provide our community with a framework to establish a large collection of virus ORF clones, an instrumental resource to determine functions, activities and binding partners of viral proteins.

Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2.

The human parvovirus adeno-associated virus type 2 (AAV2) has many features that make it attractive as a vector for gene therapy. However, the broad host range of AAV2 might represent a limitation for some applications in vivo, because recombinant AAV vector (rAAV)-mediated gene transfer would not be specific for the tissue of interest. This host range is determined by the binding of the AAV2 capsid to specific cellular receptors and/or co-receptors. The tropism of AAV2 might be changed by genetically introducing a ligand peptide into the viral capsid, thereby redirecting the binding of AAV2 to other cellular receptors. We generated six AAV2 capsid mutants by inserting a 14-amino-acid targeting peptide, L14, into six different putative loops of the AAV2 capsid protein identified by comparison with the known three-dimensional structure of canine parvovirus. All mutants were efficiently packaged. Three mutants expressed L14 on the capsid surface, and one efficiently infected wild-type AAV2-resistant cell lines that expressed the integrin receptor recognized by L14. The results demonstrate that the AAV2 capsid tolerates the insertion of a nonviral ligand sequence. This might open new perspectives for the design of targeted AAV2 vectors for human somatic gene therapy.

Mutation in the melanocortin 1 receptor is associated with amber colour in the Norwegian Forest Cat.

Amber (previously called X-Colour) is a yellow recessive coat colour observed in the Norwegian Forest Cat (NFC) population and apparently absent in other cat breeds. Until now, there has never been any scientific evidence of yellow recessive mutation (e) reported in the extension gene in Felidae. We sequenced the complete coding sequence region for the melanocortin 1 receptor in 12 amber, three carriers, two wild-type NFCs, one wild-type European Shorthair and two 'golden' Siberian cats and identified two single nucleotide polymorphisms (SNPs): a non-synonymous (FM180571: c.250G>A) and a synonymous (FM180571: c.840T>C) mutation. The c.250G>A SNP, further genotyped on 56 cats using PCR-RFLP, is associated with amber colour and only present in the amber cat lineages. It replaced an aspartic acid with a neutral polar asparagine in the second transmembrane helix (p.Asp84Asn), a position where e mutations have already been described. Three-dimensional models were built and showed electrostatic potential modification in the mutant receptor. With these results and together with those in the scientific literature, we can conclude that amber colour in NFCs is caused by a single MC1R allele called e, which has never been documented.

A common mechanism for ATP hydrolysis in ABC transporter and helicase superfamilies.

ABC (ATP-binding cassette) transporters and helicases are large superfamilies of seemingly unrelated proteins, whose functions depend on the energy provided by ATP hydrolysis. Comparison of the 3D structures of their nucleotide-binding domains reveals that, besides two well-characterized ATP-binding signatures, the folds of their nucleotide-binding sites are similar. Furthermore, there are striking similarities in the positioning of residues thought to be important for ATP binding or hydrolysis. Interestingly, structures have recently been obtained for two ABC proteins that are not involved in transport activities, but that have a function related to DNA modification. These ABC proteins, which contain a nucleotide-binding site akin to those of typical ABC transporters, might constitute the missing link between the two superfamilies.

MAGOS: multiple alignment and modelling server.

UNLABELLED: MAGOS is a web server allowing automated protein modelling coupled to the creation of a hierarchical and annotated multiple alignment of complete sequences. MAGOS is designed for an interactive approach of structural information within the framework of the evolutionary relevance of mined and predicted sequence information. AVAILABILITY: The web server is freely available at http://pig-pbil.ibcp.fr/magos.

AF15q14, a novel partner gene fused to the MLL gene in an acute myeloid leukaemia with a t(11;15)(q23;q14).

In haematopoietic malignancies the MLL gene, located on chromosome 11q23, is frequently disrupted by chromosome rearrangement, generally resulting in fusion to various partner genes. We have previously reported a t(11;15)(q23;q14) in a case of acute myeloblastic leukaemia. Here, we report the cloning of a novel MLL partner, AF15q14, at chromosome 15q14. In this translocation, the breakpoint occurred in exon 8 of MLL and exon 10 of AF15q14. The normal AF15q14 transcripts of approximately 8.5 kb in size, are expressed in different tumoral cell lines, in a variety of normal tissues, and in all the foetal tissues tested. Sequencing of AF15q14 cDNA revealed a putative open reading frame of 1833 amino acids that had no homology with any other known protein. The C-terminal end of the putative AF15q14 contained a bipartite nuclear localization site. The translocation t(11;15) preserved the open reading frame between MLL and the 3' end of AF15q14. The contribution of AF15q14 to the fusion protein was only 85 amino acids. Immunofluorescence staining experiments with expression vectors encoding these 85 amino acids confirmed the functionality of the predicted nuclear localization site.

Correlations between ATP hydrolysis, ATP synthesis, generation and utilization of delta pH in mitochondrial ATPase-ATP synthase.

Using fluorescence quenching of 9-amino-6-chloro-2- methoxyacridine induced either by ATP hydrolysis in the ATPase-ATP synthase complex or by succinate oxidation in inverted submitochondrial particles, correlation have been established between ATP hydrolysis, ATP synthesis and the generation and utilization of delta pH. The results obtained are best explained in terms of local circuits of protons.

Efficient reconstitution of mitochondrial energy-transfer reactions from depleted membranes and F1-ATPase as a function of the amount of bound oligomycin sensitivity-conferring protein (OSCP).

Pig heart mitochondrial membranes depleted of F1 and OSCP by various treatments were analyzed for their content in alpha and beta subunits of F1 and in OSCP using monoclonal antibodies. Membrane treatments and conditions of rebinding of F1 and OSCP were optimized to reconstitute efficient NADH- and ATP-dependent proton fluxes, ATP synthesis and oligomycin-sensitive ATPase activity. F1 and OSCP can be rebound independently to depleted membranes but to avoid unspecific binding of F1 to depleted membranes (ASUA) which is not efficient for ATP synthesis, F1 must be rebound before the addition of OSCP. The rebinding of OSCP to depleted membranes reconstituted with F1 inhibits the ATPase activity of rebound F1, while it restores the ATP-driven proton flux measured by the quenching of ACMA fluorescence. The rebinding of OSCP also renders the ATPase activity of bound F1 sensitive to uncouplers. The rebinding of OSCP alone or F1 alone, does not modify the NADH-dependent proton flux, while the rebinding of both F1 and OSCP controls this flux, inducing an inhibition of the rate of NADH oxidation. Similarly, oligomycin, which seals the F0 channel even in the absence of F1 and OSCP, inhibits the rate of NADH oxidation. OSCP is required to adjust the fitting of F1 to F0 for a correct channelling of protons efficient for ATP synthesis. All reconstituted energy-transfer reactions reach their optimal value for the same amount of OSCP. This amount is consistent with a stoichiometry of two OSCP per F1 in the F0-F1 complex.

Secondary structure of P-glycoprotein investigated by circular dichroism and amino acid sequence analysis.

P-glycoprotein (Pgp) is a plasma membrane protein known as an ATP-dependent drug-efflux pump that confers multidrug resistance to tumor cells. Structural analysis of Pgp was investigated by circular dichroism (CD) for the first time and in combination with amino acid sequence analysis. CD of highly purified Pgp from human, rat and murine Pgp-overexpressing drug resistant cells revealed slight variations in the spectral shape when recorded in the presence of dodecyl maltoside (DM). These species-dependent variations in CD shapes resulted from the interaction of the oligosaccharidic part with the protein core since they were abolished either in the presence of sodium dodecyl sulfate (SDS) or after deglycosylation, the latter not altering the Pgp ATP-dependent drug transport activity. Whatever the level of Pgp glycosylation and the detergent used (SDS or DM), the content in secondary structure deduced from deconvolution of CD spectra is almost the same for the three sources of Pgp and estimated to 43% alpha-helix, 16% beta-sheet, 15% beta-turn and 26% of other structures. These data, which constitute the first report of Pgp structure analysis by circular dichroism, are consistent with the 48% alpha-helix and 16% beta-sheets global contents predicted by using recently reported efficient secondary structure prediction methods. This consistency reinforces the reliability of the probable nature and localization of predicted Pgp secondary structure elements. This provides a good framework for precise 3D structure modeling of Pgp by homology with proteins of known 3D structure, as it is illustrated here for the A motifs of the ATP-binding domains of Pgp.

Dimerization kinetics of HIV-1 and HIV-2 reverse transcriptase: a two step process.

The dimerization processes of the human immunodeficiency virus (HIV) types 1 and 2 reverse transcriptase (RTs) from their subunits have been investigated using a number of complementary approaches (fluorescence spectroscopy, size exclusion-HPLC and polymerase activity assay). The formation of the native heterodimeric form of HIV-1 and HIV-2 RT occurs in a two step process. The first step is a concentration-dependent association of the two subunits (p66 and p51) to give a heterodimeric intermediate, which slowly isomerizes to the "mature" heterodimeric form of the enzyme. For both RTs, the first step behaves as a second order reaction with similar association rate constants (in the range of 2 x 10(4) to 4 x 10(4) M-1 s-1). This initial dimerization results in a 25% quenching of the intrinsic fluorescence and a 30% decrease in the accessibility of the tryptophan hydrophobic cluster to solvent as revealed by iodide quenching experiments and by monitoring the binding of 1-anilino-8-naphthalenesulphonate. The formation of the intermediate-RT form appears to involve hydrophobic regions of the subunits containing tryptophan residues. This intermediate form is devoid of polymerase activity, but is able to bind primer/template with high affinity. The final stage of the mature RT-heterodimer formation occurs in a slow first order reaction, which is 12-fold faster for HIV-2 (1.2 h-1) than HIV-1 RT (0.1 h-1). At micromolar concentrations, this slow isomerization constitutes the rate limiting step of the RT maturation and the structural change involved appears to be partly associated with the catalytic site, as shown using fluorescent labelled primer/template. On the basis of both the presently available X-ray structure of the HIV-1 RT and the predicted structure of HIV-2 RT, the thumb subdomain of the p51 subunit seems to be involved in this maturation step, which is probably the interaction of this domain with the RNAse H domain of the large subunit. The placement of the fingers subdomain of p51 in the palm subdomain of the p66 subunit may also be associated with formation of mature heterodimeric RTs.

Three-dimensional structure of the DNA-binding domain of the fructose repressor from Escherichia coli by 1H and 15N NMR.

FruR is an Escherichia coli transcriptional regulator that belongs to the LacI DNA-binding protein family. By using 1H and 15N NMR spectroscopy, we have determined the three-dimensional solution structure of the FruR N-terminal DNA-binding domain consisting of 57 amino acid residues. A total of 809 NMR-derived distances and 54 dihedral angle constraints have been used for molecular modelling with the X-PLOR program. The resulting set of calculated structures presents an average root-mean-square deviation of 0.37 A at the main-chain level for the first 47 residues. This highly defined N-terminal part of the structure reveals a similar topology for the three alpha-helices when compared to the 3D structures of LacI and PurR counterparts. The most striking difference lies in the connection between helix II and helix III, in which three additional residues are present in FruR. This connecting segment is well structured and contains a type III turn. Apart from hydrophobic interactions of non-polar residues with the core of the domain, this connecting segment is stabilised by several hydrogen bonds and by the aromatic ring stacking between Tyr19 of helix II and Tyr28 of the turn. The region containing the putative "hinge helix" (helix IV), that has been described in PurR-DNA complex to make specific base contacts in the minor groove of DNA, is unfolded. Examination of hydrogen bonds highlights the importance of homologous residues that seem to be conserved for their ability to fulfill helix N and C-capping roles in the LacI repressor family.

Identification of related proteins with weak sequence identity using secondary structure information.

Molecular modeling of proteins is confronted with the problem of finding homologous proteins, especially when few identities remain after the process of molecular evolution. Using even the most recent methods based on sequence identity detection, structural relationships are still difficult to establish with high reliability. As protein structures are more conserved than sequences, we investigated the possibility of using protein secondary structure comparison (observed or predicted structures) to discriminate between related and unrelated proteins sequences in the range of 10%-30% sequence identity. Pairwise comparison of secondary structures have been measured using the structural overlap (Sov) parameter. In this article, we show that if the secondary structures likeness is >50%, most of the pairs are structurally related. Taking into account the secondary structures of proteins that have been detected by BLAST, FASTA, or SSEARCH in the noisy region (with high E: value), we show that distantly related protein sequences (even with <20% identity) can be still identified. This strategy can be used to identify three-dimensional templates in homology modeling by finding unexpected related proteins and to select proteins for experimental investigation in a structural genomic approach, as well as for genome annotation.

Common topology within a non-collagenous domain of several different collagen types.

The secondary structure of a conserved non-collagenous module in alpha 1(V), alpha 1(XI), alpha 1(IX), alpha 1(XII), alpha 1(XIV) and alpha 1(XVI) collagen chains and in proline- and arginine-rich protein was analyzed using different algorithms. The results predict that a common anti-parallel beta-sheet structure composed of nine consensus beta-strands is present in these non-collagenous modules. A model for the packing of these beta-sheets is proposed which suggests that the predicted beta-sheet structure may be involved in molecular recognition functions.

Identification and characterization of a heparin binding site within the NC1 domain of chicken collagen XIV.

Collagen XIV is known to bind to the dermatan sulfate chain of decorin and to the heparan sulfate chain of perlecan. To study its possible interaction with glycosaminoglycans, the NC1 domain of chicken collagen XIV was overproduced in E. coli. Purified NC1*(6-119)* appears poorly organized (the asterisks indicate the presence of extension sequences), but V8-protease generated fragments containing the 84-108 basic sequence tend to fold into alpha-helix. These fragments interact specifically with heparin, which induces an alpha-helical fold with a maximum effect for equimolar heparin/peptide ratio. These data demonstrate the existence of a glycosaminoglycan binding site in NC1.

Cloning and sequence analysis of a polygalacturonase-encoding gene from the phytopathogenic fungus Sclerotinia sclerotiorum.

The phytopathogenic fungus Sclerotinia sclerotiorum produces a number of extra-cellular pectin-degrading enzymes. We have cloned and determined the complete sequence of a gene (pg1) encoding an endopolygalacturonase (PG1). The coding region consists of a non-interrupted 1143-bp open reading frame. S. sclerotiorum pg1 was compared to other fungal PG-encoding genes. Basic transcription control sequences were identified in the 5' non-coding region. The deduced amino acid (aa) sequence (380 aa) of the enzyme is compared to seven fungal PG sequences and shows a high level of identity (41.5 to 59.8%). Predicted secondary structures were compared, revealing a similar protein organization most probably in antiparallel beta sheets. Hybridization analysis using a pg1 0.65-kb BamHI fragment as a probe allowed the identification of seven different recombinant phages from a genomic library. Analysis of the hybridizing restriction fragments suggests that PG-encoding genes are organized as a family.

Characterization of a bacterial gene encoding an autophosphorylating protein tyrosine kinase.

Acinetobacter johnsonii harbors a protein tyrosine kinase activity that is able to catalyze autophosphorylation, like a number of eukaryotic tyrosine kinases. A biochemical and genetic analysis of this enzyme was performed. Maximum phosphorylation in vitro was obtained by incubating the kinase for 2 min at pH 7.0 in the presence of 5 mM magnesium chloride. In contrast to eukaryotic enzymes, no inhibitory effect of genistein and no phosphorylation of synthetic substrates such as poly (Glu80 Tyr20) or angiotensin II were observed. The analysis of the bacterial kinase by two-dimensional gel electrophoresis revealed the presence of at least five isoforms, all phosphorylated exclusively at tyrosine, which supports the concept that autophosphorylation occurs at multiple sites within the protein. The cloning and nucleotide sequencing of the gene encoding this kinase were achieved, which represents the first molecular characterization of a gene of this type in bacteria. An open reading frame of 2199 nucleotides encoding a protein of 82,373 Da was detected. The analysis of the deduced amino acid sequence suggested a possible involvement of the enzyme in cell recognition and bacterial pathogenicity. In addition, the cloning and sequencing of the region immediately upstream of the gene encoding the kinase revealed a novel open reading frame of 426 nucleotides encoding a phosphotyrosine protein phosphatase of 16,217 Da, which indicates that autophosphorylation on tyrosine is a physiologically reversible reaction.

Protein structure prediction. Implications for the biologist.

Recent improvements in the prediction of protein secondary structure are described, particularly those methods using the information contained into multiple alignments. In this respect, the prediction accuracy has been checked and methods that take into account multiple alignments are 70% correct for a three-state description of secondary structure. This quality is obtained by a 'leave-one out' procedure on a reference database of proteins sharing less than 25% identity. Biological applications such as 'protein domain design' and structural phylogeny are given. The biologist's point of view is also considered and joint predictions are encouraged in order to derive an amino acid based accuracy. All the tools described in this paper are available for biologists on the Web (http/www.ibcp.fr/predict.html).

Epitope of OSCP oligomycin sensitivity conferring protein exposed at the surface of the mitochondrial ATPase-ATPsynthase complex.

Immunological studies were designed to study the structure of the oligomycin sensitivity conferring protein (OSCP) integrated in the mitochondrial ATPase-ATPsynthase complex. The monoclonal antibody 2B1B1 used in this study could bind as well to purified or membrane bound OSCP as shown previously by Protein A-gold immunocytochemistry and by competitive immunotitration. In this paper, it is shown that 2B1B1 can also immunoprecipitate the F0F1 complex from a Triton X-100 extract. This means that not only, 2B1B1 binds to the surface of OSCP but also that the binding of 2B1B1 did not destroy the interactions between F0 and F1 and further demonstrates the external location of the 2B1B1 binding site in the ATPase-ATPsynthase complex. This antigenic site was located on the N-terminal sequence of OSCP, between residues 1 and 72, as demonstrated after chemical cleavage of OSCP with formic acid, hydroxylamine and partial cleavage with cyanogen bromide. The proximity of Tyr and Arg to the epitope was suggested by the lack of 2B1B1 binding to iodinated OSCP and by the susceptibility of this binding to trypsin or to endoproteinase Arg-C treatments of OSCP, respectively. A more precise location of the epitope has been attempted by using the method of synthesis of overlapping octapeptides on solid support. It was found that 2 groups of octapeptides could bind 2B1B1. The first group contained in common the sequence Pro7-Pro8-Val9-Gln10-Ile11-Tyr12- and the second group of peptides contained the sequence Arg62-Ser63-Val64-Lys65. Another monoclonal antibody, AF4H7, which competes with 2B1B1, also recognized the first group of peptides. The possible involvement of these 2 fragments in the epitope localized at the surface of OSCP is discussed. In addition, secondary structure theoretical analysis predicts that these 2 domains should be in a beta-strand configuration.

Structure and organization of the Bombyx mori sericin 1 gene and of the sericins 1 deduced from the sequence of the Ser 1B cDNA.

The sericin 1 primary transcript of the silkworm Bombyx mori is differentially spliced via a tissue- and developmentally-regulated process. From a middle silk gland cDNA library, we have elucidated the sequence of one of the four mRNAs, the 4.0 kb Ser1B mRNA. Determination of alternative or constitutive exons and intron-exon boundaries allowed us to establish the nine exon-eight intron structure of the Ser1 gene. From these and previous data, it was possible to deduce the sequence of the sericins 1 and to predict the secondary structure and physiochemical properties of the different regions of the proteins.

ANTHEPROT: an integrated protein sequence analysis software with client/server capabilities.

Programs devoted to the analysis of protein sequences exist either as stand-alone programs or as Web servers. However, stand-alone programs can hardly accommodate for the analysis that involves comparisons on databanks, which require regular updates. Moreover, Web servers cannot be as efficient as stand-alone programs when dealing with real-time graphic display. We describe here a stand-alone software program called ANTHEPROT, which is intended to perform protein sequence analysis with a high integration level and clients/server capabilities. It is an interactive program with a graphical user interface that allows handling of protein sequence and data in a very interactive and convenient manner. It provides many methods and tools, which are integrated into a graphical user interface. ANTHEPROT is available for Windows-based systems. It is able to connect to a Web server in order to perform large-scale sequence comparison on up-to-date databanks. ANTHEPROT is freely available to academic users and may be downloaded at http://pbil.ibcp.fr/ANTHEPROT.