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PubMed2006

Statistical, computational and visualization methodologies to unveil gene primary structure features.

OBJECTIVES: Gene sequence features such as codon bias, codon context, and codon expansion (e.g. trinucleotide repeats) can be better understood at the genomic scale level by combining statistical methodologies with advanced computer algorithms and data visualization through sophisticated graphical interfaces. This paper presents the ANACONDA system, a bioinformatics application for gene primary structure analysis. METHODS: Codon usage tables using absolute metrics and software for multivariate analysis of codon and amino acid usage are available in public databases. However, they do not provide easy computational and statistical tools to carry out detailed gene primary structure analysis on a genomic scale. We propose the usage of several statistical methods--contingency table analysis, residual analysis, multivariate analysis (cluster analysis)--to analyze the codon bias under various aspects (degree of association, contexts and clustering). RESULTS: The developed solution is a software application that provides a user-guided analysis of codon sequences considering several contexts and codon usage on a genomic scale. The utilization of this tool in our molecular biology laboratory is focused on particular genomes, especially those from Saccharomyces cerevisiae, Candida albicans and Escherichia coli. In order to illustrate the applicability and output layouts of the software these species are herein used as examples. CONCLUSIONS: The statistical tools incorporated in the system are allowing to obtain global views of important sequence features. It is expected that the results obtained will permit identification of general rules that govern codon context and codon usage in any genome. Additionally, identification of genes containing expanded codons that arise as a consequence of erroneous DNA replication events will permit uncovering new genes associated with human disease.

Algorithms↗
PubMed2005

Structural features of the lysosomal hydrolase mannose 6-phosphate uncovering enzyme.

The uncovering enzyme (UCE) removes N-acetylglucosamine from lysosomal enzymes to uncover the mannose 6-phosphate (Man-6-P) determinant necessary for targeting these enzymes to lysosomes. Failure to create the Man-6-P determinant is one cause of lysosomal storage diseases. Despite its medical importance, little structural information about UCE is available. In this report we have developed a model for the membrane proximal portion of the lumenal domain of UCE based on the structure of the EFG-3 and -4 domains of the extracellular segment of the beta chain of integrin alpha Vbeta 3. In this model the EGF-like domains of UCE (residues 285-345) are predicted to form a rod-shaped stalk region, similar to the stem region in Golgi glycosyltransferases. This stalk causes the proposed catalytic domain (residues 1-277) to be extended away from the Golgi membrane. A portion of the proposed catalytic domain (residues 85-256) resides in Cluster of Orthologous Group (COG) 4632 with four bacterial proteins but is not homologous to any known eukaryotic proteins. Thus, UCE may have evolved from the fusion of a unique catalytic domain with a common EGF-like stalk domain. We have determined by mass spectrometry that the four disulfide bonds of the proposed catalytic domain are located between Cys(2)-Cys(172), Cys(66)-Cys(99), Cys(83)-Cys(274), and Cys(258)-Cys(265). Finally, we determined that four of the six potential N-linked glycosylation sites are glycosylated (Asn 159, Asn 165, Asn 247, and Asn 317) in COS cells.

Amino Acid Sequence↗
PubMed2006

Structural features of the reprolysin atrolysin C and tissue inhibitors of metalloproteinases (TIMPs) interaction.

Atrolysin C is a P-I snake venom metalloproteinase (SVMP) from Crotalus atrox venom, which efficiently degrades capillary basement membranes, extracellular matrix, and cell surface proteins to produce hemorrhage. The tissue inhibitors of metalloproteinases (TIMPs) are effective inhibitors of matrix metalloproteinases which share some structural similarity with the SVMPs. In this work, we evaluated the inhibitory profile of TIMP-1, TIMP-2, and the N-terminal domain of TIMP-3 (N-TIMP-3) on the proteolytic activity of atrolysin C and analyzed the structural requirements and molecular basis of inhibitor-enzyme interaction using molecular modeling. While TIMP-1 and TIMP-2 had no inhibitory activity upon atrolysin C, the N-terminal domain of TIMP-3 (N-TIMP-3) was a potent inhibitor with a K(i) value of approximately 150nM. The predicted docking structures of atrolysin C and TIMPs were submitted to molecular dynamics simulations and the complex atrolysin C/N-TIMP-3 was the only one that maintained the inhibitory conformation. This study is the first to shed light on the structural determinants required for the interaction between a SVMP and a TIMP, and suggests a structural basis for TIMP-3 inhibitory action and related proteins such as the ADAMs.

Amino Acid Sequence↗
PubMed2004

Crystallographic studies on structural features that determine the enzymatic specificity and potency of human angiogenin: Thr44, Thr80, and residues 38-41.

Human angiogenin (Ang) is a potent inducer of blood vessel formation and is a member of the pancreatic ribonuclease superfamily. Its enzymatic activity is unusually weak and biased toward cleavage after cytidine nucleotides. As part of an ongoing investigation into the structural basis of Ang's characteristic activity, we have determined the crystal structures of three Ang variants having novel activity. (i) The structure of T44D-Ang indicates that Asp44 can participate directly in pyrimidine binding and that the intrinsic hydrogen-bonding capability of this residue largely governs the pyrimidine specificity of this variant. Unexpectedly, the mutation also causes the most extensive disruption of the C-terminus seen in any Ang variant thus far. This allows the side chain of Arg101 to penetrate the B(1) site, raising the possibility that it participates in substrate binding as occurs in ribonuclease 4. (ii) The structure of T80A-Ang supports the view that Thr80 plays little role in maintaining the obstructive conformation of the C-terminus and that its participation in a hydrogen bond with Thr44 selectively weakens the interaction between Thr44 and N3 of cytosine. (iii) ARH-II is an angiogenin/RNase A chimera in which residues 38-41 of Ang are replaced with the corresponding residues (38-42) of RNase A. Its structure suggests that the guest segment influences catalysis by subtle means, possibly by reducing the pK(a) of the catalytic lysine. The loss of angiogenic activity is not attributable to disruption of known cell-binding or nuclear translocation sites but may be a consequence of the chimera's enhanced ribonucleolytic activity.

Alanine↗
PubMed1999

Characterization of structural features important for T7 RNAP elongation complex stability reveals competing complex conformations and a role for the non-template strand in RNA displacement.

We have characterized the roles of the phage T7 RNA polymerase (RNAP) thumb subdomain and the RNA binding activity of the N-terminal domain in elongation complex (EC) stability by evaluating how disrupting these structures affects the dissociation rates of halted ECs. Our results reveal distinct roles for these elements in EC stabilization. On supercoiled or partially single-stranded templates the enzyme with a deletion of the thumb subdomain is exceptionally unstable. However, on linear duplex templates the polymerase which has been proteolytically cleaved within the N-terminal domain is the most unstable. The differences in the effects of these RNAP modifications on the stability of ECs on the different templates appear to be due to differences in EC structure: on the linear duplex templates the RNA is properly displaced from the DNA, but on the supercoiled or partially single-stranded templates an extended RNA:DNA hybrid makes a larger contribution to the conformational state of the EC. The halted EC can therefore exist either in a conformation in which the RNA is displaced from the DNA and forms an interaction with the RNAP, or in a conformation in which a more extended RNA:DNA hybrid is present and the RNA:RNAP interaction is less extensive. The partitioning between these competing conformations appears to be a function of the energetics of template reannealing and the relative strengths of the RNA:RNAP interaction and the RNA:DNA hybrid.

Bacteriophage T7↗
PubMed2003

Unique structural features of a BCL-2 family protein CED-9 and biophysical characterization of CED-9/EGL-1 interactions.

The interactions between B-cell lymphoma 2 (BCL-2) family members are known to be mediated through the binding of the BH3 domain of a proapoptotic member to the BH3-binding groove of an antiapoptotic member. We determined the crystal structure of antiapoptotic CED-9, which reveals a unique C-terminal helix altering the common BH3-binding region. A coexpression system to produce CED-9 in complex with proapoptotic EGL-1 enabled us to show that the binding of EGL-1 to CED-9 is extremely stable, raising the melting temperature (T(M)) of CED-9 by 25 degrees C, and that the binding surface of CED-9 extends beyond the BH3-binding region and reaches the BH4 domain. Consistently, the T(M) and a 1H-15N correlation NMR spectrum of CED-9 in complex with EGL-1 are drastically different from those of CED-9 in complex with the EGL-1 BH3 peptide. The data suggest that the recognition between other BCL-2 family members may also involve much wider protein surfaces than is previously thought.

Animals↗
PubMed1996

Characterization of carbohydrate structural features recognized by anti-arabinogalactan-protein monoclonal antibodies.

Arabinogalactan-proteins (AGPs) are a diverse class of plant cell surface proteoglycans implicated in a range of fundamental processes associated with plant cell development. Anti-AGP monoclonal antibodies have been used extensively for the investigation of the developmental regulation of AGPs although virtually nothing is known about the structure of the carbohydrate epitopes recognised by these antibodies. In this report, a series of methyl glycosides of monosaccharides and a range of oligosaccharides that are elements of the carbohydrate component of AGPs have been investigated for recognition by previously derived anti-AGP monoclonal antibodies. No clear evidence was obtained for the involvement of terminal arabinofuranosides, nor of the galactan backbone, in the recognition of the glycan structure of AGPs by any of the antibodies used in this study. Interestingly, the most effective inhibitor of the binding of the monoclonal antibodies MAC207, JIM4 and JIM13 to exudate gum antigens was an acidic trisaccharide, isolated from a partial acid hydrolysate of gum karaya which has the structure: GlcA beta(1-->3) GalA alpha(1-->2)Rha, determined by a combination of FAB-MS, GC-MS and NMR spectroscopy.

Antibodies, Monoclonal↗
PubMed2000

Major groove recognition by three-stranded beta-sheets: affinity determinants and conserved structural features.

We present the results of a rational mutagenesis and binding-affinity study of the three-stranded beta-sheet-DNA interface in the complex formed by the amino-terminal DNA-binding domain of the Tn916 integrase protein and its cognate binding site. The relative importance of interfacial contacts present in its NMR-derived solution structure have been tested through mutagenesis, fluorescence anisotropy, and intrinsic quenching DNA-binding assays. We find that seven protein-DNA hydrogen bonds (two base-specific and five to phosphate groups) significantly contribute to the level of affinity. These interactions span the entire DNA-binding surface on the protein, but primarily originate from residues in only two strands of the sheet and loop L2. Interestingly, we show that highly populated, precisely defined intermolecular hydrogen bonds in the ensemble of conformers are invariably important for DNA-binding, implying that NMR-derived solution structures provide direct insight into the energetics of recognition. Unusual three-stranded beta-sheet-DNA interfaces have recently been discovered in three unrelated protein-DNA complexes. A comparative analysis of these structures reveals similar sheet positioning, the presence of two invariant interfacial contacts to the phosphodiester backbone, and two semi-conserved base-specific hydrogen bonds. Two of these conserved contacts significantly contribute to the affinity of the integrase-DNA complex, suggesting that the three-stranded beta-sheet DNA-binding motif exhibits conserved principles of recognition.

Amino Acid Motifs↗
PubMedDate not supplied

Focus on modified microcins: structural features and mechanisms of action.

Microcins are gene-encoded antimicrobial (poly)peptides secreted by Enterobacteriaceae. Produced under conditions of nutrient depletion, they are active against phylogenetically related microbial strains. Therefore, they are considered to play an important role in the microbial competitions within the intestinal flora. Among the limited sample of nine microcins hitherto described, a wide variety of structures and modes of action could be identified. The knowledge on microcins is very uneven, some being extensively studied, and others remaining uncharacterized. In this article, we have focused on a subgroup of highly modified microcins that show very original structures. We present an updated overview on the structures and mechanisms of action of microcins B17, C7 and J25, and on the associated effector proteins, also encoded by the microcin genetic system, which include specific modification enzymes, export proteins, and immunity factors.

Bacteriocins↗
PubMed2003

Structural features of arabinoxylans extracted with water at different temperatures from two rye flours of diverse breadmaking quality.

The water extractable (WE) arabinoxylans from two rye flours differing in baking quality were studied following sequential extraction with water at 4, 40, and 100 degrees C. Ammonium sulfate fractionation of the resulting WE fractions and subsequent analysis revealed substantial differences in the structure of the isolated materials. Furthermore, it allowed us to identify the factors contributing to arabinoxylan water extractability. Our results provide compelling evidence for the existence of separate polymers in rye WE arabinoxylans with different substitution degrees, ranging from quantitatively dominating, lowly substituted populations (arabinose to xylose ratio, Ara/Xyl approximately 0.5) to comparatively less abundant, highly substituted analogues (Ara/Xyl approximately 1.3). Generally, arabinoxylan water extractability was governed by the relative proportion of lowly and highly branched structures. A gradually increasing proportion of highly substituted populations was observed from cold to hot WE fractions. This was associated with the lower proportion of monosubstituted xylopyranosyl residues in the backbone, the higher proportion of disubstituted xylopyranosyl residues, and the higher level of substitution with feruloyl residues. Notable differences in the ratio of phenolic compounds to arabinose residues were observed between corresponding polymers isolated from rye flours of high and low baking quality, whereas the differences in their molecular weights were much less pronounced.

Arabinose↗
PubMedDate not supplied

The multi-structural feature of the multidrug resistance gene product P-glycoprotein: implications for its mechanism of action (hypothesis).

P-glycoprotein is a member of the ATP-binding cassette (ABC) transport superfamily. It plays an important role in the development of multidrug resistance in cancers by effluxing a wide variety of anticancer drugs. A large amount of information on the structure and function of P-glycoprotein has been accumulated over recent years from studies using molecular, biochemical, and biophysical approaches. It remains unclear, however, how this protein folds in membranes and how it transports such a wide variety of hydrophobic compounds. This paper highlights the recent progress in the structural and biogenesis aspects of P-glycoprotein. A model mechanism of P-glycoprotein action is proposed as a hypothesis that is based on recent progress in studying the topological folding of P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Limited proteolytic digestions identify common structural features of HIV-1 Tat proteins expressed during infection from alternatively spliced mRNAs.

Although multiple forms of the HIV-1 Tat protein are synthesized during infection from alternatively spliced mRNAs, only 72 amino acid residues encoded in the first Tat exon are necessary for full transactivation activity. We used limited proteolytic digestions of proteins expressed in vitro to study the structures of three Tat proteins from isolate HXB2: 72R Tat (first Tat exon); 86R Tat (first and second Tat exons), Tev or TNV (first Tat exon plus Env and Rev exons). For the 86R and Tev proteins, either trypsin or chymotrypsin cleaved the majority of carboxyl residues from the first exon. Moreover, when released from carboxyl residues, the first exon of 86R and Tev was relatively resistant to subsequent proteolysis. The entire 72R Tat protein was relatively resistant to proteolysis. The protease-resistant first exon in all Tat proteins was abolished by EDTA treatment, suggesting that divalent cations are required for its conformation. Our results suggest that the first exon in the 86R, 72R, and Tev proteins is folded into a similar structure which, as defined by partial proteolysis, acts as a single biochemical domain.

Amino Acid Sequence↗
PubMed2005

Association of the cystic fibrosis transmembrane regulator with CAL: structural features and molecular dynamics.

The association of the cystic fibrosis transmembrane regulator (CFTR) with two PDZ-containing molecular scaffolds (CAL and EBP50) plays an important role in CFTR trafficking and membrane maintenance. The CFTR-molecular scaffold interaction is mediated by the association of the C-terminus of the transmembrane regulator with the PDZ domains. Here, we characterize the structure and dynamics of the PDZ of CAL and the complex formed with CFTR employing high-resolution NMR. On the basis of NMR relaxation data, the alpha2 helix as well as the beta2-beta3 loop of CAL PDZ domain undergoes rapid dynamics. Molecular dynamics simulations suggest a concerted motion between the alpha2 helix and the beta1-beta2 and beta2-beta3 loops, elements which define the binding pocket, suggesting that dynamics may play a role in PDZ-ligand specificity. The C-terminus of CFTR binds to CAL with the final four residues (-D(-)(3)-T-R-L(0)) within the canonical PDZ-binding motif, between the beta2 strand and the alpha2 helix. The R(-)(1) and D(-)(3) side chains make a number of contacts with the PDZ domain; many of these interactions differ from those in the CFTR-EBP50 complex, suggesting sites that can be targeted in the development of PDZ-selective inhibitors that may help modulate CFTR function.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Structural features of mouse telomerase RNA are responsible for the lower activity of mouse telomerase versus human telomerase.

Human and mouse telomerases show a high degree of similarity in both the protein and RNA components. Human telomerase is more active and more processive than the mouse telomerase. There are two key differences between hTR [human TR (telomerase RNA)] and mTR (mouse TR) structures. First, the mouse telomerase contains only 2 nt upstream of its template region, whereas the human telomerase contains 45 nt. Secondly, the template region of human telomerase contains a 5-nt alignment domain, whereas that of mouse has only 2 nt. We hypothesize that these differences are responsible for the differential telomerase activities. Mutations were made in both the hTR and mTR, changing the template length and the length of the RNA upstream of the template, and telomerase was reconstituted in vitro using mouse telomerase reverse transcriptase generated by in vitro translation. We show that the sequences upstream of the template region, with a potential to form a double-stranded helix (the P1 helix) as in hTR, increase telomerase activity. The longer alignment domain increases telomerase activity only in the context of the P1 helix. Thus the TR contributes to regulating the level of activity of mammalian telomerases.

Animals↗