PredCSF: an integrated feature-based approach for predicting conotoxin superfamily

Conotoxins are small disulfide-rich peptides that are invaluable channel-targeted peptides and target neuronal receptors. They show prospects for being potent pharmaceuticals in the treatment of Alzheimer's disease, Parkinson's disease, and epilepsy. Accurate and fast prediction of conotoxin superfamily is very helpful towards the understanding of its biological and pharmacological functions especially in the post-genomic era. In the present study, we have developed a novel approach called PredCSF for predicting the conotoxin superfamily from the amino acid sequence directly based on fusing different kinds of sequential features by using modified one-versus-rest SVMs. The input features to the PredCSF classifiers are composed of physicochemical properties, evolutionary information, predicted second structure and amino acid composition, where the most important features are further screened by random forest feature selection to improve the prediction performance. The prediction results show that PredCSF can obtain an overall accuracy of 90.65% based on a benchmark dataset constructed from the most recent database, which consists of 4 main conotoxin superfamilies and 1 class of non-conotoxin class. Systematic experiments also show that combing different features is helpful for enhancing the prediction power when dealing with complex biological problems. PredCSF is expected to be a powerful tool for in silico identification of novel conotonxins and is freely available for academic use at http://www.csbio.sjtu.edu.cn/bioinf/PredCSF.     

Pseudo amino acid composition and multi-class support vector machines approach for conotoxin superfamily classification

Conotoxins are disulfide rich small peptides that target a broad spectrum of ion-channels and neuronal receptors. They offer promising avenues in the treatment of chronic pain, epilepsy and cardiovascular diseases. Assignment of newly sequenced mature conotoxins into appropriate superfamilies using a computational approach could provide valuable preliminary information on the biological and pharmacological functions of the toxins. However, creation of protein sequence patterns for the reliable identification and classification of new conotoxin sequences may not be effective due to the hypervariability of mature toxins. With the aim of formulating an in silico approach for the classification of conotoxins into superfamilies, we have incorporated the concept of pseudo-amino acid composition to represent a peptide in a mathematical framework that includes the sequence-order effect along with conventional amino acid composition. The polarity index attribute, which encodes information such as residue surface buriability, polarity, and hydropathy, was used to store the sequence-order effect. Several methods like BLAST, ISort (Intimate Sorting) predictor, least Hamming distance algorithm, least Euclidean distance algorithm and multi-class support vector machines (SVMs), were explored for superfamily identification. The SVMs outperform other methods providing an overall accuracy of 88.1% for all correct predictions with generalized squared correlation of 0.75 using jackknife cross-validation test for A, M, O and T superfamilies and a negative set consisting of short cysteine rich sequences from different eukaryotes having diverse functions. The computed sensitivity and specificity for the superfamilies were found to be in the range of 84.0-94.1% and 80.0-95.5%, respectively, attesting to the efficacy of multi-class SVMs for the successful in silico classification of the conotoxins into their superfamilies.     

Molecular Evolution and Diversity of Conus Peptide Toxins,as Revealed by Gene Structure and Intron Sequence Analyses

Cone snails, which are predatory marine gastropods, produce a cocktail of venoms used for predation, defense and competition. The major venom component, conotoxin, has received significant attention because it is useful in neuroscience research, drug development and molecular diversity studies. In this study, we report the genomic characterization of nine conotoxin gene superfamilies from 18 Conus species and investigate the relationships among conotoxin gene structure, molecular evolution and diversity. The I1, I2, M, O2, O3, P, S, and T superfamily precursors all contain three exons and two introns, while A superfamily members contain two exons and one intron. The introns are conserved within a certain gene superfamily, and also conserved across different Conus species, but divergent among different superfamilies. The intronic sequences contain many simple repeat sequences and regulatory elements that may influence conotoxin gene expression. Furthermore, due to the unique gene structure of conotoxins, the base substitution rates and the number of positively selected sites vary greatly among exons. Many more point mutations and trinucleotide indels were observed in the mature peptide exon than in the other exons. In addition, the first example of alternative splicing in conotoxin genes was found. These results suggest that the diversity of conotoxin genes has been shaped by point mutations and indels, as well as rare gene recombination or alternative splicing events, and that the unique gene structures could have made a contribution to the evolution of conotoxin genes.     

Definition of the M-conotoxin superfamily: characterization of novel peptides from molluscivorous Conus venoms

Most of the >50,000 different pharmacologically active peptides in Conus venoms belong to a small number of gene superfamilies. In this work, the M-conotoxin superfamily is defined using both biochemical and molecular criteria. Novel excitatory peptides purified from the venoms of the molluscivorous species Conus textile and Conus marmoreus all have a characteristic pattern of Cys residues previously found in the mu-, kappaM-, and psi-conotoxins (CC-C-C-CC). The new peptides are smaller (12-19 amino acids) than the mu-, kappaM-, and psi-conotoxins (22-24 amino acids). One peptide, mr3a, was chemically synthesized in a biologically active form. Analysis of the disulfide bridges of a natural peptide tx3c from C. textile and synthetic peptide mr3a from C. marmoreus showed a novel pattern of disulfide connectivity, different from that previously established for the mu- and psi-conotoxins. Thus, these peptides belong to a new group of structurally and pharmacologically distinct conotoxins that are particularly prominent in the venoms of mollusc-hunting Conus species. Analysis of cDNA clones encoding the novel peptides as well as those encoding mu-, kappaM-, and psi-conotoxins revealed highly conserved amino acid residues in the precursor sequences; this conservation in both amino acid sequence and in the Cys pattern defines a gene superfamily, designated the M-conotoxin superfamily. The peptides characterized can be provisionally assigned to four distinct groups within the M-superfamily based on sequence similarity within and divergence between each group. A notable feature of the superfamily is that two distinct structural frameworks have been generated by changing the disulfide connectivity on an otherwise conserved Cys pattern.     

I-conotoxin superfamily revisited.

The I-conotoxin superfamily (I-Ctx) is known to have four disulfide bonds with the cysteine arrangement C-C-CC-CC-C-C, and the members inhibit or modify ion channels of nerve cells. Recently, Olivera and co-workers (FEBS J. 2005; 272: 4178-4188) have suggested that the previously described I-Ctx should now be divided into two different gene superfamilies, namely, I1 and I2, in view of their having two different types of signal peptides and exhibiting distinct functions. We have revisited the 28 entries presently grouped as I-Ctx in UniProt Swiss-Prot knowledgebase, and on the basis of in silico analysis have divided them into I1 and I2 superfamilies. The sequence analysis has provided a framework for in silico annotation enabling us to carry out computer-based functional characterization of the UniProtKB/TrEMBL entry Q59AA4 from Conus miles and to predict it as a member of the I2 superfamily. Furthermore, we have predicted the mature toxin of this entry and have proposed that it may be an inhibitor of voltage-gated potassium channels.     

Conotoxin GI: disulfide bridges, synthesis, and preparation of iodinated derivatives

The 13 amino acid toxic peptide from the marine snail Conus geographus, conotoxin GI, blocks the acetylcholine receptor at the neuromuscular junction. In this report, we describe a method for analyzing disulfide bonding in nanomole amounts of small cystine-rich peptides. The procedure involves partial reduction and a double-label alkylation of cysteine residues. Using this method, we show that the natural conotoxin GI has a (2-7, 3-13) disulfide configuration. The structure of conotoxin GI has been confirmed by chemical synthesis. The preparation and purification of molecularly homogeneous, iodinated derivatives of this toxin are also described. All derivatives, including the [diiodohistidine,diiodotyrosine]conotoxin GI, retained at least half of the biological activity of unmodified toxin. Since the tetraiodinated toxin, which is greater than 25% by weight iodine, retains considerable toxicity, unmodified histidine and tyrosine residues in conotoxin GI are not crucial for biological activity.     

Three-dimensional structure of conotoxin tx3a: An m-1 branch peptide of the M-superfamily

The M-superfamily, one of eight major conotoxin superfamilies found in the venom of the cone snail, contains a Cys framework with disulfide-linked loops labeled 1, 2, and 3 (-CC (1) C (2) C (3) CC-). M-Superfamily conotoxins can be divided into the m-1, -2, -3, and -4 branches, based upon the number of residues located in the third Cys loop between the fourth and fifth Cys residues. Here we provide a three-dimensional solution structure for the m-1 conotoxin tx3a found in the venom of Conus textile. The 15-amino acid peptide, CCSWDVCDHPSCTCC, has disulfide bonds between Cys (1) and Cys (14), Cys (2) and Cys (12), and Cys (7) and Cys (15) typical of the C1-C5, C2-C4, and C3-C6 connectivity pattern seen in m-1 branch peptides. The tertiary structure of tx3a was determined by two-dimensional (1)H NMR in combination with the combined assignment and dynamics algorithm for nuclear magnetic resonance (NMR) applications CYANA program. Input for structure calculations consisted of 62 inter- and intraproton, five phi angle, and four hydrogen bond constraints. The root-mean-square deviation values for the 20 final structures are 0.32 +/- 0.07 and 0.84 +/- 0.11 A for the backbone and heavy atoms, respectively. Surprisingly, the structure of tx3a has a "triple-turn" motif seen in the m-2 branch conotoxin mr3a, which is absent in mr3e, the only other member of the m-1 branch of the M-superfamily whose structure is known. Interestingly, injection of tx3a into mice elicits an excitatory response similar to that of the m-2 branch peptide mr3a, even though the conotoxins have different disulfide connectivity patterns.     

The A-superfamily of conotoxins: structural and functional divergence

The generation of functional novelty in proteins encoded by a gene superfamily is seldom well documented. In this report, we define the A-conotoxin superfamily, which is widely expressed in venoms of the predatory cone snails (Conus), and show how gene products that diverge considerably in structure and function have arisen within the same superfamily. A cDNA clone encoding alpha-conotoxin GI, the first conotoxin characterized, provided initial data that identified the A-superfamily. Conotoxin precursors in the A-superfamily were identified from six Conus species: most (11/16) encoded alpha-conotoxins, but some (5/16) belong to a family of excitatory peptides, the kappaA-conotoxins that target voltage-gated ion channels. alpha-Conotoxins are two-disulfide-bridged nicotinic antagonists, 13-19 amino acids in length; kappaA-conotoxins are larger (31-36 amino acids) with three disulfide bridges. Purification and biochemical characterization of one peptide, kappaA-conotoxin MIVA is reported; five of the other predicted conotoxins were previously venom-purified. A comparative analysis of conotoxins purified from venom, and their precursors reveal novel post-translational processing, as well as mutational events leading to polymorphism. Patterns of sequence divergence and Cys codon usage define the major superfamily branches and suggest how these separate branches arose.     

Structure-Based Prediction of the Peptide Sequence Space Recognized by Natural and Synthetic PDZ Domains

Protein-protein recognition, frequently mediated by members of large families of interaction domains, is one of the cornerstones of biological function. Here, we present a computational, structure-based method to predict the sequence space of peptides recognized by PDZ domains, one of the largest families of recognition proteins. As a test set, we use a considerable amount of recent phage display data that describe the peptide recognition preferences for 169 naturally occurring and engineered PDZ domains. For both wild-type PDZ domains and single point mutants, we find that 70-80% of the most frequently observed amino acids by phage display are predicted within the top five ranked amino acids. Phage display frequently identified recognition preferences for amino acids different from those present in the original crystal structure. Notably, in about half of these cases, our algorithm correctly captures these preferences, indicating that it can predict mutations that increase binding affinity relative to the starting structure. We also find that we can computationally recapitulate specificity changes upon mutation, a key test for successful forward design of protein-protein interface specificity. Across all evaluated data sets, we find that incorporation backbone sampling improves accuracy substantially, irrespective of using a crystal or NMR structure as the starting conformation. Finally, we report successful prediction of several amino acid specificity changes from blind tests in the DREAM4 peptide recognition domain specificity prediction challenge. Because the foundational methods developed here are structure based, these results suggest that the approach can be more generally applied to specificity prediction and redesign of other protein-protein interfaces that have structural information but lack phage display data.     

A new level of conotoxin diversity,a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity

alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NMR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.     

Use of proline-specific endopeptidase in the isolation of all four "native" disulfides of hen egg white lysozyme

The disulfide peptides from the tryptic digestion of cyanogen bromide-treated hen egg white lysozyme (HEWL) were isolated by reverse phase high performance liquid chromatography (HPLC) and identified by amino acid analysis. Three peptides containing the I-VIII, II-VII, and III-V + IV-VI disulfide bonds were obtained. The two-disulfide peptide was further digested with proline-specific endopeptidase (PCE) (EC 3.4.21.26). Amino acid analysis of digest peptides separated by HPLC showed four peptides with the IV-VI disulfide bond as well as a peptide with the III-V disulfide bond. The IV-VI peptides were produced by hydrolysis of several alanine-X bonds as well as the prolyl-cystine bond. Our studies show that alanyl peptide bonds to lysyl, seryl, and leucyl residues are susceptible to hydrolysis by PCE preparations, thus substantially extending its known specificity range. The two-disulfide peptide was also digested sequentially with thermolysin and PCE; the resulting IV-VI and III-V peptides were identified by HPLC and amino acid analysis. PCE showed substantial activity at pH 5.3 as well as at pH 8.3. The lower pH is useful in studies of proteins or peptides where base-catalyzed reactions must be limited.     

Characterization of D-amino-acid-containing excitatory conotoxins and redefinition of the I-conotoxin superfamily

Post-translational isomerization of l-amino acids to d-amino acids is a subtle modification, not detectable by standard techniques such as Edman sequencing or MS. Accurate predictions require more sequences of modified polypeptides. A 46-amino-acid-long conotoxin, r11a, belonging to the I-superfamily was previously shown to have a d-Phe residue at position 44. In this report, we characterize two related peptides, r11b and r11c, with d-Phe and d-Leu, respectively, at the homologous position. Electrophysiological tests show that all three peptides induce repetitive activity in frog motor nerve, and epimerization of the single amino acid at the third position from the C-terminus attenuates the potency of r11a and r11b, but not that of r11c. Furthermore, r11c (but neither r11a nor r11b) also acts on skeletal muscle. We identified more cDNA clones encoding conopeptide precursors with Cys patterns similar to r11a/b/c. Although the predicted mature toxins have the same cysteine patterns, they belong to two different gene superfamilies. A potential correlation between the identity of the gene superfamily to which the I-conotoxin belongs and the presence or absence of a d-amino acid in the primary sequence is discussed. The great diversity of I-conopeptide sequences provides a rare opportunity for defining parameters that may be important for this most stealthy of all post-translational modifications. Our results indicate that neither the chemical nature of the side chain nor the precise vicinal sequence around the modified residue seem to be critical, but there may be favored loci for isomerization to a d-amino acid.     

Alpha S-conotoxin RVIIIA: a structurally unique conotoxin that broadly targets nicotinic acetylcholine receptors

We report the purification and characterization of a new conotoxin from the venom of Conus radiatus. The peptide, alphaS-conotoxin RVIIIA (alphaS-RVIIIA), is biochemically unique with respect to its amino acid sequence, post-translational modification, and molecular targets. In comparison to other nicotinic antagonists from Conus venoms, alphaS-RVIIIA exhibits an unusually broad targeting specificity for nicotinic acetylcholine receptor (nAChR) subtypes, as assayed by electrophysiology. The toxin is paralytic to mice and fish, consistent with its nearly irreversible block of the neuromuscular nAChR. Similar to other antagonists of certain neuronal nAChRs, the toxin also elicits seizures in mice upon intracranial injection. The only previously characterized conotoxin from the S superfamily, sigma-conotoxin GVIIIA, is a specific competitive antagonist of the 5-HT3 receptor; thus, alphaS-RVIIIA defines a novel family of nicotinic antagonists within the S superfamily. All previously characterized competitive conotoxin nAChR antagonists have been members of the A superfamily of conotoxins. Our working hypothesis is that the particular group of fish-hunting Conus species that includes Conus radiatus uses the alphaS-conotoxin family to target the muscle nAChR and paralyze prey.     

Superfamily active site templates

We show that three-dimensional signatures consisting of only a few functionally important residues can be diagnostic of membership in superfamilies of enzymes. Using the enolase superfamily as a model system, we demonstrate that such a signature, or template, can identify superfamily members in structural databases with high sensitivity and specificity. This is remarkable because superfamilies can be highly diverse, with members catalyzing many different overall reactions; the unifying principle can be a conserved partial reaction or chemical capability. Our definition of a superfamily thus hinges on the disposition of residues involved in a conserved function, rather than on fold similarity alone. A clear advantage of basing structure searches on such active site templates rather than on fold similarity is the specificity with which superfamilies with distinct functional characteristics can be identified within a large set of proteins with the same fold, such as the (beta/alpha)8 barrels. Preliminary results are presented for an additional group of enzymes with a different fold, the haloacid dehalogenase superfamily, suggesting that this approach may be generally useful for assigning reading frames of unknown function to specific superfamilies and thereby allowing inference of some of their functional properties.     

A novel conotoxin inhibitor of Kv1.6 channel and nAChR subtypes defines a new superfamily of conotoxins

Using assay-directed fractionation of the venom from the vermivorous cone snail Conus planorbis, we isolated a new conotoxin, designated pl14a, with potent activity at both nicotinic acetylcholine receptors and a voltage-gated potassium channel subtype. pl14a contains 25 amino acid residues with an amidated C-terminus, an elongated N-terminal tail (six residues), and two disulfide bonds (1-3, 2-4 connectivity) in a novel framework distinct from other conotoxins. The peptide was chemically synthesized, and its three-dimensional structure was demonstrated to be well-defined, with an alpha-helix and two 3(10)-helices present. Analysis of a cDNA clone encoding the prepropeptide precursor of pl14a revealed a novel signal sequence, indicating that pl14a belongs to a new gene superfamily, the J-conotoxin superfamily. Five additional peptides in the J-superfamily were identified. Intracranial injection of pl14a in mice elicited excitatory symptoms that included shaking, rapid circling, barrel rolling, and seizures. Using the oocyte heterologous expression system, pl14a was shown to inhibit both a K+ channel subtype (Kv1.6, IC50 = 1.59 microM) and neuronal (IC50 = 8.7 microM for alpha3beta4) and neuromuscular (IC50 = 0.54 microM for alpha1beta1 epsilondelta) subtypes of the nicotinic acetylcholine receptor (nAChR). Similarities in sequence and structure are apparent between the middle loop of pl14a and the second loop of a number of alpha-conotoxins. This is the first conotoxin shown to affect the activity of both voltage-gated and ligand-gated ion channels.     

Enhancing In Silico Protein-Based Vaccine Discovery for Eukaryotic Pathogens Using Predicted Peptide-MHC Binding and Peptide Conservation Scores

Given thousands of proteins constituting a eukaryotic pathogen, the principal objective for a high-throughput in silico vaccine discovery pipeline is to select those proteins worthy of laboratory validation. Accurate prediction of T-cell epitopes on protein antigens is one crucial piece of evidence that would aid in this selection. Prediction of peptides recognised by T-cell receptors have to date proved to be of insufficient accuracy. The in silico approach is consequently reliant on an indirect method, which involves the prediction of peptides binding to major histocompatibility complex (MHC) molecules. There is no guarantee nevertheless that predicted peptide-MHC complexes will be presented by antigen-presenting cells and/or recognised by cognate T-cell receptors. The aim of this study was to determine if predicted peptide-MHC binding scores could provide contributing evidence to establish a protein’s potential as a vaccine. Using T-Cell MHC class I binding prediction tools provided by the Immune Epitope Database and Analysis Resource, peptide binding affinity to 76 common MHC I alleles were predicted for 160 Toxoplasma gondii proteins: 75 taken from published studies represented proteins known or expected to induce T-cell immune responses and 85 considered less likely vaccine candidates. The results show there is no universal set of rules that can be applied directly to binding scores to distinguish a vaccine from a non-vaccine candidate. We present, however, two proposed strategies exploiting binding scores that provide supporting evidence that a protein is likely to induce a T-cell immune response–one using random forest (a machine learning algorithm) with a 72% sensitivity and 82.4% specificity and the other, using amino acid conservation scores with a 74.6% sensitivity and 70.5% specificity when applied to the 160 benchmark proteins. More importantly, the binding score strategies are valuable evidence contributors to the overall in silico vaccine discovery pool of evidence.     

Secondary structure formations of conotoxin genes: a possible role in mediating variability

Small venomous peptides called conotoxins produced by the predatory marine snail (genus Conus) present an interesting case for mutational studies. They have a high degree of amino acid variability among them yet they possess highly conserved structural elements that are defined by cysteine residues forming disulfide bridges along the length of the mature peptide. It has been observed that codons specifying these cysteines are also highly conserved. It is unknown how such codon conservation is maintained within the mature conotoxin gene since this entire region undergoes an accelerated rate of mutation. There is evidence suggesting that nucleic acids wield some influence in mechanisms that dictate the region and frequency where mutations occur in DNA. Nucleic acids exert this effect primarily through secondary structures that bring about local peaks and troughs in the energy relief of these transient formations. Secondary structure predictions of several conotoxin genes were analyzed to see if there was any correspondence between the highly variable regions of the conotoxin. Regions of the DNA encompassing the conserved Cys codons (and several other conserved amino acid codons) have been found to correspond to predicted secondary structures of higher stabilities. In stark contrast the regions of the conotoxin that have a higher degree of variation correlate to regions of lower stability. This striking co-relation allows for a simple model of inaccessibility of a mutator to these highly conserved regions of the conotoxin gene allowing them a relative degree of resistance towards change.