Concerted evolution of sea anemone neurotoxin genes is revealed through analysis of the Nematostella vectensis genome

Gene families, which encode toxins, are found in many poisonous animals, yet there is limited understanding of their evolution at the nucleotide level. The release of the genome draft sequence for the sea anemone Nematostella vectensis enabled a comprehensive study of a gene family whose neurotoxin products affect voltage-gated sodium channels. All gene family members are clustered in a highly repetitive approximately 30-kb genomic region and encode a single toxin, Nv1. These genes exhibit extreme conservation at the nucleotide level which cannot be explained by purifying selection. This conservation greatly differs from the toxin gene families of other animals (e.g., snakes, scorpions, and cone snails), whose evolution was driven by diversifying selection, thereby generating a high degree of genetic diversity. The low nucleotide diversity at the Nv1 genes is reminiscent of that reported for DNA encoding ribosomal RNA (rDNA) and 2 hsp70 genes from Drosophila, which have evolved via concerted evolution. This evolutionary pattern was experimentally demonstrated in yeast rDNA and was shown to involve unequal crossing-over. Through sequence analysis of toxin genes from multiple N. vectensis populations and 2 other anemone species, Anemonia viridis and Actinia equina, we observed that the toxin genes for each sea anemone species are more similar to one another than to those of other species, suggesting they evolved by manner of concerted evolution. Furthermore, in 2 of the species (A. viridis and A. equina) we found genes that evolved under diversifying selection, suggesting that concerted evolution and accelerated evolution may occur simultaneously.     

Peptide Toxins in Sea Anemones: Structural and Functional Aspects

Sea anemones are a rich source of two classes of peptide toxins, sodium channel toxins and potassium channel toxins, which have been or will be useful tools for studying the structure and function of specific ion channels. Most of the known sodium channel toxins delay channel inactivation by binding to the receptor site 3 and most of the known potassium channel toxins selectively inhibit Kv1 channels. The following peptide toxins are functionally unique among the known sodium or potassium channel toxins: APETx2, which inhibits acid-sensing ion channels in sensory neurons; BDS-I and II, which show selectivity for Kv3.4 channels and APETx1, which inhibits human ether-a-go-go-related gene potassium channels. In addition, structurally novel peptide toxins, such as an epidermal growth factor (EGF)-like toxin (gigantoxin I), have also been isolated from some sea anemones although their functions remain to be clarified.     

Hg1, novel peptide inhibitor specific for Kv1.3 channels from first scorpion Kunitz-type potassium channel toxin family

The potassium channel Kv1.3 is an attractive pharmacological target for autoimmune diseases. Specific peptide inhibitors are key prospects for diagnosing and treating these diseases. Here, we identified the first scorpion Kunitz-type potassium channel toxin family with three groups and seven members. In addition to their function as trypsin inhibitors with dissociation constants of 140 nM for recombinant LmKTT-1a, 160 nM for LmKTT-1b, 124 nM for LmKTT-1c, 136 nM for BmKTT-1, 420 nM for BmKTT-2, 760 nM for BmKTT-3, and 107 nM for Hg1, all seven recombinant scorpion Kunitz-type toxins could block the Kv1.3 channel. Electrophysiological experiments showed that six of seven scorpion toxins inhibited ~50-80% of Kv1.3 channel currents at a concentration of 1 μM. The exception was rBmKTT-3, which had weak activity. The IC(50) values of rBmKTT-1, rBmKTT-2, and rHg1 for Kv1.3 channels were ~129.7, 371.3, and 6.2 nM, respectively. Further pharmacological experiments indicated that rHg1 was a highly selective Kv1.3 channel inhibitor with weak affinity for other potassium channels. Different from classical Kunitz-type potassium channel toxins with N-terminal regions as the channel-interacting interfaces, the channel-interacting interface of Hg1 was in the C-terminal region. In conclusion, these findings describe the first scorpion Kunitz-type potassium channel toxin family, of which a novel inhibitor, Hg1, is specific for Kv1.3 channels. Their structural and functional diversity strongly suggest that Kunitz-type toxins are a new source to screen and design potential peptides for diagnosing and treating Kv1.3-mediated autoimmune diseases.     

Potentiation and cellular phenotypes of the insecticidal Toxin complexes of Photorhabdus bacteria

The toxin complex (tc) genes of bacteria comprise a large and growing family whose mode of action remains obscure. In the insect pathogen Photorhabdus, tc genes encode high molecular weight insecticidal toxins with oral activity against caterpillar pests. One protein, TcdA, has recently been expressed in transgenic plants and shown to confer insect resistance. These toxins therefore represent alternatives to toxins from Bacillus thuringiensis (Bt) for deployment in transgenic crops. Levels of TcdA expression in transgenic plants were, however, low and the full toxicity associated with the native toxin was not reconstituted. Here we show that increased activity of the toxin TcdA1 requires potentiation by either of two pairs of gene products, TcdB1 and TccC1 or TcdB2 and TccC3. Moreover, these same pairs of proteins can also cross-potentiate a second toxin, TcaA1B1. To elucidate the likely functional domains present in these large proteins, we expressed fragments of each 'toxin' or 'potentiator' gene within mammalian cells. Several domains produced abnormal cellular morphologies leading to cell death, while others showed specific phenotypes such as nuclear translocation. Our results prove that the Tc toxins are complex proteins with multiple functional domains. They also show that both toxin genes and their potentiator pairs will need to be expressed to reconstitute full activity in insect-resistant transgenic plants. Moreover, they suggest that the same potentiator pair will be able to cross-potentiate more than one toxin in a single plant.     

Genomic and Structural Characterization of Kunitz-Type Peptide LmKTT-1a Highlights Diversity and Evolution of Scorpion Potassium Channel Toxins

Background

Recently, a new subfamily of long-chain toxins with a Kunitz-type fold was found in scorpion venom glands. Functionally, these toxins inhibit protease activity and block potassium channels. However, the genomic organization and three-dimensional (3-D) structure of this kind of scorpion toxin has not been reported.

Principal Findings

Here, we characterized the genomic organization and 3-D nuclear magnetic resonance structure of the scorpion Kunitz-type toxin, LmKTT-1a, which has a unique cysteine pattern. The LmKTT-1a gene contained three exons, which were interrupted by two introns located in the mature peptide region. Despite little similarity to other Kunitz-type toxins and a unique pattern of disulfide bridges, LmKTT-1a possessed a conserved Kunitz-type structural fold with one α-helix and two β-sheets. Comparison of the genomic organization, 3-D structure, and functional data of known toxins from the α-KTx, β-KTx, γ-KTx, and κ-KTx subfamily suggested that scorpion Kunitz-type potassium channel toxins might have evolved from a new ancestor that is completely different from the common ancestor of scorpion toxins with a CSα/β fold. Thus, these analyses provide evidence of a new scorpion potassium channel toxin subfamily, which we have named δ-KTx.

Conclusions/Significance

Our results highlight the genomic, structural, and evolutionary diversity of scorpion potassium channel toxins. These findings may accelerate the design and development of diagnostic and therapeutic peptide agents for human potassium channelopathies.     

BTK-2, a new inhibitor of the Kv1.1 potassium channel purified from Indian scorpion Buthus tamulus

A novel inhibitor of voltage-gated potassium channel was isolated and purified to homogeneity from the venom of the red scorpion Buthus tamulus. The primary sequence of this toxin, named BTK-2, as determined by peptide sequencing shows that it has 32 amino acid residues with six conserved cysteines. The molecular weight of the toxin was found to be 3452 Da. It was found to block the human potassium channel hKv1.1 (IC(50)=4.6 microM). BTK-2 shows 40-70% sequence similarity to the family of the short-chain toxins that specifically block potassium channels. Multiple sequence alignment helps to categorize the toxin in the ninth subfamily of the K+ channel blockers. The modeled structure of BTK-2 shows an alpha/beta scaffold similar to those of the other short scorpion toxins. Comparative analysis of the structure with those of the other toxins helps to identify the possible structure-function relationship that leads to the difference in the specificity of BTK-2 from that of the other scorpion toxins. The toxin can also be used to study the assembly of the hKv1.1 channel.     

Identification of a new RTX-like gene cluster in Vibrio cholerae

A gene cluster containing two genes in tandem has been identified in Vibrio cholerae ElTor N16961. Each has more than one cadherin domain and is homologous to the RTX toxin family and was common in various V. cholerae strains. Insertional mutagenesis demonstrated that each gene has a role in Hep-2 cell rounding, hemolytic activity towards human and sheep RBCs and biofilm formation. The mutants showed reduced adherence to intestinal epithelial cells as well as reduction of in vivo colonization in suckling mice. These two genes thus code for RTX-like toxins in V. cholerae and are associated with the pathogenecity of this organism.     

Localization of the voltage-sensor toxin receptor on KvAP

A variety of venomous animals produce small protein toxins that impair the function of voltage-dependent cation channels by affecting the motions of the voltage-sensor domains and altering the energetics of the opening of the channel. In this study, we investigate the location of the receptor for tarantula venom voltage-sensor toxins on the voltage-dependent K+ channel from Aeropyrum pernix (KvAP), an archeabacterial channel that is functionally inhibited by members of this toxin family. We show that it is possible to purify the same set of toxins from venom of the tarantula Grammostola spatulata using either the purified KvAP voltage-sensor domain or the full-length KvAP channel. The equivalence of toxin retention profiles for the two channel proteins implies that the tarantula voltage-sensor toxin receptor resides exclusively on the voltage-sensor domain and that the pore is not required for the toxin-channel interaction. We have identified and characterized the functional properties of a subset of the tarantula toxins that bind to the KvAP voltage-sensor domain. Some of these toxins, VSTX1 and GSMTX4, have been previously isolated, while others, VSTX2 and VSTX3, are new members of the tarantula voltage-sensor toxin family. Some but not all toxins that bind to the voltage-sensor domain affect voltage-dependent gating of KvAP channels in lipid membranes.     

A single conserved basic residue in the potassium channel filter region controls KCNQ1 insensitivity toward scorpion toxins

Although many studies concerning the sensitivity mechanism of scorpion toxin-potassium channel interactions have been reported, few have explored the biochemical insensitivity mechanisms of potassium channel receptors toward natural scorpion toxin peptides, such as the KCNQ1 channel. Here, by sequence alignment analyses of the human KCNQ1 channel and scorpion potassium channel MmKv2, which is completely insensitive to scorpion toxins, we proposed that the insensitivity mechanism of KCNQ1 toward natural scorpion toxins might involve two functional regions, the turret and filter regions. Based on this observation, a series of KCNQ1 mutants were constructed to study molecular mechanisms of the KCNQ1 channel insensitivity toward natural scorpion toxins. Electrophysiological studies of chimera channels showed that the channel filter region controls KCNQ1 insensitivity toward the classical scorpion toxin ChTX. Interestingly, further residue mutant experiments showed that a single basic residue in the filter region determined the insensitivity of KCNQ1 channels toward scorpion toxins. Our present work showed that amino acid residue diversification at common sites controls the sensitivity and insensitivity of potassium channels toward scorpion toxins. The unique insensitivity mechanism of KCNQ1 toward natural scorpion toxins will accelerate the rational design of potent peptide inhibitors toward this channel.     

Kv1.3 potassium channel-blocking toxin Ctri9577, novel gating modifier of Kv4.3 potassium channel from the scorpion toxin family

Scorpion toxin Ctri9577, as a potent Kv1.3 channel blocker, is a new member of the α-KTx15 subfamily which are a group of blockers for Kv4.x potassium channels. However, the pharmacological function of Ctri9577 for Kv4.x channels remains unknown. Scorpion toxin Ctri9577 was found to effectively inhibit Kv4.3 channel currents with IC₅₀ value of 1.34 ± 0.03 μM. Different from the mechanism of scorpion toxins as the blocker recognizing channel extracellular pore entryways, Ctri9577 was a novel gating modifier affecting voltage dependence of activation, steady-state inactivation, and the recovery process from the inactivation of Kv4.3 channel. However, Ctri9755, as a potent Kv1.3 channel blocker, was found not to affect voltage dependence of activation of Kv1.3 channel. Interestingly, pharmacological experiments indicated that 1 μM Ctri9755 showed less inhibition on Kv4.1 and Kv4.2 channel currents. Similar to the classical gating modifier of spider toxins, Ctri9577 was shown to interact with the linker between the transmembrane S3 and S4 helical domains through the mutagenesis experiments. To the best of our knowledge, Ctri9577 was the first gating modifier of potassium channels among scorpion toxin family, and the first scorpion toxin as both gating modifier and blocker for different potassium channels. These findings further highlighted the structural and functional diversity of scorpion toxins specific for the potassium channels.     

Structural and functional consequences of the presence of a fourth disulfide bridge in the scorpion short toxins: solution structure of the potassium channel inhibitor HsTX1

We have determined the three-dimensional structure of the potassium channel inhibitor HsTX1, using nuclear magnetic resonance and molecular modeling. This protein belongs to the scorpion short toxin family, which essentially contains potassium channel blockers of 29 to 39 amino acids and three disulfide bridges. It is highly active on voltage-gated Kv1.3 potassium channels. Furthermore, it has the particularity to possess a fourth disulfide bridge. We show that HsTX1 has a fold similar to that of the three-disulfide-bridged toxins and conserves the hydrophobic core found in the scorpion short toxins. Thus, the fourth bridge has no influence on the global conformation of HsTX1. Most residues spatially analogous to those interacting with voltage-gated potassium channels in the three-disulfide-bridged toxins are conserved in HsTX1. Thus, we propose that Tyr21, Lys23, Met25, and Asn26 are involved in the biological activity of HsTX1. As an additional positively charged residue is always spatially close to the aromatic residue in toxins blocking the voltage-gated potassium channels, and as previous mutagenesis experiments have shown the critical role played by the C-terminus in HsTX1, we suggest that Arg33 is also important for the activity of the four disulfide-bridged toxin. Docking calculations confirm that, if Lys23 and Met25 interact with the GYGDMH motif of Kv1.3, Arg33 can contact Asp386 and, thus, play the role of the additional positively charged residue of the toxin functional site. This original configuration of the binding site of HsTX1 for Kv1.3, if confirmed experimentally, offers new structural possibilities for the construction of a molecule blocking the voltage-gated potassium channels.     

Dynamic Diversification from a Putative Common Ancestor of Scorpion Toxins Affecting Sodium, Potassium, and Chloride Channels

Scorpions have survived successfully over millions of years without detectable changes in their morphology. Instead, they have developed an efficient alomonal machinery and a stinging device supporting their needs for prey and defense. They produce a large variety of polypeptidic toxins that bind and modulate ion channel conductance in excitable tissues. The binding site, mode of action, and chemical properties of many toxins have been studied extensively, but little is known about their genomic organization and diversity. Genes representing each of the major classes of Buthidae scorpion toxins, namely, ``long' toxins, affecting sodium channels (alpha, depressant, and excitatory), and ``short' toxins, affecting potassium and chloride channels, were isolated from a single scorpion segment and analyzed. Each toxin type was found to be encoded by a gene family. Regardless of toxin length, 3-D structure, and site of action, all genes contain A+T-rich introns that split, at a conserved location, an amino acid codon of the signal sequence. The introns vary in length and sequence but display identical boundaries, agree with the GT/AG splice junctions, and contain T-runs downstream of a putative branch point, 5′-TAAT-3′. Despite little sequence similarity among all toxin classes, the conserved gene organization, intron features, and common cysteine-stabilized α-helical (CSH) core connecting an α-helix to a three-stranded β-sheet suggest, that they all evolved from an ancestral common progenitor. Furthermore, the vast diversity found among genomic copies, cDNAs, and their protein products for each toxin suggests an extensive evolutionary process of the scorpion ``pharmaceutical factory,' whose success is due, most likely, to the inherent permissiveness of the toxin exterior to structural alterations. Received: 16 March 1998 / Accepted: 30 July 1998     

Solution structure of APETx2, a specific peptide inhibitor of ASIC3 proton-gated channels

Acid-sensing ion channels (ASIC) are proton-gated sodium channels that have been implicated in pain transduction associated with acidosis in inflamed or ischemic tissues. APETx2, a peptide toxin effector of ASIC3, has been purified from an extract of the sea anemone Anthopleura elegantissima. APETx2 is a 42-amino-acid peptide cross-linked by three disulfide bridges. Its three-dimensional structure, as determined by conventional two-dimensional 1H-NMR, consists of a compact disulfide-bonded core composed of a four-stranded beta-sheet. It belongs to the disulfide-rich all-beta structural family encompassing peptide toxins commonly found in animal venoms. The structural characteristics of APETx2 are compared with that of PcTx1, another effector of ASIC channels but specific to the ASIC1a subtype and to APETx1, a toxin structurally related to APETx2, which targets the HERG potassium channel. Structural comparisons, coupled with the analysis of the electrostatic characteristics of these various ion channel effectors, led us to suggest a putative channel interaction surface for APETx2, encompassing its N terminus together with the type I-beta turn connecting beta-strands III and IV. This basic surface (R31 and R17) is also rich in aromatic residues (Y16, F15, Y32, and F33). An additional region made of the type II'-beta turn connecting beta-strands I and II could also play a role in the specificity observed for these different ion effectors.     

New binding site on common molecular scaffold provides HERG channel specificity of scorpion toxin BeKm-1

The scorpion toxin BeKm-1 is unique among a variety of known short scorpion toxins affecting potassium channels in its selective action on ether-a-go-go-related gene (ERG)-type channels. BeKm-1 shares the common molecular scaffold with other short scorpion toxins. The toxin spatial structure resolved by NMR consists of a short alpha-helix and a triple-stranded antiparallel beta-sheet. By toxin mutagenesis study we identified the residues that are important for the binding of BeKm-1 to the human ERG K+ (HERG) channel. The most critical residues (Tyr-11, Lys-18, Arg-20, Lys-23) are located in the alpha-helix and following loop whereas the "traditional" functional site of other short scorpion toxins is formed by residues from the beta-sheet. Thus the unique location of the binding site of BeKm-1 provides its specificity toward the HERG channel.     

Voltage-dependent potassium channels: minK and Shaker families

In the last 4 years, the molecular identity of several types of voltage-dependent potassium channels has been discovered. These include channels that terminate action potentials and control repetitive neuronal firing, as well as channels whose biological role is not yet understood. The majority of these are encoded by genes related to the Drosophila Shaker gene. The large number of genes comprising the Shaker gene family, coupled with the existence of different channels that result from alternatively spliced messages from the same gene, provide both vertebrates and invertebrates with a wide selection of channels whose voltage-dependence and kinetics can be tailored to the needs of a specific cell. Mutagenesis experiments on such channels are providing new information on those regions of the protein that govern essential aspects of channel activity, such as gating by voltage and ion permeation. Another gene, unrelated to the Shaker family, encodes a voltage-dependent potassium channel that activates much more slowly than the Shaker channels. This has been termed the MinK channel.     

Computational simulations of interactions of scorpion toxins with the voltage-gated potassium ion channel

Based on a homology model of the Kv1.3 potassium channel, the recognitions of the six scorpion toxins, viz. agitoxin2, charybdotoxin, kaliotoxin, margatoxin, noxiustoxin, and Pandinus toxin, to the human Kv1.3 potassium channel have been investigated by using an approach of the Brownian dynamics (BD) simulation integrating molecular dynamics (MD) simulation. Reasonable three-dimensional structures of the toxin-channel complexes have been obtained employing BD simulations and triplet contact analyses. All of the available structures of the six scorpion toxins in the Research Collaboratory for Structural Bioinformatics Protein Data Bank determined by NMR were considered during the simulation, which indicated that the conformations of the toxin significantly affect both the molecular recognition and binding energy between the two proteins. BD simulations predicted that all the six scorpion toxins in this study use their beta-sheets to bind to the extracellular entryway of the Kv1.3 channel, which is in line with the primary clues from the electrostatic interaction calculations and mutagenesis results. Additionally, the electrostatic interaction energies between the toxins and Kv1.3 channel correlate well with the binding affinities (-logK(d)s), R(2) = 0.603, suggesting that the electrostatic interaction is a dominant component for toxin-channel binding specificity. Most importantly, recognition residues and interaction contacts for the binding were identified. Lys-27 or Lys-28, residues Arg-24 or Arg-25 in the separate six toxins, and residues Tyr-400, Asp-402, His-404, Asp-386, and Gly-380 in each subunit of the Kv1.3 potassium channel, are the key residues for the toxin-channel recognitions. This is in agreement with the mutation results. MD simulations lasting 5 ns for the individual proteins and the toxin-channel complexes in a solvated lipid bilayer environment confirmed that the toxins are flexible and the channel is not flexible in the binding. The consistency between the results of the simulations and the experimental data indicated that our three-dimensional models of the toxin-channel complex are reasonable and can be used as a guide for future biological studies, such as the rational design of the blocking agents of the Kv1.3 channel and mutagenesis in both toxins and the Kv1.3 channel. Moreover, the simulation result demonstrates that the electrostatic interaction energies combined with the distribution frequencies from BD simulations might be used as criteria in ranking the binding configuration of a scorpion toxin to the Kv1.3 channel.     

Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes.

The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance.     

The tc genes of Photorhabdus: a growing family

The toxin complex (tc) genes of Photorhabdus encode insecticidal, high molecular weight Tc toxins. These toxins have been suggested as useful alternatives to those derived from Bacillus thuringiensis for expression in insect-resistant transgenic plants. Although Photorhabdus luminescens is symbiotic with nematodes that kill insects, tc genes have recently been described from other insect-associated bacteria such as Serratia entomophila, an insect pathogen, and Yersinia pestis, the causative agent of bubonic plague, which has a flea vector. Here, recent advances in our understanding of the tc gene family are reviewed in view of their potential development as insect-control agents.