Short Scorpion Toxins Research Articles

Structural analysis of the unique insecticidal activity of novel mungbean defensin VrD1 reveals possibility of homoplasy evolution between plant defensins and scorpion neurotoxins

A variety of evolutionarily related defensin molecules is found in plants and animals. Plant gamma-thionins and scorpion neurotoxins, for instance, may be categorized in this functional group, although each class recognizes a distinct receptor binding site. Such molecules are also categorized into the superfamily of cysteine-rich proteins. Plant defensins were generally believed to be involved in antimicrobial or antifungal mechanisms and, unlike scorpion toxins, little is known about whether these molecules are also endowed with the function of insect resistance. We have previously reported the isolation of a cDNA encoding a small cysteine-rich protein designated VrD1 (VrCRP) from a bruchid-resistant mungbean, which is apparently the first discovered plant defensin exhibiting in vitro and in vivo both insecticidal and antifungal activities. Our previous data also successfully demonstrated that VrD1 is toxic to E. coli and able to completely arrest the growth of Sf-21 insect cells at low concentration. However, the molecular and structural basis of this unique insecticidal activity of VrD1 is not clear. Therefore, in the present study, we use structural approach and phylogenic analysis to investigate the evolutionary and functional relations for such unique insecticidal activity. From our results, it is suggested that VrD1, in addition to gamma-thionins and several amylase inhibitors, is highly homologous to scorpion toxins, especially the short toxins. Moreover, based on the observation from our homology structures, VrD1 may utilize a newly found cluster of basic residues to achieve its insecticidal function, whereas all the other plant gamma-thionins were known to use a previously identified basic cluster conserved for gamma-thionins. Considering the general feature of short scorpion toxins to act on insect cell membranes with K(+)- or Cl(-)-channels as molecular targets, our analysis of interaction and recognition modes provides reasonable correlations between this newly found basic cluster and the insecticidal activity of VrD1, which is also comprehended as a possible link for "homoplasy evolution" between plant and animal defensin molecules.

Caste-based differences in gene expression in the polyembryonic wasp Copidosoma floridanum

The polyembryonic parasitoid Copidosoma floridanum produces two larval castes, soldiers and reproductives, during development within its host. Soldier larvae defend the brood against competitors while reproductive larvae develop into adult wasps. As with other caste-forming insects, the distinct morphological and behavioral features of soldier and reproductive larvae likely involve differential gene expression. In this study we used a bi-directional suppression subtractive hybridization (SSH) approach to isolate differentially expressed genes from C. floridanum soldier and reproductive larvae. We isolated 230 novel expressed sequence tags (ESTs) from the two subtractions (114 soldier/116 reproductive ESTs). Among these ESTs were sequences with significant similarity to genes coding for serine proteinases, proteinase inhibitors, odorant-binding and chemosensory proteins, and cuticular proteins. Also, three novel genes were isolated that resemble one another in conceptual translation and share the cysteine spacing pattern of short scorpion toxins and insect defensins. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of 20 ESTs from the two libraries indicated that 85% were differentially expressed in one caste or the other. We conclude that our SSH strategy was effective in identifying a number of genes differentially expressed in soldier and reproductive larvae and that several of these genes will be useful in characterizing caste-specific gene networks in C. floridanum.

Unique interaction of scorpion toxins with the hERG channel.

ERG potassium channels specify one component of the delayed rectifier in the heart and are likely to play an important functional role in other excitable cells. Compared to other K+ channels, the human ERG (hERG) channel possesses an unusually long S5-P linker that presumably forms an alpha-helix important for channel function. hERG-specific toxins bind to the outer mouth of the hERG channel. Channel residues in the middle of the S5-P linker and at the pore entrance are critical for toxin binding. One of these scorpion toxins is BeKm-1. Residues critical for BeKm-1 binding to the hERG channel are located in the alpha-helix and the following loop, whereas the "traditional" interaction surface of other short scorpion toxins is formed by residues on the beta-sheet. This unique localization of BeKm-1's interaction surface and its specific action on the hERG channel suggest a unique outer mouth structure of the hERG channel. We used the mutant cycle analysis approach to define contacts in the toxin-channel complex. This information provides critical constraints and is important for molecular modeling of the hERG pore structure.

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 μM). 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 α/β 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.

Primary structure of a short toxin from the venom of a vietnamese scorpion (Buthus sp.)

The complete amino acid sequence of an important toxin (toxin 14) from the venom of a Vietnamese scorpion (Buthus occitanus sp.) has been determined, which includes 35 amino acid residues and three disulfide bridges (molecular weight, 3843 Da). The comparison of the sequence with sequences of short scorpion toxins led us to conclude that toxin 14 belongs to a novel group of toxins affecting the excitability of myelinated nerves.

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.

Role of disulfide bonds in folding and activity of leiurotoxin I: just two disulfides suffice.

The aim of this study is to investigate the contribution of each disulfide bond in the folding and function of leiurotoxin I, a short scorpion toxin that blocks small conductance K(+) channels. The structure of leiurotoxin I contains a motif conserved in all scorpion toxins, formed by a helix and a double-stranded beta-sheet and stabilized by three disulfide bridges. We synthesized three analogues, each presenting two alpha-aminobutyric acid (Abu) moieties replacing two bridged cysteine residues: LeTx1 ([Abu 3,21] Leiurotoxin I), LeTx2 ([Abu 8,26] Leiurotoxin I), and LeTx3 ([Abu 12,28] Leiurotoxin I). All three analogues fold into a major product containing two native disulfide bonds, while LeTx3 forms an additional isomer, containing non-native disulfides. In denaturing conditions, analogues LeTx2 and LeTx3 yield non-native isomers, while LeTx1 only forms the isomer with native disulfides. All isomers with native disulfides contain nativelike alpha-helical conformations and bind to synaptosomal membranes with affinities within a log of that shown by the native toxin. By contrast, the non-native LeTx3A analogue exhibits a disordered conformation and a decreased biological potency. Our results indicate that the "CxxxC, CxC" cysteine spacing, conserved in all scorpion toxins and preserved in LeTx1, may play an active role in folding, and that only two native disulfide bonds in leiurotoxin I are sufficient to preserve a nativelike and active conformation. Thus, in the scorpion toxin scaffold, modifications of conserved and interior cysteine residues may permit modulation of function, without significantly affecting folding efficiency and structure.

Variability in automated assignment of NOESY spectra and three-dimensional structure determination: a test case on three small disulfide-bonded proteins.

Three independent runs of automatic assignment and structure calculations were performed on three small proteins, calcicludine from the venom of the green mamba Dendroaspis angusticeps, kappa-conotoxin PVIIA from the purple cone Conus purpurascens and HsTX1, a short scorpion toxin from the venom of Heterometrus spinnifer. At the end of all the runs, the number of cross peaks which remained unassigned (0.6%, 1.4% and 2% for calcicludine, kappa-conotoxin and HsTX1, respectively), as well as the number of constraints which were rejected as producing systematic violations (2.7%, 1.0%, and 1.4% for calcicludine, kappa-conotoxin and HsTX1, respectively) were low. The conformation of the initial model used in the procedure (linear model or constructed by homology) has no influence on the final structures. Mainly two parameters control the procedure: the chemical shift tolerance and the cut-off distance. Independent runs of structure calculations, using the same parameters, yield structures for which the rmsd between averaged structures and the rmsd around each averaged structure were of the same order of magnitude. A different cut-off distance and a different chemical shift tolerance yield rmsd values on final average structures which did not differ more than 0.5 A compared to the rmsd obtained around the averaged structure for each calculation. These results show that the procedure is robust when applied to such a small disulfide-bonded protein.

Isolation and characterization of a novel lepidopteran-selective toxin from the venom of South Indian red scorpion, Mesobuthus tamulus

BackgroundScorpion venom contains insect and mammal selective toxins. We investigated the venom of the South Indian red scorpion, Mesobuthus tamulus for the purpose of identifying potent insecticidal peptide toxins.ResultsA lepidopteran-selective toxin (Buthus tamulus insect toxin; ButaIT) has been isolated from this venom. The primary structure analysis reveals that it is a single polypeptide composed of 37 amino acids cross-linked by four disulfide bridges with high sequence homology to other short toxins such as Peptide I, neurotoxin P2, Lqh-8/6, chlorotoxin, insectotoxin I5A, insect toxin 15 and insectotoxin I1. Three dimensional modeling using Swiss automated protein modeling server reveals that this toxin contains a short α-helix and three antiparallel β-strands, similar to other short scorpion toxins. This toxin is selectively active on Heliothis virescens causing flaccid paralysis but was non-toxic to blowfly larvae and mice.ConclusionThis is the first report of a Heliothine selective peptide toxin. Identification of diverse insect selective toxins offer advantages in employing these peptides selectively for pest control.

Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein.

Protein-protein interacting surfaces are usually large and intricate, making the rational design of small mimetics of these interfaces a daunting problem. On the basis of a structural similarity between the CDR2-like loop of CD4 and the beta-hairpin region of a short scorpion toxin, scyllatoxin, we transferred the side chains of nine residues of CD4, central in the binding to HIV-1 envelope glycoprotein (gp120), to a structurally homologous region of the scorpion toxin scaffold. In competition experiments, the resulting 27-amino acid miniprotein inhibited binding of CD4 to gp120 with a 40 microM IC(50). Structural analysis by NMR showed that both the backbone of the chimeric beta-hairpin and the introduced side chains adopted conformations similar to those of the parent CD4. Systematic single mutations suggested that most CD4 residues from the CDR2-like loop were reproduced in the miniprotein, including the critical Phe-43. The structural and functional analysis performed suggested five additional mutations that, once incorporated in the miniprotein, increased its affinity for gp120 by 100-fold to an IC(50) of 0.1-1.0 microM, depending on viral strains. The resulting mini-CD4 inhibited infection of CD4(+) cells by different virus isolates. Thus, core regions of large protein-protein interfaces can be reproduced in miniprotein scaffolds, offering possibilities for the development of inhibitors of protein-protein interactions that may represent useful tools in biology and in drug discovery.

Chemical synthesis and structure-activity relationships of Ts kappa, a novel scorpion toxin acting on apamin-sensitive SK channel.

Tityus kappa (Ts kappa), a novel toxin from the venom of the scorpion Tityus serrulatus, is a 35-residue polypeptide cross-linked by three disulphide bridges and acts on small-conductance calcium-activated potassium channels (SK channels). Ts K was chemically synthesized using the solid-phase method and characterized. The synthetic product, sTs kappa, was indistinguishable from the natural toxin when tested in vitro in competition assay with radiolabelled apamin for binding to rat brain synaptosomes (IC50 = 3 nM). The sTs kappa was further tested in vivo for lethal activity to mice following intracerebroventricular inoculation (LD50 = 70 ng per mouse). The half-cystine pairings were formerly established by enzyme-based cleavage of sTs kappa; they were between Cys7-Cys28, Cys13-CyS33 and Cys17-Cys35, which is a disulphide bridge pattern similar to that of other short scorpion toxins. According to previous studies on SK channel-acting toxins, the putative influence of certain basic residues of Ts kappa (i.e. Arg6, Arg9, Lys18, Lys19) in its pharmacological activity was investigated using synthetic point-mutated analogues of the toxin with an Ala substitution at these positions. Data from binding assay, together with conformational analysis of the synthetic analogues by 1H-NMR, suggest that Arg6, and to a lesser extent Arg9, are important residues for an high-affinity interaction of this toxin with SK channels; interestingly these residues are located outside the alpha-helical structure, whereas the pharmacologically important basic residues from other SK channel-specific toxins had been located inside the alpha-helix.

The first chemical synthesis of a short scorpion toxin cross-linked by four disulfide bridges, maurotoxin

The first chemical synthesis of a short scorpion toxin cross-linked by four disulfide bridges, maurotoxin

Solution structure of maurotoxin, a scorpion toxin from Scorpio maurus, with high affinity for voltage‐gated potassium channels

Maurotoxin (MTX), purified from the scorpionid Scorpio maurus is a potent ligand for potassium channels. It shows a broad specificity as being active on Kv1.1 (Kd = 37 nM), Kv1.2 (Kd = 0.8 nM), Kv1.3 (Kd = 150 nM) voltage-gated potassium channels, as well as on small-conductance calcium-activated potassium channels. It has a unique disulfide pairing among the scorpion toxins family. The solution structure of MTX has been determined by 2D-NMR techniques, which led to the full description of its 3D conformation: a bended helix from residues 6 to 16 connected by a loop to a two-stranded antiparallel β sheet (residues 23 to 26 and 28 to 31). The interaction of MTX with the pore region of the Kv1.2 potassium channel has been modeled according to their charge anisotropy. The structure of MTX is similar to other short scorpion toxins despite its peculiar disulfide pairing. Its interaction with the Kv1.2 channel involves a dipole moment, which guides and orients the toxin onto the pore, toward the binding site, and which thus is responsible for the specificity. Proteins 29:321–333, 1997. © 1997 Wiley-Liss, Inc.

Solution structure of maurotoxin, a scorpion toxin fromScorpio maurus, with high affinity for voltage-gated potassium channels

Maurotoxin (MTX), purified from the scorpionid Scorpio maurus is a potent ligand for potassium channels. It shows a broad specificity as being active on Kv1.1 (Kd = 37 nM), Kv1.2 (Kd = 0.8 nM), Kv1.3 (Kd = 150 nM) voltage-gated potassium channels, as well as on small-conductance calcium-activated potassium channels. It has a unique disulfide pairing among the scorpion toxins family. The solution structure of MTX has been determined by 2D-NMR techniques, which led to the full description of its 3D conformation: a bended helix from residues 6 to 16 connected by a loop to a two-stranded antiparallel beta sheet (residues 23 to 26 and 28 to 31). The interaction of MTX with the pore region of the Kv1.2 potassium channel has been modeled according to their charge anisotropy. The structure of MTX is similar to other short scorpion toxins despite its peculiar disulfide pairing. Its interaction with the Kv1.2 channel involves a dipole moment, which guides and orients the toxin onto the pore, toward the binding site, and which thus is responsible for the specificity.

Characterization of a new family of toxin-like peptides from the venom of the scorpion Leiurus quinquestriatus hebraeus. 1H-NMR structure of leiuropeptide II.

To extend our knowledge about the structural features of short scorpion toxins, the ion-exchange fractions obtained from Leiurus quinquestriatus hebraeus venom were investigated by plasma desorption mass spectrometry in order to select low molecular mass polypeptides. Three toxin-like peptides with molecular mass close to 3 kDa, named leiuropeptides I, II and III, were purified and found devoid of any significant toxicity against mammals and insects. Their amino acid sequences revealed a cysteine pattern analogous to that of short-chain scorpion toxins. The solution structure of leiuropeptide II was determined by 2D 1H-NMR spectroscopy and indicated the presence of a helix accommodating a proline, connected to a two-standard beta-sheet by three disulfide bonds. The overall fold of leiuropeptide II is found to be similar to that of leiurotoxin I, a 31-residue toxin present in the same scorpion venom which acts on K+ channels. In order to rationalize the absence of toxicity, the electrostatic potential of leiuropeptide II was compared to that of leiurotoxin I. The peptide is characterized by a large negative zone around Glu4, Asp5 and Asp8 residues, beginning in the neighbourhood of the beta-turn and extending along the helix. In the same area, leiurotoxin I exhibits a positive surface, around Arg6 and Arg13 basic residues, which are essential for its receptor affinity.

Chemical synthesis and characterization of maurotoxin, a short scorpion toxin with four disulfide bridges that acts on K+ channels.

Maurotoxin is a toxin isolated from the venom of the Tunisian chactoid scorpion Scorpio maurus. It is a 34-amino-acid peptide cross-linked by four disulfide bridges. Maurotoxin competes with radiolabeled apamin and kaliotoxin for binding to rat-brain synaptosomes. Due to its very low concentration in venom (0.6% of the proteins), maurotoxin was chemically synthesized by means of an optimized solid-phase technique. The synthetic maurotoxin was characterized. It was lethal to mice following intracerebroventricular injection (LD50, 80 ng/mouse). The synthetic maurotoxin competed with 125I-apamin and 125I-kaliotoxin for binding to rat-brain synaptosomes with half-maximal effects at concentrations of 5 nM and 0.2 nM, respectively. Synthetic maurotoxin was tested on K+ channels and was found to block the Kv1.1, Kv1.2, and Kv1.3 currents with half-maximal blockage (IC50) at 37, 0.8 and 150 nM, respectively. Thus, maurotoxin is a scorpion toxin with four disulfide bridges that acts on K+ channels. The half-cystine pairings of synthetic maurotoxin were identified by enzymatic cleavage. The pairings were Cys3-Cys24, Cys9-Cys29, Cys13-Cys19 and Cys31-Cys34. This disulfide organization is unique among known scorpion toxins. The physicochemical and pharmacological properties of synthetic maurotoxin were indistinguishable from those of natural maurotoxin, which suggests that natural maurotoxin adopts the same half-cystine pairing pattern. The conformation of synthetic maurotoxin was investigated by means of circular dichroism spectroscopy and molecular modeling. In spite of its unusual half-cystine pairings, the synthetic-maurotoxin conformation appears to be similar to that of other short scorpion toxins.

Chemical Synthesis, Structural and Functional Characterization of Noxiustoxin, a Powerful Blocker of Lymphocyte Voltage-Dependent K + Channels

Two forms of the Centruduoides noxius scorpion noxiustoxin, containing an amidated and an acid C-terminus, were synthesized on a solid support by using Fmoc-chemistry and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) coupling. Comparison of the two synthetic forms with the native toxin by tryptic mapping and CD spectroscopy shows that noxiustoxin possesses an amidated C-terminus and the same fold as all short scorpion toxins. Patch-clamp assays on B lymphocytes demonstrate that noxiustoxin inhibits the voltage-dependent K + channels with 2 nM affinity, but does not affect the Ca 2+-activated K + channels. This toxin, because of its high affinity and specificity for voltage-gated K + channel, may provide a powerful tool in the investigation of the role(s) of these channels in the T and B lymphocyte activation and proliferation.

NMR sequential assignments and solution structure of chlorotoxin, a small scorpion toxin that blocks chloride channels.

The solution structure of chlorotoxin, a small toxin purified from the venom of the Leiurus quinquestriatus scorpion, has been determined using 2D 1H NMR spectroscopy. Analysis of the NMR data shows that the structure consists of a small three-stranded antiparallel beta-sheet packed against an alpha-helix, thereby adopting the same fold as charybdotoxin and other members of the short scorpion toxin family [Arseniev et al. (1984) FEBS Lett. 165, 57-62; Martins et al. (1990) FEBS Lett. 260, 249-253; Bontems et al. (1991) Science 254, 1521-1523]. Three disulfide bonds of chlorotoxin (Cys5-Cys28, Cys16-Cys33, and Cys20-Cys35), cross-linking the alpha-helix to the beta-sheet, follow the common pattern found in the other short scorpion toxins. The fourth disulfide bridge (Cys2-Cys19) links the small N-terminal beta strand to the rest of the molecule, in contrast to charybdotoxin where this disulfide bridge is absent and the first strand interacts with the rest of the molecule by several contacts between hydrophobic residues. Another structural difference between chlorotoxin and charybdotoxin is observed at the level of the alpha-beta turn. This difference is accompanied by a change in the electrostatic potential surface, which is largely positive at the level of this turn in chlorotoxin, whereas no such positive potential surface can be found at the same position in charybdotoxin. In the latter protein, the positive surface is formed by different charged residues situated on the solvent-exposed site of the C-terminal beta-sheet.(ABSTRACT TRUNCATED AT 250 WORDS)

Three‐dimensional structure of natural charybdotoxin in aqueous solution by 1H‐NMR Charybdotoxin possesses a structural motif found in other scorpion toxins

A 600-MHz proton NMR study of natural charybdotoxin, a toxin acting on K+ channels, is reported. The unambiguous sequential assignment of all the protons of the toxin was achieved. The analysis of NOEs and of backbone coupling constants showed the existence of an alpha-helix (residues 10-19) and of an antiparallel beta-sheet in the 26-35 part. Three-dimensional structures were generated by distance geometry, using a set of 114 interresidual calibrated constraints (63 sequential, 47 medium and long range, 4 hydrogen bonds) and 29 phi angles. These structures show that charybdotoxin is composed of a beta-sheet linked to an alpha-helix by two disulphide bridges and to an extended fragment by the third disulphide bridge. Comparison with the other known structures of long and short scorpion toxins shows that this structural motif is common to all these proteins.