Solution structure of an insect-specific neurotoxin from the New World scorpion Centruroides sculpturatus Ewing.

We report the high-resolution solution structure of the 6.3 kDa neurotoxic protein CsE-v5 from the scorpion Centruroides sculpturatus Ewing (CsE, range southwestern U.S.). This protein is the second example of an Old World-like neurotoxin isolated from the venom of this New World scorpion. However, unlike CsE-V, which is the first Old World-like toxin isolated and shows both anti-insect and anti-mammal activity, CsE-v5 shows high specificity for insect sodium channels. Sequence-specific proton NMR assignments and distance and angle constraints were obtained from 600 MHz 2D-NMR data. Distance geometry and dynamical simulated annealing refinements were performed to produce a final family of 20 structures without constraint violations, along with an energy-minimized average structure. The protein structure is well-defined (0.66 and 0.97 D rmsd for backbone and all heavy atoms, respectively) with a compact hydrophobic core and several extending loops. A large hydrophobic patch, containing four aromatic rings and other aliphatic residues, makes up a large area of one side of the protein. CsE-v5 shows secondary structural features characteristic of long-chain scorpion toxins: a two and a half-turn alpha-helix, a three-strand antiparallel beta-sheet, and four beta-turns. Among the proteins studied to date from the CsE venom, CsE-v5 is the most compact protein with nearly 50% of the amide protons having long exchange lifetimes, but CsE-v5 is unusual in that it has loop structures similar to both Old and New World toxins. Further, it also lacks prolines in its C-terminal 14 residues. It shows some important differences with respect to CsE-V not only in its primary sequence, but also in its electrostatic potential surface, especially around areas in register with residues 8, 9, 17, 18, 32, 43, and 57. The loss of anti-mammal activity in CsE-v5 and the differences in its anti-insect activity compared to that of other proteins such as CsE-V, v1, and v3 from this New World scorpion may be related to residue variations at these locations.     

NMR solution structure of BmK-betaIT, an excitatory scorpion beta-toxin without a 'hot spot' at the relevant position

BmK-betaIT (previously named as Bm32-VI in the literature), an excitatory scorpion beta-toxin, is purified from the venom of the Chinese scorpion Buthus martensii Karsch. It features a primary sequence typical of the excitatory anti-insect toxins: two contiguous Cys residues (Cys37-Cys38) and a shifted location of the fourth disulfide bridges (Cys38-Cys64), and demonstrates bioactivity characteristic of the excitatory beta-toxins. However, it is noteworthy that BmK-betaIT is not conserved with a glutamate residue at the preceding position of the third Cys residue, and is the first example having a non-glutamate residue at the relevant position in the excitatory scorpion beta-toxin subfamily. The 3D structure of BmK-betaIT is determined with 2D NMR spectroscopy and molecular modeling. The solution structure of BmK-betaIT is closely similar to those of BmK IT-AP and Bj-xtrIT, only distinct from the latter by lack of an alpha(0)-helix. The surface functional patch comparison with those of BmK IT-AP and Bj-xtrIT reveals their striking similarity in the spatial arrangement. These results infer that the functional surface of beta-toxins is composed of two binding regions and a functional site. The main binding site is consisted of hydrophobic residues surrounding the alpha(1)-helix and its preceding loop, which is common to all beta-type scorpion toxins affecting Na(+) channels. The second binding site, which determines the specificity of the toxin, locates at the C-terminus for excitatory insect beta-toxin, while rests at the beta-sheet and its linking loop for anti-mammal toxins. The functional site involved in the voltage sensor-trapping model, which characterizes the function of all beta-toxins, is the negatively charged residue Glu15.     

Primary structure of scorpion anti-insect toxins isolated from the venom of Leiurus quinquestriatus quinquestriatus

The amino acid sequences of insect-selective scorpion toxins, purified from the venom of Leiurus quinquestriatus quinquestriatus, have been determined by automatic phenyl isothiocyanate degradation of the S-carboxymethylated proteins and derived proteolytic peptides. The excitatory toxin Lqq IT1 and Lqq IT1' (70 residues) show the shift of one half-cystine from an external position, which is characteristic of anti-mammal toxins, to an internal sequence position. Lqq IT2 (61 residues) displays the half-cystine residue in position 12, common to the sequence of all known anti-mammal toxins; it induces flaccid paralysis on insects but is non-toxic for the mouse. Lqq IT2 structurally defines a new type of anti-insect toxins from scorpion venoms. CD spectra and immunological data are in agreement with this finding.     

An anti-insect toxin purified from the scorpion Androctonus australis Hector also acts on the alpha- and beta-sites of the mammalian sodium channel: sequence and circular dichroism study

A new anti-insect neurotoxin, AaH IT4, has been isolated from the venom of the North African scorpion Androctonus australis Hector. This polypeptide has a toxic effect on insects and mammals and is capable of competing with anti-insect scorpion toxins for binding to the sodium channel of insects; it also modulates the binding of alpha-type and beta-type anti-mammal scorpion toxins to the mammal sodium channel. This is the first report of a scorpion toxin able to exhibit these three kinds of activity. The molecule is composed of 65 amino acid residues and lacks methionine and, more unexpectedly, proline, which until now has been considered to play a role in the folded structure of all scorpion neurotoxins. The primary structure showed a poor homology with the sequences of other scorpion toxins; however, it had features in common with beta-type toxins. In fact, radioimmunoassays using antibodies directed to scorpion toxins representative of the main structural groups showed that there is a recognition of AaH IT4 via anti-beta-type toxin antibodies only. A circular dichroism study revealed a low content of regular secondary structures, particularly in beta-sheet structures, when compared to other scorpion toxins. This protein might be the first member of a new class of toxins to have ancestral structural features and a wide toxic range.     

Unusual binding mode of scorpion toxin BmKTX onto potassium channels relies on its distribution of acidic residues

Besides classical scorpion toxin–potassium channel binding modes, novel modes remain unknown. Here, we report a novel binding mode of native toxin BmKTX towards Kv1.3 channel. The combined experimental and computational data indicated that BmKTX-D33H analog used the classical anti-parallel β-sheet domain as the channel-interacting interface together with the conserved channel pore-blocking Lys²⁶. However, the wild-type BmKTX was found to use Arg²³ rather than Lys²⁶ as the new pore-blocking residue, and mainly adopt the turn motif between the α-helix and antiparallel β-sheet domains to recognize K_v1.3 channel. Together, these findings not only reveal that scorpion toxin–potassium channel interaction modes are more diverse than thought, but also highlight the functional role of toxin acidic residues in mediating diverse toxin–potassium channel binding modes.     

Solution structure of P01, a natural scorpion peptide structurally analogous to scorpion toxins specific for apamin-sensitive potassium channel

The venom of the North African scorpion Androctonus mauretanicus mauretanicus possesses numerous highly active neurotoxins that specifically bind to various ion channels. One of these, P05, has been found to bind specifically to calcium-activated potassium channels and also to compete with apamin, a toxin extracted from bee venom. Besides the highly potent ones, several of these peptides (including that of P01) have been purified and been found to possess only a very weak, although significant, activity in competition with apamin. The amino acid sequence of P01 shows that it is shorter than P05 by two residues. This deletion occurs within an α-helix stretch (residues 5–12). This α-helix has been shown to be involved in the interaction of P05 with its receptor via two arginine residues. These two arginines are absent in the P01 sequence. Furthermore, a proline residue in position 7 of the P01 sequence may act as an α-helix breaker. We have determined the solution structure of P01 by conventional two-dimensional ¹H nuclear magnetic resonance and show that 1) the proline residue does not disturb the α-helix running from residues 5 to 12; 2) the two arginines are topologically replaced by two acidic residues, which explains the drop in activity; 3) the residual binding activity may be due to the histidine residue in position 9; and 4) the overall secondary structure is conserved, i.e., an α-helix running from residues 5 to 12, two antiparallel stretches of β-sheet (residues 15–20 and 23–27) connected by a type I′ β-turn, and three disulfide bridges connecting the α-helix to the β-sheet.     

Functional evolution of scorpion venom peptides with an inhibitor cystine knot fold

The ICK (inhibitor cystine knot) defines a large superfamily of polypeptides with high structural stability and functional diversity. Here, we describe a new scorpion venom-derived K⁺ channel toxin (named λ-MeuKTx-1) with an ICK fold through gene cloning, chemical synthesis, nuclear magnetic resonance spectroscopy, Ca²⁺ release measurements and electrophysiological recordings. λ-MeuKTx-1 was found to adopt an ICK fold that contains a three-strand anti-parallel β-sheet and a 3₁₀-helix. Functionally, this peptide selectively inhibits the Drosophila Shaker K⁺ channel but is not capable of activating skeletal-type Ca²⁺ release channels/ryanodine receptors, which is remarkably different from the previously known scorpion venom ICK peptides. The removal of two C-terminal residues of λ-MeuKTx-1 led to the loss of the inhibitory activity on the channel, whereas the C-terminal amidation resulted in the emergence of activity on four mammalian K⁺ channels accompanied by the loss of activity on the Shaker channel. A combination of structural and pharmacological data allows the recognition of three putative functional sites involved in channel blockade of λ-MeuKTx-1. The presence of a functional dyad in λ-MeuKTx-1 supports functional convergence among scorpion venom peptides with different folds. Furthermore, similarities in precursor organization, exon–intron structure, 3D-fold and function suggest that scorpion venom ICK-type K⁺ channel inhibitors and Ca²⁺ release channel activators share a common ancestor and their divergence occurs after speciation between buthidae and non-buthids. The structural and functional characterizations of the first scorpion venom ICK toxin with K⁺ channel-blocking activity sheds light on functionally divergent and convergent evolution of this conserved scaffold of ancient origin.     

Dipole moments of scorpion toxins direct the interaction towards small- or large-conductance Ca2+-activated K+ channels

Summary Ca²⁺-activated K⁺ channels consist of a large family of membrane proteins, among which two groups have been characterized by electrophysiological criteria, the small conductance (SK) and the large conductance (BK) Ca²⁺-activated K⁺ channels. Scorpion toxins that block K⁺ channels exhibit a common three-dimensional structure constituted of a short α-helix connected by disulfide bonds to a β-sheet. The leiurotoxin I (LTX1) related toxins interact specifically with the SK channel via basic residues of their α-helix, while the charybdotoxin (ChTX) family recognizes the BK channel with basic residues of their β-sheet. In an attempt to better understand the structure-activity relationships of these toxins and the characteristics of the electrostatic interactions with the receptor site, we investigated the electrostatic potential supported by natural toxins and a synthetic analogue to find out if it may help in understanding the molecular mechanisms involved in this peptide-protein interaction.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

NMR Studies of the Variant-3 Neurotoxin From Centruroides Sculpturatus Ewing

Abstract

We report a preliminary high-resolution proton nuclear magnetic resonance characterization of the variant-3 toxin from the scorpion Centruroides sculpturatus Ewing (range Southwestern USA). This toxin assumes a well defined folded conformation in aqueous solutions at room temperature and undergoes reversible thermal denaturation. A number of amide hydrogens exhibit exchange life times varying from several minutes to several hours. A few tentative assignments of the low field aromatic CH resonances has been made on the basis of 2D-COSY and NOE experiments. The upfield shifts exhibited by Trp-47 suggest a unique microenvironment for this residue. The NMR data suggest that there is some degree of correlation between the solution structure of the variant-3 toxin and its crystallographic structure. Our studies provide a basis for a detailed elucidation of the structure-function relationships of these interesting scorpion toxins which bind to the sodium channels of excitable membranes and delay sodium current inactivation.     

Solution structure of native and recombinant expressed toxin CssII from the venom of the scorpion Centruroides suffusus suffusus, and their effects on Nav1.5 Sodium channels

The three-dimensional structures of the long-chain mammalian scorpion β-toxin CssII from Centruroides suffusus suffusus and of its recombinant form, HisrCssII, were determined by NMR. The neurotoxin CssII (nCssII) is a 66 amino acid long peptide with four disulfide bridges; it is the most abundant and deadly toxin from the venom of this scorpion. Both native and recombinant CssII structures were determined by nuclear magnetic resonance using a total of 828 sequential distance constraints derived from the volume integration of the cross peaks observed in 2D NOESY spectra. Both nCssII and HisrCssII structures display a mixed α/β fold stabilized by four disulfide bridges formed between pairs of cysteines: C1-C8, C2-C5, C3-C6, and C4-C7 (the numbers indicate the relative positions of the cysteine residues in the primary structure), with a distortion induced by two cis-prolines in its C-terminal part. The native CssII electrostatic surface was compared to both the recombinant one and to the Cn2 toxin, from the scorpion Centruroides noxius, which is also toxic to mammals. Structural features such N- and C-terminal differences could influence toxin specificity and affinity towards isoforms of different sub-types of Na_v channels.     

AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells

Prostate cancer is the most highly diagnosed cancer in men worldwide. It is characterized by high proliferation, great invasion and metastatic potential. Sodium channel subtypes have been identified as highly expressed in different prostate cancer cell lines. In this study, we have screened the negatively charged fractions of Androctonus australis (Aa) scorpion venom to identify active peptides on DU145 prostate cancer cells proliferation. The most active compound was identified to be the sodium channel peptide AaHIV with an IC₅₀ value of 15 μM. At this concentration, AaHIV had low effect on the adhesion of DU145 cells to fibronectin. When compared to other Na⁺ channel Aa toxins, AaHIV was found to be 2 times more active than AaHI and AaHII on DU145 cells proliferation and slightly less active than AaHII on their adhesion. The three peptides are inactive on DU145 cells migration. AaHIV was found to be 16 times more active than veratridine, asteroidal alkaloid from plants of the lily family widely used as a sodium channel activator. Electrophysiological experiments showed that the AaHIV toxin activates Nav1.6 channel, suggesting that this sodium channel subtype is implicated in the proliferation of DU145 prostate cancer cells.     

Secondary structure of peptides. 18. 15N-nmr and 13C-nmr CP/MAS investigation of the primary and secondary structure of (Ala/Val) copolypeptides

Various copolypeptides were prepared by benzylamine or tertiary amine-initiated copolymerizations of alanine–N-carboxyanhydride (Ala-NCA) and valine–N-carboxyanhydride (Val-NCA). The number-average molecular weights of these copolypeptides were detemined by ¹H-nmr spectroscopic end-group analyses and viscosity measurements. The sequences were characterized by ¹⁵N-nmr spectra in solution, and the average lengths of the homogeneous blocks were determined from the signal intensities. The 50.3-and 75.4-MHz ¹³C-nmr CP/MAS spectra of the solid copolypeptides are not sensitive to sequence effects, but allow qualitative and quantitative analyses of the secondary structures. In contrast to other methods, the ¹³C-nmr spectra allow determination of the extent to which individual amino acids are incorporated into β-sheet or α-helix phases. Depending on primary structure and molecular weight, the secondary structure of (Ala/Val) copolypeptides may vary significantly. Both monomer units may be predominantly helical or predominantly β-sheet structure, or the Val units may prefer the β-sheet structure with most Ala-units forming β-helices. However, these secondary structures are more or less thermodynamically unstable and revert to the stable conformations on reprecipitation from trifluoroacetic acid/water.     

ATP-induced noncooperative thermal unfolding of hen lysozyme

To understand the role of ATP underlying the enhanced amyloidosis of hen egg white lysozyme (HEWL), the synchrotron radiation circular dichroism, combined with tryptophan fluorescence, dynamic light-scattering, and differential scanning calorimetry, is used to examine the alterations of the conformation and thermal unfolding pathway of the HEWL in the presence of ATP, Mg²⁺-ATP, ADP, AMP, etc. It is revealed that the binding of ATP to HEWL through strong electrostatic interaction changes the secondary structures of HEWL and makes the exposed residue W62 move into hydrophobic environments. This alteration of W62 decreases the β-domain stability of HEWL, induces a noncooperative unfolding of the secondary structures, and produces a partially unfolded intermediate. This intermediate containing relatively rich α-helix and less β-sheet structures has a great tendency to aggregate. The results imply that the ease of aggregating of HEWL is related to the extent of denaturation of the amyloidogenic region, rather than the electrostatic neutralizing effect or monomeric β-sheet enriched intermediate.     

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.     

Ts17, a Tityus serrulatus β-toxin structurally related to α-scorpion toxins

Ts17 was purified from the venom of the scorpion Tityus serrulatus, the most dangerous scorpion species in Brazil. The activity on Na_v1.1-Na_v1.7 channels was electrophysiologically characterized by patch-clamp technique. Ts17 amino acid sequence indicated high similarity to alpha-scorpion toxins; however, it presented beta-toxin activity, altering the kinetics of the Na⁺-channels. The most affected subtypes during activation (with and without prepulse) and inactivation phases were Na_v1.2 and Na_v1.5, respectively. For recovery from inactivation, the most affected voltage-gated sodium channel was Na_v1.5. Circular dichroism spectra showed that Ts17 presents mainly β-sheet and unordered structures at all analyzed pHs, and the maximum value of α-helix was found at pH 4.0 (13.3 %). Based on the results, Ts17 might be used as a template to develop a new cardiac drug.Key contributionPurification of Ts17 from Tityus serrulatus, electrophysiological characterization of Ts17 on voltage-gated sodium channel subtypes, β-toxin classification.     

Stimulation of sodium and calcium uptake by scorpion toxin in chick embryo heart cells

Scorpion toxins, the basic miniprotiens of scorpion venom, stimulated the passive uptake of Na⁺ and Ca²⁺ in chick ermbryo heart cells. Half-maximum stimulation was obtained for 20–30 nM Na⁺ and 40–50 nM Ca²⁺. Scorpion toxin-activated Na⁺ and Ca²⁺ uptakes were fully inhibited by tetrodotoxin, a specific inhibitor of the action potential Na⁺ ionophore in excitable membranes. Half-maximum inhibition was obtained with the same concentration of tetrodotoxin (10 nm) for both Na⁺ and Ca²⁺. Scorpion toxin-stimulated Ca²⁺ uptake was dependent on extracellular Na⁺ concentration and was not inhibited by Ca²⁺ channel blocking drugs which are inactive on heart cell action potential. Thus, in heart cells scorpion toxin affects the passive Ca²⁺ transport, which is coupled to passive Na⁺ ionphore. Other results suggest that (1) tetrodotoxin and scorpion toxin bind to different sites of the sarcolemma and (2) binding of scorpion toxin to its specific sites may unmask latent tetrodotoxin — sensitive fast channels.     

The complete amino acid sequence of toxin TsTX-VI isolated from the venom of the scorpion Tityus serrulatus

The complete sequence of the toxin TsTX-VI from the venom of the scorpionTityus serrulatus Lutz and Mello is presented. The sequence has been determined by automated Edman analysis of the reduced and carboxymethylated protein as well as of the resulting peptides, obtained fromS. aureus protease and tryptic digestions. TsTX-VI is composed of 62 residues and has a calculated molecular weight of 6717. Homology studies with other scorpion toxins show that TsTX-VI is more similar to the Old World than to the North American scorpion toxins. The hydropathic index indicates that TsTX-VI is more hydrophobic than Ts-. Toxicity studies carried out in mice demonstrate that i.v. injection of TsTX-VI is unable to evoke the usual symptoms induced by the typical neurotoxins of this venom, but only a generalized allergic reaction. These properties are important in clarifying the relationship between primary structure and biological function of scorpion toxins.     

Prediction of the structure of the replication initiator protein DnaA

The secondary structure of DnaA protein and its interaction with DNA and ribonucleotides has been predicted using biochemical, biophysical techniques, and prediction methods based on multiple-sequence alignment and neural networks. The core of all proteins from the DnaA family consists of an “open twisted α/β structure,” containing five α-helices alternating with five β-strands. In our proposed structural model the interior of the core is formed by a parallel β-sheet, whereas the α-helices are arranged on the surface of the core. The ATP-binding motif is located within the core, in a loop region following the first β-strand. The N-terminal domain (80 aa) is composed of two α-helices, the first of which contains a potential leucine zipper motif for mediating protein-protein interaction, followed by a β-strand and an additional α-helix. The N-terminal domain and the α/β core region of DnaA are connected by a variable loop (45–70 aa); major parts of the loop region can be deleted without loss of protein activity. The C-terminal DNA-binding domain (94 aa) is mostly α-helical and contains a potential helix-loop-helix motif. DnaA protein does not dimerize in solution; instead, the two longest C-terminal α-helices could interact with each other, forming an internal “coiled coil” and exposing highly basic residues of a small loop region on the surface, probably responsible for DNA backbone contacts. © 1997 Wiley-Liss Inc.