Solution structure of a K+-channel blocker from the scorpion Tityus cambridgei

A new K(+)-channel blocking peptide identified from the scorpion venom of Tityus cambridgei (Tc1) is composed of 23 amino acid residues linked with three disulfide bridges. Tc1 is the shortest known toxin from scorpion venom that recognizes the Shaker B K(+) channels and the voltage-dependent K(+) channels in the brain. Synthetic Tc1 was produced using solid-phase synthesis, and its activity was found to be the same as that of native Tc1. The pairings of three disulfide bridges in the synthetic Tc1 were identified by NMR experiments. The NMR solution structures of Tc1 were determined by simulated annealing and energy-minimization calculations using the X-PLOR program. The results showed that Tc1 contains an alpha-helix and a 3(10)-helix at N-terminal Gly(4)-Lys(10) and a double-stranded beta-sheet at Gly(13)-Ile(16) and Arg(19)-Tyr(23), with a type I' beta-turn at Asn(17)-Gly(18). Superposition of each structure with the best structure yielded an average root mean square deviation of 0.26 +/- 0.05 A for the backbone atoms and of 1.40 +/- 0.23 A for heavy atoms in residues 2 to 23. The three-dimensional structure of Tc1 was compared with two structurally and functionally related scorpion toxins, charybdotoxin (ChTx) and noxiustoxin (NTx). We concluded that the C-terminal structure is the most important region for the blocking activity of voltage-gated (Kv-type) channels for scorpion K(+)-channel blockers. We also found that some of the residues in the larger scorpion K(+)-channel blockers (31 to 40 amino acids) are not involved in K(+)-channel blocking activity.     

Solution structure of BmBKTx1, a new BKCa1 channel blocker from the Chinese scorpion Buthus martensi Karsch

BmBKTx1 is a 31-amino acid peptide identified from the venom of the Chinese scorpion Buthus martensi Karsch, blocking high-conductance calcium-activated potassium channels. Sequence homology analysis indicates that BmBKTx1 is a new subfamily of short-chain alpha-KTx toxins of the potassium channel, which we term alpha-KTx19. Synthetic BmBKTx1 was prepared by using solid-phase peptide synthesis. Two-dimensional NMR spectroscopy techniques were used to determine the solution structure of BmBKTx1. The results show that the BmBKTx1 forms a typical cysteine-stabilized alpha/beta scaffold adopted by most short-chain scorpion toxins. The structure of BmBKTx1 consists of a two-stranded antiparallel beta-sheet (residues 20-29) and an alpha-helix (residues 5-15). The three-dimensional structure of BmBKTx1 was also compared with those of two function-related scorpion toxins, charybdotoxin (ChTx) and BmTx1, and their structural and functional implications are discussed.     

Solution structure of BmP08, a novel short-chain scorpion toxin from Buthus martensi Karsch

A novel short-chain scorpion toxin BmP08 was purified from the venom of the Chinese scorpion Buthus martensi Karsch by a combination of gel-filtration, ion exchange, and reversed-phase chromatography. The primary sequence of BmP08 was determined using the tandem MS/MS technique and Edman degradation, as well as results of NMR sequential assignments. It is composed of 31 amino acid residues including six cysteine residues and shares less than 25% sequence identity with the known alpha-KTx toxins. BmP08 shows no inhibitory activity on all tested voltage-dependent and Ca(2+)-activated potassium channels. The 3D-structure of BmP08 has been determined by 2D-NMR spectroscopy and molecular modeling techniques. This toxin adopts a common alpha/beta-motif, but shows a distinctive local conformation and features a 3(10)-helix and a shorter beta-sheet. The unique structure is closely related to the distinct primary sequence of the toxin, especially to the novel arrangement of S-S linkages in the molecule, in which two disulfide bridges (C(i)-C(j) and C(i+3)-C(j+3)) link covalently the 3(10)-helix with one strand of the beta-sheet structure. The electrostatic potential surface analysis of the toxin reveals salt bridges and hydrogen bonds between the basic residues and negatively charged residues nearby in BmP08, which may be unfavorable for its binding with the known voltage-dependent and Ca(2+)-activated potassium channels. Thus, finding the target for this toxin should be an interesting task in the future.     

Solution structure of BmP02, a new potassium channel blocker from the venom of the Chinese scorpion Buthus martensi Karsch

BmP02 is a 28-amino acid residue peptide purified from the venom of the Chinese scorpion Buthus martensi Karsch, which had been demonstrated to be a weak blocker of apamin-sensitive calcium-activated potassium channels. Two-dimensional NMR spectroscopy techniques were used to determine the solution structure of BmP02. The results show that BmP02 formed a alpha/beta scorpion fold, the typical three-dimensional structure adopted by most short chain scorpion toxins whose structures have been determined. However, in BmP02 this alpha/beta fold was largely distorted. The alpha-helix was shortened to only one turn, and the loop connecting the helix to the first beta-strand exhibited conformational heterogeneity. The instability of BmP02 could be attributed to a proline at position 17, which is usually a glycine. Because the residue at this position makes intense contact with the alpha-helix, it was supposed that the bulky side chain of proline had pushed the helix away from the beta-sheet. This had a significant influence on the structure and function of BmP02. The alpha-helix rotated by about 40 degrees to avoid Pro17 while forming two disulfides with the second beta-strand. The rotation further caused both ends of the helix to be unwound due to covalent restrictions. According to its structure, BmP02 was supposed to interact with its target via the side chains of Lys11 and Lys13.     

Solution structure of BmKalphaIT01, an alpha-insect toxin from the venom of the Chinese scorpion Buthus martensii Karsch

The solution structure of an alpha-insect toxin from Buthus martensii Karsch, BmKalphaIT01, has been determined by two-dimensional NMR spectroscopy and molecular modeling techniques. Combining the sequence homology comparison and toxicity bioassays, BmKalphaIT01 has been suggested to be a natural mutant of alpha-insect toxins and so can serve as a tool to study the relationship of structure-function among this group of toxins. The overall structure of BmKalphaIT01 shares a common core structure consisting of an alpha-helix packed against a three-stranded antiparallel beta-sheet, which exhibits distinctive local conformations within the loops connecting these secondary structure elements. The solution structure of BmKalphaIT01 features a non-proline cis peptide bond between Asn9 and Tyr10, which is proposed to mediate the spatial closing of the five-residue turn (Gln8-Cys12) and the C-terminal segment (Arg58-His64) to form the NC domain and confer the toxin insect-specific bioactivity. Conformational heterogeneity is observed in the solution of BmKalphaIT01 and could be attributed to the cis-trans isomerization of the peptide bond between residues 9 and 10. The minor conformation of BmKalphaIT01 with a trans peptide bond between Asn9 and Tyr10 may be responsible for its moderate bioactivity against mammals. The cis-trans isomerization of the peptide bond between residues 9 and 10 may be the structural basis of dual pharmacological activities of alpha-insect and alpha-like scorpion toxins, which is supported by the fact that conformational heterogeneity occurs in the solution structures of LqhalphaIT, LqqIII, and LqhIII and by comparison of the solution structure of BmKalphaIT01 with those of some relevant alpha-type toxins.     

BmBKTx1, a novel Ca2+-activated K+ channel blocker purified from the Asian scorpion Buthus martensi Karsch

BmBKTx1 is a novel short chain toxin purified from the venom of the Asian scorpion Buthus martensi Karsch. It is composed of 31 residues and is structurally related to SK toxins. However, when tested on the cloned rat SK2 channel, it only partially inhibited rSK2 currents, even at a concentration of 1 microm. To screen for other possible targets, BmBKTx1 was then tested on isolated metathoracic dorsal unpaired median neurons of Locusta migratoria, in which a wide variety of ion channels are expressed. The results suggested that BmBKTx1 could specifically block voltage-gated Ca(2+)-activated K(+) currents (BK-type). This was confirmed by testing the BmBKTx1 effect on the alpha subunits of BK channels of the cockroach (pSlo), fruit fly (dSlo), and human (hSlo), heterologously expressed in HEK293 cells. The IC(50) for channel blocking by BmBKTx1 was 82 nm for pSlo and 194 nm for dSlo. Interestingly, BmBKTx1 hardly affected hSlo currents, even at concentrations as high as 10 microm, suggesting that the toxin might be insect specific. In contrast to most other scorpion BK blockers that also act on the Kv1.3 channel, BmBKTx1 did not affect this channel as well as other Kv channels. These results show that BmBKTx1 is a novel kind of blocker of BK-type Ca(2+)-activated K(+) channels. As the first reported toxin active on the Drosophila Slo channel dSlo, it will also greatly facilitate studying the physiological role of BK channels in this model organism.     

Solution structure of BmKK2, a new potassium channel blocker from the venom of chinese scorpion Buthus martensi Karsch

A natural K+ channel blocker, BmKK2 (a member of scorpion toxin subfamily alpha-KTx 14), which is composed of 31 amino acid residues and purified from the venom of the Chinese scorpion Buthus martensi Karsch, was characterized using whole-cell patch-clamp recording in rat hippocampal neurons. The three dimensional structure of BmKK2 was determined with two-dimensional NMR spectroscopy and molecular modelling techniques. In solution this toxin adopted a common alpha/beta-motif, but showed distinct local conformation in the loop between alpha-helix and beta-sheet in comparison with typical short-chain scorpion toxins (e.g., CTX and NTX). Also, the alpha helix is shorter and the beta-sheet element is smaller (each strand consisted only two residues). The unusual structural feature of BmKK2 was attributed to the shorter loop between the alpha-helix and beta-sheet and the presence of two consecutive Pro residues at position 21 and 22 in the loop. Moreover, two models of BmKK2/hKv1.3 channel and BmKK2/rSK2 channel complexes were simulated with docking calculations. The results demonstrated the existence of a alpha-mode binding between the toxin and the channels. The model of BmKK2/rSK2 channel complex exhibited favorable contacts both in electrostatic and hydrophobic, including a network of five hydrogen bonds and bigger interface containing seven pairs of inter-residue interactions. In contrast, the model of BmKK2/hKv1.3 channel complex, containing only three pairs of inter-residue interactions, exhibited poor contacts and smaller interface. The results well explained its lower activity towards Kv channel, and predicted that it may prefer a type of SK channel with a narrower entryway as its specific receptor.     

东亚钳蝎K+通道阻断肽cDNA的克隆与表达

目的：克隆东亚钳蝎毒素基因,以进一步研究其生物学和药理学功能。方法：利用已知蝎神经毒素基因序列,设计引物,用RT-PCR方法克隆从蝎毒腺组织蝎毒素cDNA。结果：成功地克隆了一个新的东亚钳蝎毒素基因,该基因开放阅读框架编码59个氨基酸残基,其中前22个为信号肽,成熟肽为37个氨基酸残基,经PCR扩增除去信号肽序列,克隆到pTreHisA质粒中,在E．coli中表达了分子质量为7ku左右融合蛋白,表达产物占菌体总蛋白的21％左右。结论：其结构中含有三对二硫链,6个Cys残基组成蝎K^ 通道毒素共同特征序列-CXXXC-、-GXC-、-CXC-,推断其为K^ 通道阻断肽,命名为KChTX1。已被Gene-bank收录,收录号为AY129234。     

Solution structure of BmP01 from the venom of scorpion Buthus martensii Karsch

From the venom of scorpion Buthus martensii Karsch,a short peptide (BmP01, 29 amino acid residues) was isolated and characterized as previously reported (Lebren, R. R., et al. (1997) Eur. J. Biochem. 245, 457-464). It was shown to reduce 33% outward K(+) channel (hippocampal neurons) currents at 10 microM. The solution structure of BmP01 was determined by 2D (1)H NMR spectroscopy. The NOEs, coupling constants, and H-D exchange obtained from NMR spectroscopy were used in structural calculations. The conformation of BmP01 is composed of a short alpha-helix (Cys 3-Thr 12) and a two-stranded antiparallel beta-sheet (Ala 15-Asp 20 and Lys 23-Pro 28). There are three disulfide bridges (Cys 3-Cys 19, Cys 6-Cys 24 and Cys 10-Cys 26) connecting the alpha-helix and beta-sheet. Asp 20 to Lys 23 form a type II turn linking the two strands. Structural and electrostatic potential comparison between BmP01 and its analogues are also presented.     

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.     

Purification, characterization and sequence determination of BmKK4, a novel potassium channel blocker from Chinese scorpion Buthus martensi Karsch

The scorpion neurotoxin BmKK4 was purified from the venom of the Chinese scorpion Buthus martensi Karsch by a combination of gel-filtration, ion exchange and reversed phase chromatography. The primary sequence of BmKK4 was determined using the tandem MS/MS technique and the cDNA database searching as followings: ZTQCQ SVRDC QQYCL TPDRC SYGTC YCKTT (NH(2)). BmKK4 is the first isolated member of a new subfamily alpha-KTx17 of scorpion K(+) toxins.     

Characterization of a new Kv1.3 channel-specific blocker, J123, from the scorpion Buthus martensii Karsch

The potassium channel Kv1.3 is an attractive pharmacological target for T-cell-mediated autoimmune diseases, and specific and selective peptidic blockers of Kv1.3 channels have served as valuable therapeutic leads for treating these diseases. Here, we found a new peptide toxin, J123, with 43 amino acids including six cysteine residues by screening the venomous cDNA library of scorpion Buthus martensii Karsch, which has been used as traditional medicine in China for more than 2000 years. The sequence analysis suggested that peptide J123 constituted a new member of the alpha-KTx toxins. The electrophysiological experiments further indicated that peptide J123 has a novel pharmacological profiles: it blocked Kv1.3 channel with high potency (IC(50)=0.79nM), and exhibited good selectivity on Kv1.3 over Kv1.1 (>1000-fold) and Kv1.2 (about 30-fold), respectively. Furthermore, peptide J123 had no activity on SKCa2 and SKCa3 channels at micromolar concentration level. Based on the pharmacological activities, the possible channel-interacting surface of peptide J123 was also predicted by molecular modeling and docking. All these data not only enrich the knowledge of the structure-function relationship of the new Kv1.3-speicific peptide but also present a potential drug candidate for selectively targeting Kv1.3 channels.     

Cloning and characterization of BmK86, a novel K+ -channel blocker from scorpion venom

Scorpion venom represents a tremendous hitherto unexplored resource for understanding ion channels. BmK86 is a novel K+ -channel toxin gene isolated from a cDNA library of Mesobuthus martensii Karsch, which encodes a signal peptide of 22 amino acid residues and a mature toxin of 35 residues with three disulfide bridges. The genomic sequence of BmK86 consists of two exons disrupted by an intron of 72 bp. Comparison with the other scorpion toxins BmK86 shows low sequence similarity. The GST-BmK86 fusion protein was successfully expressed in Escherichia coli. The fusion protein was cleaved by enterokinase and the recombinant BmK86 was purified by HPLC. Using whole-cell patch-clamp recording, the recombinant BmK86 was found to inhibit the potassium current of mKv1.3 channel expressed in COS7 cells. These results indicated that BmK86 belongs to a representative member of a novel subfamily of alpha-KTxs. The systematic number assigned to BmK86 is alpha-KTx26.1.     

OcyKTx2, a new K+-channel toxin characterized from the venom of the scorpion Opisthacanthus cayaporum

Opisthacanthus cayaporum belongs to the Liochelidae family, and the scorpions from this genus occur in southern Africa, Central America and South America and, therefore, can be considered a true Gondwana heritage. In this communication, the isolation, primary structure characterization, and K⁺-channel blocking activity of new peptide from this scorpion venom are reported. OcyKTx2 is a 34 amino acid long peptide with four disulfide bridges and molecular mass of 3807 Da. Electrophysiological assays conducted with pure OcyKTx2 showed that this toxin reversibly blocks Shaker B K⁺-channels with a Kd of 82 nM, and presents an even better affinity toward hKv1.3, blocking it with a Kd of ∼18 nM. OcyKTx2 shares high sequence identity with peptides belonging to subfamily 6 of α-KTxs that clustered very closely in the phylogenetic tree included here. Sequence comparison, chain length and number of disulfide bridges analysis classify OcyKTx2 into subfamily 6 of the α-KTx scorpion toxins (systematic name, α-KTx6.17).     

A depressant insect toxin with a novel analgesic effect from scorpion Buthus martensii Karsch

A new peptide named BmK dITAP3 from scorpion Buthus martensii Karsch (BmK) has been identified to possess a dual bioactivity, a depressant neurotoxicity on insects and an analgesic effect on mice. The bioassays also showed that the peptide was definitely devoid of the neurotoxicity on mammals, which indicated that the analgesic effect of BmK dITAP3 could not be ascribed to the syndromic effects of a mammalian neurotoxicity. BmK dITAP3 exhibited 43.0% inhibition efficiency of the analgesic effect on mice at a dose of 5 mg/kg and the FPU value of 0.5 microg/body (approximately 30 mg) on the fly larvae. The pI value and the molecular mass determined by MALDI-TOF MS for dITAP3 were 6.5 and 6722.7, respectively. Its first 15 N-terminal residues were determined by Edman degradation, based on which the full amino acid sequence was deduced from the cDNA sequence encoding the peptide with 3'-RACE. Circular dichroism and sequence based prediction analyses showed dITAP3 may have a similar molecular scaffold as the most scorpion toxins but with features of the more beta structures and much less of alpha helix. The details of the purification, characterization and sequencing as well as the sequence comparison with other depressant insect toxins and the correlation between the analgesic effect and the insect toxicity will be reported and discussed, respectively.     

Solution structure of potassium channel-inhibiting scorpion toxin Lq2

Lq2 is a unique scorpion toxin. Acting from the extracellular side, Lq2 blocks the ion conduction pore in not only the voltage- and Ca2+ -activated channels, but also the inward-rectifier K+ channels. This finding argues that the three-dimensional structures of the pores in these K+ channels are similar. However, the amino acid sequences that form the external part of the pore are minimally conserved among the various classes of K+ channels. Because Lq2 can bind to all the three classes of K+ channels, we can use Lq2 as a structural probe to examine how the non-conserved pore-forming sequences are arranged in space to form similar pore structures. In the present study, we determined the three-dimensional structure of Lq2 using nuclear magnetic resonance (NMR) techniques. Lq2 consists of an alpha-helix (residues S10 to L20) and a beta-sheet, connected by an alphabeta3 loop (residues N22 to N24). The beta-sheet has two well-defined anti-parallel strands (residues G26 to M29 and residues K32 to C35), which are connected by a type I' beta-turn centered between residues N30 and K31. The N-terminal segment (residues Z1 to T8) appears to form a quasi-third strand of the beta-sheet.     

Solution spatial structure of 'long' neurotoxin M9 from the scorpion Buthus eupeus by 1H-NMR spectroscopy

1H-NMR spectra of Buthus eupeus neurotoxin M9 (66 amino acid residues, four disulfide bonds) reveal two slowly exchangeable conformations at acidic pH. The spatial structure of the conformer prevailing under physiologically relevant conditions has been determined from two-dimensional 1H-NMR data treated by means of a distance geometry algorithm and refined by molecular modelling. Interrelation between the structure and function of mammalian neurotoxin M9 is discussed by comparing its conformation with those of the scorpion insectotoxins which exhibit different biological specificity (insectotoxins v-2, v-3 and I5A).     

Solution structure of BTK-2, a novel hK(v)1.1 inhibiting scorpion toxin, from the eastern Indian scorpion Mesobuthus tamulus

The three dimensional structure of a 32 residue three disulfide scorpion toxin, BTK-2, from the Indian red scorpion Mesobuthus tamulus has been determined using isotope edited solution NMR methods. Samples for structural and electrophysiological studies were prepared using recombinant DNA methods. Electrophysiological studies show that the peptide is active against hK(v)1.1 channels. The structure of BTK-2 was determined using 373 distance restraints from NOE data, 66 dihedral angle restraints from NOE, chemical shift and scalar coupling data, 6 constraints based on disulfide linkages and 8 constraints based on hydrogen bonds. The root mean square deviation (r.m.s.d) about the averaged co-ordinates of the backbone (N, C(α), C') and all heavy atoms are 0.81 ± 0.23? and 1.51 ± 0.29? respectively. The backbone dihedral angles (? and ψ) for all residues occupy the favorable and allowed regions of the Ramachandran map. The three dimensional structure of BTK-2 is composed of three well defined secondary structural regions that constitute the α-β-β structural motif. Comparisons between the structure of BTK-2 and other closely related scorpion toxins pointed towards distinct differences in surface properties that provide insights into the structure-function relationships among this important class of voltage-gated potassium channel inhibiting peptides.     

Characterisation of a novel toxin active on potassium apanaine channels, purified from Buthus occitanus tunetanus scorpion venom

A new peptidyl inhibitor of the small-conductance Ca(2+)-activated K+ channels (SKca) was purified to homogeneity from the venom of the Tunisian scorpion Buthus occitanus tunetanus. The molecular mass determined by SDS-PAGE, shows that it's a short peptide (3300 Da). The primary sequence of this toxin shows that it is a 31-residue polypeptide cross-linked by three disulfide bridges and structurally related to subfamily 5 of short scorpion toxins. This molecule shows similar pharmacological properties with this group of peptides inducing high toxicity in mice after intracerebro-ventricular injection, and competing with iodinated apamin for binding to its receptor site from rat brain synaptosomes (K0.5 = 4 nM).