Crystal structures of two alpha-like scorpion toxins: non-proline cis peptide bonds and implications for new binding site selectivity on the sodium channel.

The crystal structures of two group III alpha-like toxins from the scorpion Buthus martensii Karsch, BmK M1 and BmK M4, were determined at 1.7 A and 1.3 A resolution and refined to R factors of 0.169 and 0.166, respectively. The first high-resolution structures of the alpha-like scorpion toxin show some striking features compared with structures of the "classical" alpha-toxin. Firstly, a non-proline cis peptide bond between residues 9 and 10 unusually occurs in the five-member reverse turn 8-12. Secondly, the cis peptide 9-10 mediates the spatial relationship between the turn 8-12 and the C-terminal stretch 58-64 through a pair of main-chain hydrogen bonds between residues 10 and 64 to form a unique tertiary arrangement which features the special orientation of the terminal residues 62-64. Finally, in consequence of the peculiar orientation of the C-terminal residues, the functional groups of Arg58, which are crucial for the toxin-receptor interaction, are exposed and accessible in BmK M1 and M4 rather than buried as in the classical alpha-toxins. Sequence alignment and characteristics analysis suggested that the above structural features observed in BmK M1 and M4 occur in all group III alpha-like toxins. Recently, some group III alpha-like toxins were demonstrated to occupy a receptor site different from the classical alpha-toxin. Therefore, the distinct structural features of BmK M1 and M4 presented here may provide the structural basis for the newly recognized toxin-receptor binding site selectivity. Besides, the non-proline cis peptide bonds found in these two structures play a role in the formation of the structural characteristics and in keeping accurate positions of the functionally crucial residues. This manifested a way to achieve high levels of molecular specificity and atomic precision through the strained backbone geometry.     

Pharmacological comparison of two different insect models using the scorpion alpha-like toxin BmK M1 from Buthus martensii Karsch

In this study, the effect of the scorpion alpha-like toxin BmK M1 was investigated on isolated DUM neurons from Locusta migratoria and compared with the effect on para/tipE voltage-gated Na(+) channels (VGSC), cloned from Drosophila melanogaster. The two insects display different pharmacological properties regarding alpha-like toxins. Moreover, with the aid of the alpha-like toxin BmK M1 and 5 of its mutants, the importance of aromatic residues for the interaction of the toxin with the VGSC in L. migratoria and D. melanogaster, is shown.     

Scorpion toxins from Buthus martensii Karsch all possess a predicted alpha-tight-turn

We have purified a new toxin (BmK 17[4]) from Asian scorpion (Buthus martensii Karsch) venom that possesses a distinctive structural motif in its N-terminal (positions 8-12) that is similarly found in two other previously described alpha-like toxins. BmK 17[4] prolongs action potentials (APs) in frog nerve and was purified using gel filtration, ion exchange, fast protein liquid chromatography (FPLC), and high-performance liquid chromatography (HPLC). BmK 17[4] significantly prolonged frog APs but it did not alter APs from an insect ventral nerve cord at similar doses. When applied to voltage-clamped frog muscle single fibers, BmK 17[4] prolonged fast inactivation. Because the polypeptide prolongs APs when both K+ and Ca2+ channels were blocked, BMK 17[4] acts to selectively alter Na+ channel inactivation. The N-terminal sequence of BmK 17[4] was found to be VRDAYIAKPENCVYXC --. The molar mass of BmK 17[4] was determined by LC/MS/MS to be 7097 Daltons. The N- terminal motif (KPENC), which introduces a reverse turn in residues 8-12, does not appear in previously characterized BmK alpha-toxins and may be characteristic of alpha-like toxins. Sequence similarity database searches were used to test whether the N-terminal sequences of alpha-like polypeptide toxins from B. martensii Karsch possess a distinctive structural motif in its 5-residue reverse turn (alpha-turn) that is conserved. Sequence similarities with putative polypeptides encoded by cDNAs obtained from a cDNA library [Zhu, S. Y., Li, W. X., Zenq, X. C., et al. (2000) Nine novel precursors of Buthus martensii scorpiox alpha-toxin homologues. Toxicon 38, 1653-1661] from BmK venom glands showed that an active polypeptide toxin cleaved from the putative propolypeptide toxin BmK M9 is likely identical to BmK 17[4]. Sequence comparisons with toxins and putative toxins from B. martensii Karsch and other species revealed that a group of these toxins possess a common structural motif in their alpha-turn. A neighbor-joining phylogenetic analysis suggests that there are two phylogenetic sister groups of related BmK polypeptides; one possesses the KPENC motif and the other possesses a modifed version (KPHNC) of it.     

Binding characteristics of BmK I, an alpha-like scorpion neurotoxic polypeptide, on cockroach nerve cord synaptosomes.

In this study, the binding characteristics of BmK I, an alpha-like neurotoxic polypeptide purified from the venom of the Chinese scorpion Buthus martensi Karsch, were investigated on rat brain and cockroach nerve cord synaptosomes. The results showed that BmK I can bind to a single class of noninteracting binding sites on cockroach nerve cord synaptosomes with medium affinity (Kd = 16.5 +/ - 4.4 nM) and low binding capacity (Bmax = 1.05 +/- 0.23 pmol/mg protein), but lacks specific binding on rat brain synaptosomes. BmK AS, BmK AS-1 (two novel sodium channel-blocking ligands), BmK IT (an excitatory insect-selective toxin) and BmK IT2 (a depressant insect-selective toxin) from the same venom were found to be capable of depressing BmK I binding in cockroach nerve cord synaptosomes, which might be attributed to either allosteric modulation of voltage-gated Na+ channels by these toxic polypeptides or partial overlapping between the receptor binding sites of BmK I and these toxins. This thus supported the notion that alpha-like scorpion neurotoxic polypeptides bind to a distinct receptor site on sodium channels, which might be similar to the binding receptor site of alpha-type insect toxins, and also related to those of BmK AS type and insect-selective scorpion toxins on insect sodium channels.     

Structural mechanism governing cis and trans isomeric states and an intramolecular switch for cis/trans isomerization of a non-proline peptide bond observed in crystal structures of scorpion toxins

Non-proline cis peptide bonds have been observed in numerous protein crystal structures even though the energetic barrier to this conformation is significant and no non-prolyl-cis/trans-isomerase has been identified to date. While some external factors, such as metal binding or co-factor interaction, have been identified that appear to induce cis/trans isomerization of non-proline peptide bonds, the intrinsic structural basis for their existence and the mechanism governing cis/trans isomerization in proteins remains poorly understood. Here, we report the crystal structure of a newly isolated neurotoxin, the scorpion alpha-like toxin Buthus martensii Karsch (BmK) M7, at 1.4A resolution. BmK M7 crystallizes as a dimer in which the identical non-proline peptide bond between residues 9 and 10 exists either in the cis conformation or as a mixture of cis and trans conformations in either monomer. We also determined the crystal structures of several mutants of BmK M1, a representative scorpion alpha-like toxin that contains an identical non-proline cis peptide bond as that observed in BmK M7, in which residues within or neighboring the cis peptide bond were altered. Substitution of an aspartic acid residue for lysine at residue 8 in the BmK M1 (K8D) mutant converted the cis form of the non-proline peptide bond 9-10 into the trans form, revealing an intramolecular switch for cis-to-trans isomerization. Cis/trans interconversion of the switch residue at position 8 appears to be sequence-dependent as the peptide bond between residues 9 and 10 retains its wild-type cis conformation in the BmK M1 (K8Q) mutant structure. The structural interconversion of the isomeric states of the BmK M1 non-proline cis peptide bond may relate to the conversion of the scorpion alpha-toxins subgroups.     

Electrophysiological characterization of BmK M1, an alpha-like toxin from Buthus martensi Karsch venom

The present study investigates the electrophysiological actions of BmK M1, an alpha-like toxin purified from the venom of the scorpion Buthus martensi Karsch, on voltage-gated Na+ channels. Using the voltage clamp technique, we assessed the BmK M1 activity on the cardiac Na+ channel (hH1) functionally expressed in Xenopus oocytes. The main actions of the toxin are a concentration-dependent slowing of the inactivation process and a hyperpolarizing shift of the steady-state inactivation. This work is the first electrophysiological characterization of BmK M1 on a cloned Na+ channel, demonstrating that this toxin belongs to the class of scorpion alpha-toxins. Our results also show that BmK M1 can be considered as a cardiotoxin.     

Structural basis for the voltage-gated Na+ channel selectivity of the scorpion alpha-like toxin BmK M1

Scorpion alpha-like toxins are proteins that act on mammalian and insect voltage-gated Na+ channels. Therefore, these toxins constitute an excellent target for examining the foundations that underlie their target specificity. With this motive we dissected the role of six critical amino acids located in the five-residue reverse turn (RT) and C-tail (CT) of the scorpion alpha-like toxin BmK M1. These residues were individually substituted resulting in 11 mutants and were subjected to a bioassay on mice, an electrophysiological characterization on three cloned voltage-gated Na+ channels (Nav1.2, Nav1.5 and para), a CD analysis and X-ray crystallography. The results reveal two molecular sites, a couplet of residues (8-9) in the RT and a hydrophobic surface consisting of residues 57 and 59-61 in the CT, where the substitution with specific residues can redirect the alpha-like characteristics of BmK M1 to either total insect or much higher mammal specificity. Crystal structures reveal that the pharmacological ramification of these mutants is accompanied by the reshaping of the 3D structure surrounding position 8. Furthermore, our results also reveal that residues 57 and 59-61, located at the CT, enclose the critical residue 58 in order to form a hydrophobic "gasket". Mutants of BmK M1 that interrupt this hydrophobic surface significantly gain insect selectivity.     

Characterization of Na(+) currents in isolated dorsal unpaired median neurons of Locusta migratoria and effect of the alpha-like scorpion toxin BmK M1

A primary cell culture was developed for efferent dorsal unpaired median (DUM) neurons of the locust. The isolated somata were able to generate Tetrodotoxin (TTX)-sensitive action potentials in vitro. The alpha-like scorpion toxin BmK M1, from the Asian scorpion Buthus martensi Karsch, prolonged the duration of the action potential up to 50 times. To investigate the mechanism of action of BmK M1, the TTX-sensitive voltage gated Na(+) currents were studied in detail using the whole cell patch clamp technique. BmK M1 slowed down and partially inhibited the inactivation of the TTX-sensitive Na(+) current in a dose dependent manner (EC50=326.8+/-34.5 nM). Voltage and time dependence of the Na(+) current were described in terms of the Hodgkin-Huxley model and compared in control conditions and in the presence of 500 nM BmK M1. The BmK M1 shifted steady state inactivation by 10.8 mV to less negative potentials. The steady state activation was shifted by 5.5 mV to more negative potentials, making the activation window larger. Moreover, BmK M1 increased the fast time constant of inactivation, leaving the activation time constant unchanged. In summary, BmK M1 primarily affected the inactivation parameters of the voltage gated Na(+) current in isolated locust DUM neurons.     

Purification, cDNA cloning and function assessment of BmK abT, a unique component from the Old World scorpion species

A new neurotoxic component named BmK abT was purified from the venom of Chinese scorpion Buthus martensi Karsch. The molecular weight of BmK abT was determined to be 7212 Da on a mass spectrum. The minimum lethal dose of BmK abT was tested to be about 1.5 microg per mouse by intracerebroventricular injection, and the dose induced significant paralysis effect on cockroach was about 5 microg by i.p. injection. The partial amino acid sequence indicated that it was a distinctive polypeptide in the scorpion neurotoxin family. Thereafter, the whole amino acid sequence of mature BmK abT was deduced from cDNA sequence by 5'- and 3'-rapid amplification of cDNA ends. Finally, it was defined to be composed of 63 residues with amidation at the C-terminal residue. By sequence comparison, BmK abT was found to be most similar to Ts VII, a beta-toxin from the New World scorpion. The patch-clamp recording on DRG neurons, unexpectedly, showed this toxin could prolong the action potential and increase the amplitude of the peak Na+ currents, which are the typical characters of alpha-toxin. These results suggested that BmK abT was a new toxic component found in the Old World scorpion species structurally similar to beta-toxins, but functionally similar to alpha-toxins.     

Purification,crystallization and initial structural solution of a new alpha-like toxin with cardiac toxicity from scorpion Buthus martensii Karsch

An alpha-like toxin named BmK M7 active on both mammals and insects has been purified from the venom of scorpion Buthus martensii Karsch (BmK) recently. The electrophysiological experiments showed that M7 can bind to human cardiac Na+-channel and modify its normal properties, hence can be considered as a cardiotoxin. Single crystals of M7 have been obtained by hanging-drop vapor diffusion method using ammonium sulfate as precipitant in Tris-HCl buffer at pH 8.5. A data set to 1.40 A resolution was collected using synchrotron radiation and CCD detector in Photon Factory in Japan. Data analysis showed that the crystals belonged to space group P3(1)21/P3(1)21, with cell dimensions a=b=32.76 A, c=176.82 A. Assuming two molecules per asymmetric unit, the Vm value is 1.92 A3/Da. The initial structural analysis was carried out by molecular replacement, which showed the correct space group (P3(1)21), and the orientations and positions of the two molecules in the asymmetric unit.     

Homology modeling and molecular dynamics simulation of odonthubuthus doriae (Od1) scorpion toxin in comparison to the BmK M1

All of the α-subgroups share similarity in their sequence and structure but different in the toxicity to various voltage-gated sodium channels (VGSCs). We modeled the first 3D structural model of the Od1 based on BmK M1 using homology modeling. The reliability of model for more investigation and compare to BmK M1 has been examined and confirmed. Then the model structure is further refined by energy minimization and molecular dynamics methods. The purpose of this modeling and simulation is comparison toxicity of two mentioned toxins by investigation structural feature of functional regions including core domain, 5-turn and C-terminal which make NC domain. In the one hand, it is intriguing that Od1 in comparison to BmK M1 shows same solvent accessible surface area (SASA) in 5-turn region but a little more exposed and feasibility (more SASA) in C-terminal region and key functional residues of C-terminal such as positive residues Arg58, lys62 and Arg (His)64. These data suggested that Od1 has similarity with BmK M1 but has more toxicity to sodium channel. In the other hand 5-turn proximity of C-terminal to 5-turn in BmK M1with cis peptide bond is less than Od1 without cis peptide bond which is a confirmation with experimental data about BmK M1.A better understanding of the 3-D structure of Od1and comparison to BmK M1 will be helpful for more investigation of functional characters action of natural toxins with a specialized role for VGSCs.     

Expression and purification of the BmK M1 neurotoxin from the scorpion Buthus martensii Karsch

The gene encoding a neurotoxin (BmK M1) from the scorpion Buthus martensii Karsch was expressed in Saccharomyces cerevisiae at a high level with the alcohol dehydrogenase promoter. SDS-PAGE of the culture confirmed expression and showed secretion into medium from yeast. Recombinant BmK M1 was purified rapidly and efficiently by ion exchange and gel filtration chromatography to homogeneity, produced a single band on tricine-SDS-PAGE, and processed the homologous N-terminus. Amino acid analysis and N-terminal sequencing demonstrated that the recombinant toxin was processed correctly from the alpha-mating factor leader sequence and was chemically identical to the native form. The expressed recombinant BmK M1 was toxic for mice, which indicated that it was biologically active. Quantitative estimation showed that recombinant BmK M1 had an LD(50) similar to that of the native toxin.     

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.     

Site-Directed Mutagenesis of BmK AGP-SYPU1: The Role of Two Conserved Tyr (Tyr5 and Tyr42) in Analgesic Activity

In this study, the role of two conversed tyrosines (Tyr5 and Tyr42) from the scorpion toxin BmK AGP-SYPU1 was investigated with an effective Escherichia coli expression system. Site-directed mutagenesis was used to individually substitute Tyr5 and Tyr42 with hydrophobic or hydrophilic amino acids, and the extent to which these scorpion toxin BmK AGP-SYPU1 tyrosines contribute to analgesic activity was evaluated. The results of the mouse-twisting test showed that Tyr5 and Tyr42 are associated with the analgesic activity of the toxin because the analgesic activities of Y5F and Y42F were significantly increased compared with the rBmK AGP-SYPU1; however, the Y5W had decreased activity. The results of molecular simulation reveal the following: (1) for analgesic activity, the core domain of the scorpion toxin BmK AGP-SYPU1 is key and (2) for pharmacological function, Tyr42 is most likely involved when the core domain conformation is altered. These studies identify a new relationship between the structure and analgesic activity of the scorpion toxin BmK AGP-SYPU1 and are significant for further research and the application of analgesic peptides.     

Molecular interaction of delta-conotoxins with voltage-gated sodium channels

Various neurotoxic peptides modulate voltage-gated sodium (Na(V)) channels and thereby affect cellular excitability. Delta-conotoxins from predatory cone snails slow down inactivation of Na(V) channels, but their interaction site and mechanism of channel modulation are unknown. Here, we show that delta-conotoxin SVIE from Conus striatus interacts with a conserved hydrophobic triad (YFV) in the domain-4 voltage sensor of Na(V) channels. This site overlaps with that of the scorpion alpha-toxin Lqh-2, but not with the alpha-like toxin Lqh-3 site. Delta-SVIE functionally competes with Lqh-2, but exhibits strong cooperativity with Lqh-3, presumably by synergistically trapping the voltage sensor in its "on" position.     

Crystal structure of a highly acidic neurotoxin from scorpion Buthus tamulus at 2.2A resolution reveals novel structural features

The crystal structure of a highly acidic neurotoxin from the scorpion Buthus tamulus has been determined at 2.2A resolution. The amino acid sequence determination shows that the polypeptide chain has 64 amino acid residues. The pI measurement gave a value of 4.3 which is one of the lowest pI values reported so far for a scorpion toxin. As observed in other alpha-toxins, it contains four disulphide bridges, Cys12-Cys63, Cys16-Cys36, Cys22-Cys46, and Cys26-Cys48. The crystal structure reveals the presence of two crystallographically independent molecules in the asymmetric unit. The conformations of two molecules are identical with an r.m.s. value of 0.3A for their C(alpha) tracings. The overall fold of the toxin is very similar to other scorpion alpha-toxins. It is a betaalphabetabeta protein. The beta-sheet involves residues Glu2-Ile6 (strand beta1), Asp32-Trp39 (strand beta3) and Val45-Val55 (strand beta4). The single alpha-helix formed is by residues Asn19-Asp28 (alpha2). The structure shows a trans peptide bond between residues 9 and 10 in the five-membered reverse turn Asp8-Cys12. This suggests that this toxin belongs to classical alpha-toxin subfamily. The surface features of the present toxin are highly characteristic, the first (A-site) has residues, Phe18, Trp38 and Trp39 that protrude outwardly presumably to interact with its receptor. There is another novel face (N-site) of this neurotoxin that contains several negatively charged residues such as, Glu2, Asp3, Asp32, Glu49 and Asp50 which are clustered in a small region of the toxin structure. On yet another face (P-site) in a triangular arrangement, with respect to the above two faces there are several positively charged residues, Arg58, Lys62 and Arg64 that also protrude outwardly for a potentially potent interaction with other molecules. This toxin with three strong features appears to be one of the most toxic molecules reported so far. In this sense, it may be a new subclass of neurotoxins with the largest number of hot spots.     

Soluble expression, purification and the role of C-terminal glycine residues in scorpion toxin BmK AGP-SYPU2

The existence of glycine residues in long-chain scorpion toxins has been well documented. However, their role as analgesics has not been evaluated. To address this issue, we investigated the functional role of glycines in the C-terminal end of Chinese-scorpion toxin from Buthus martensii Karsch (BmK AGP-SYPU2) using site-directed mutagenesis and analgesic activity assays. Recombinant BmK AGP-SYPU2 and its mutants were efficiently expressed in E. coli and purified to homogeneity using immobilized metal ion affinity chromatography (IMAC) and cation exchange chromatography. The mouse-twisting test was used to detect the analgesic activity of BmK AGP-SYPU2 and its mutants. As a result, we identified glycines at the C-terminal end that, when altered, significantly affected analgesic activity. Also, Mut6566 was significantly decreased compared to BmK AGP-SYPU2. These data indicate that the glycines at the C-terminal end are important for the analgesic activity of BmK AGP-SYPU2.     

Chinese-scorpion (Buthus martensi Karsch) toxin BmK alphaIV, a novel modulator of sodium channels: from genomic organization to functional analysis

In the present study, BmK alphaIV, a novel modulator of sodium channels, was cloned from venomous glands of the Chinese scorpion (Buthus martensi Karsch) and expressed successfully in Escherichia coli. The BmK alphaIV gene is composed of two exons separated by a 503 bp intron. The mature polypeptide contains 66 amino acids. BmK alphaIV has potent toxicity in mice and cockroaches. Surface-plasmon-resonance analysis found that BmK alphaIV could bind to both rat cerebrocortical synaptosomes and cockroach neuronal membranes, and shared similar binding sites on sodium channels with classical AaH II (alpha-mammal neurotoxin from the scorpion Androctonus australis Hector), BmK AS (beta-like neurotoxin), BmK IT2 (the depressant insect-selective neurotoxin) and BmK abT (transitional neurotoxin), but not with BmK I (alpha-like neurotoxin). Two-electrode voltage clamp recordings on rNav1.2 channels expressed in Xenopus laevis oocytes revealed that BmK alphaIV increased the peak amplitude and prolonged the inactivation phase of Na+ currents. The structural and pharmacological properties compared with those of other scorpion alpha-toxins suggests that BmK alphaIV represents a novel subgroup or functional hybrid of alpha-toxins and might be an evolutionary intermediate neurotoxin for alpha-toxins.     

Investigation of the role of disulphide bond in modulating internal motions of BmK AGAP protein by molecular dynamics simulation

Elucidating structural determinants in the functional regions of toxins can provide useful knowledge for designing novel analgesic peptides. A series of 100 ns MD simulations were performed on the scorpion toxin BmK AGAP in native and disulphide bond broken states. The comparison of disulphide bond broken states with the native state showed the α-helix was found to be the key to the analgesic activity. Furthermore, our results revealed disulphide bonds have considerable influence on the functionally important essential modes of motions and the correlations between the motions of the Core domain and the C-terminal region which are involved in the analgesic activity. Therefore, we can conclude that disulphide bonds have a crucial role in modulating the function via adjusting the dynamics of scorpion toxin BmK AGAP molecule.     

Molecular characterization of an anti-epilepsy peptide from the scorpion Buthus martensi Karsch.

For a long time Asian scorpion Buthus martensi Karsch (BmK) has been used in Chinese traditional medicine to cure many diseases of nervous system. Here we report the purification and characterization of a pharmacologically active neurotoxin from the scorpion BmK. This toxin had little toxicity in mice and insects but was found to have an anti-epilepsy effect in rats, and is thus named as BmK anti-epilepsy peptide (BmK AEP). Its amino-acid sequence was determined by lysylendopeptidase digestion, Edman degradation and mass spectrographic analysis. Based on the determined sequence, the gene coding for this peptide was also cloned and sequenced by the 3' and 5' RACE methods. It encodes a precursor of 85 amino-acid residues including a signal peptide of 21 residues, a mature peptide of 61 residues and three additional residues Gly-Lys-Lys at the C-terminus. The additional Gly sometimes followed by one or two basic residues is prerequisite for the amidation of its C-terminus. C-terminal amidation was also verified by the molecular-mass determination of BmK AEP. This anti-epilepsy peptide toxin shares homology with other depressant insect toxins. The remarkable difference between them was mainly focused at residues 6, 7 and 39; these residues might relate to the unique action of BmK AEP.