Bacterial expression, NMR, and electrophysiology analysis of chimeric short/long-chain alpha-neurotoxins acting on neuronal nicotinic receptors

Different snake venom neurotoxins block distinct subtypes of nicotinic acetylcholine receptors (nAChR). Short-chain alpha-neurotoxins preferentially inhibit muscle-type nAChRs, whereas long-chain alpha-neurotoxins block both muscle-type and alpha7 homooligomeric neuronal nAChRs. An additional disulfide in the central loop of alpha- and kappa-neurotoxins is essential for their action on the alpha7 and alpha3beta2 nAChRs, respectively. Design of novel toxins may help to better understand their subtype specificity. To address this problem, two chimeric toxins were produced by bacterial expression, a short-chain neurotoxin II Naja oxiana with the grafted disulfide-containing loop from long-chain neurotoxin I from N. oxiana, while a second chimera contained an additional A29K mutation, the most pronounced difference in the central loop tip between long-chain alpha-neurotoxins and kappa-neurotoxins. The correct folding and structural stability for both chimeras were shown by (1)H and (1)H-(15)N NMR spectroscopy. Electrophysiology experiments on the nAChRs expressed in Xenopus oocytes revealed that the first chimera and neurotoxin I blockalpha7 nAChRs with similar potency (IC(50) 6.1 and 34 nM, respectively). Therefore, the disulfide-confined loop endows neurotoxin II with full activity of long-chain alpha-neurotoxin and the C-terminal tail in neurotoxin I is not essential for binding. The A29K mutation of the chimera considerably diminished the affinity for alpha7 nAChR (IC(50) 126 nM) but did not convey activity at alpha3beta2 nAChRs. Docking of both chimeras toalpha7 andalpha3beta2 nAChRs was possible, but complexes with the latter were not stable at molecular dynamics simulations. Apparently, some other residues and dimeric organization of kappa-neurotoxins underlie their selectivity for alpha3beta2 nAChRs.     

"Weak toxin" from Naja kaouthia is a nontoxic antagonist of alpha 7 and muscle-type nicotinic acetylcholine receptors

A novel "weak toxin" (WTX) from Naja kaouthia snake venom competes with [(125)I]alpha-bungarotoxin for binding to the membrane-bound Torpedo californica acetylcholine receptor (AChR), with an IC(50) of approximately 2.2 microm. In this respect, it is approximately 300 times less potent than neurotoxin II from Naja oxiana and alpha-cobratoxin from N. kaouthia, representing short-type and long-type alpha-neurotoxins, respectively. WTX and alpha-cobratoxin displaced [(125)I]alpha-bungarotoxin from the Escherichia coli-expressed fusion protein containing the rat alpha7 AChR N-terminal domain 1-208 preceded by glutathione S-transferase with IC(50) values of 4.3 and 9.1 microm, respectively, whereas for neurotoxin II the IC(50) value was >100 microm. Micromolar concentrations of WTX inhibited acetylcholine-activated currents in Xenopus oocyte-expressed rat muscle AChR and human and rat alpha7 AChRs, inhibiting the latter most efficiently (IC(50) of approximately 8.3 microm). Thus, a virtually nontoxic "three-fingered" protein WTX, although differing from alpha-neurotoxins by an additional disulfide in the N-terminal loop, can be classified as a weak alpha-neurotoxin. It differs from the short chain alpha-neurotoxins, which potently block the muscle-type but not the alpha7 AChRs, and is closer to the long alpha-neurotoxins, which have comparable potency against the above-mentioned AChR types.     

Benzophenone-type photoactivatable derivatives of alpha-neurotoxins and alpha-conotoxins in studies on Torpedo nicotinic acetylcholine receptor

By chemical modification of different lysine residues, benzoylbenzoyl (BzBz) groups were introduced into neurotoxin II Naja naja oxiana (NT-II), a short-chain snake venom alpha-neurotoxin, while p-benzoylphenylalanyl (Bpa) residue was incorporated in the course of peptide synthesis at position 11 of alpha-conotoxin G1, a neurotoxic peptide from marine snails. Although the crosslinking yields for iodinated BzBz derivatives of NT-II and for Bpa analogue of G1 to the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR) are relatively low, the subunit labeling patterns confirm the earlier conclusions, derived from arylazide or diazirine photolabels, that alpha-neurotoxins and alpha-conotoxins bind at the subunit interfaces. Detecting the labeled alpha-subunit with iodinated Bpa analogue of G1 provided a direct proof for the contact between this subunit and alpha-conotoxin molecule.     

Role of arginine and histidine residues in the biological activity of botulinic neurotoxin A

Treatment of botulinic neurotoxin A with cyclohexanedione demonstrated that modification of 5 to 10 arginine residues does not change the neurotoxin toxicity, while after modification of 15-20 arginine residues the toxicity is decreased by 40-50% of the original value. Butanedione exerts a stronger detoxicating effect on neurotoxin than cyclohexanedione. The molecular conformation of the modified toxin derivatives and their precipitability upon interaction with antisera against toxin and toxin fragments does not change thereby. The non-toxic derivatives of toxin containing 40 modified arginine residues possess a partial serological affinity for the original toxin in a reaction with antiserum against toxin but do not interact with the antifragment sera. The molecular conformation of these preparations is changed considerably. It is assumed that one or two arginine residues are located near the toxic site of the neurotoxin molecule and are also components of its antigenic determinants. Modification of histidine residues in the neurotoxin molecule by diethylpyrocarbonate is accompanied by a decrease of its toxicity. An additional 10% toxicity is revealed upon modification of 11-13 histidine residues. The molecular conformation of the modified derivatives of neurotoxin and their precipitability do not change thereby. It is probable that 1 or 2 histidine residues are located at or near the toxic site. The data obtained suggest that histidine residues are not localized in antigenic determinants of the neurotoxin molecule.     

Effect of photooxidation on biological properties of botulinum neurotoxin A

Photooxidation of botulinic neurotoxin A in the presence of methylene blue is associated with a decrease in toxicity down to complete detoxication. During neurotoxin photooxidation, when the toxicity makes up to 1 to 3% of the original one, the conformation of the neurotoxin molecule and its antigenic properties remain unchanged. Under these conditions, using diethylpyrocarbonate, a specific reagent for histidine, the photooxidized neurotoxin was found to contain 5-6 oxidized histidine residues per molecule of neurotoxin; this was accompanied by changes in the UV absorbance spectrum around 280 nm. It was assumed that the main decrease in neurotoxin toxicity during photooxidation is probably due to oxidation of tryptophane, since the differential UV spectra suggest that the higher the extremum around 280 nm, the greater the decrease of toxicity; chemical modification of histidine residues alone causes no noticeable detoxication.     

How do acetylcholine receptor ligands reach their binding sites?

The access pathway to the binding sites for large competitive antagonists of the nicotinic acetylcholine receptor from Torpedo californica electric tissue was analyzed by binding and photolabeling experiments with alpha-neurotoxins. Binding assays with [125I]alpha-bungarotoxin showed an increase in the number of accessible binding sites upon stepwise solubilization of the receptor-rich membranes. Similarily, ligand binding is facilitated upon fluidization of the membrane by increasing the temperature. The access to the binding sites seems to be sterically 'hindered' in the densely packed membrane state. Using a novel series of large biotinylated photoactivatable derivatives of neurotoxin II, we observed that the accessibility to the alpha/gamma- but not to the alpha/delta-binding site was considerably decreased for some derivatives under native conditions. This effect was less apparent at higher temperatures and could be abolished by complete solubilization. These observations support the nonequivalence of the receptor's binding sites. Together, our data suggest (a) that alpha-neurotoxins approach their binding sites from the membrane-facing periphery of the receptor's extramembrane domain rather than through the channel mouth and (b) that different entrance pathways to each binding site exist which vary in their sensitivity to the physical state of the plasma membrane.     

Toxic components of the venom of the Central Asian scorpion, Orthochirus scrobiculosus

Four polypeptide neurotoxins, possessing paralytic activity for mice, were isolated from the venom of the Central Asian black scorpion Orthochirus scrobiculosus. All these toxins, Os-1 - Os-4, were shown to be homogeneous by disc-electrophoresis and N-terminal group analyses. The amino acid composition of the toxins was determined, methionine residues being found in toxin Os-1. The neurotoxin Os-3 was subjected to tryptic and chymotryptic hydrolyses and its total amino acid sequence was established. It was shown that neurotoxin Os-3 consists of 67 amino acid residues with four intramolecular disulfide bonds.     

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.     

5-fluoro-tryptophan-containing N-terminal domain of the alpha-subunit of the Torpedo californica acetylcholine receptor: preparation in E. coli and 19F NMR study

A protein corresponding to the extracellular 1-209 domain of the alpha-subunit of the nicotine acetylcholine receptor from the electric organ of Torpedo californica was prepared using the corresponding cDNA domain by culturing Escherichia coli cells on a synthetic medium supplemented with 5-fluoro-L-tryptophan. The presence of a (His)6 fragment preceding the 1-209 sequence allowed purification of the protein isolated from inclusion bodies by affinity chromatography on Ni-NTA Agarose. The incorporation of 5-fluorotryptophan residues was found by 19F NMR to be approximately 50%. The spectrum of the protein reduced under denaturing conditions and subsequently reoxidized in a dilute solution under denaturing conditions in the presence of 0.05% SDS was sufficiently resolved, which allowed partial assignment of 19F resonances using the Trp60Phe mutant protein. The ability of the prepared domains to specifically bind snake alpha-neurotoxins was demonstrated with the use of radioiodinated alpha-bungarotoxin and trifluoroacetylated alpha-cobratoxin.     

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).     

Determination of alpha-conotoxin binding modes on neuronal nicotinic acetylcholine receptors

alpha-Conotoxins, from cone snails, and alpha-neurotoxins, from snakes, are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) that have overlapping binding sites in the ACh binding pocket. These disulphide-rich peptides are used extensively as tools to localize and pharmacologically characterize specific nAChRs subtypes. Recently, a homology model based on the high-resolution structure of an ACh binding protein (AChBP) allowed the three-fingered alpha-neurotoxins to be docked onto the alpha7 nAChR. To investigate if alpha-conotoxins interact with the nAChR in a similar manner, we built homology models of human alpha7 and alpha3beta2 nAChRs, and performed docking simulations of alpha-conotoxins ImI, PnIB, PnIA and MII using the program GOLD. Docking revealed that alpha-conotoxins have a different mode of interaction compared with alpha-neurotoxins, with surprisingly few nAChR residues in common between their overlapping binding sites. These docking experiments show that ImI and PnIB bind to the ACh binding pocket via a small cavity located above the beta9/beta10 hairpin of the (+)alpha7 nAChR subunit. Interestingly, PnIB, PnIA and MII were found to bind in a similar location on alpha7 or alpha3beta2 receptors mostly through hydrophobic interactions, while ImI bound further from the ACh binding pocket, mostly through electrostatic interactions. These findings, which distinguish alpha-conotoxin and alpha-neurotoxin binding modes, have implications for the rational design of selective nAChR antagonists.     

Action of glycosyl transferases upon "Bombay" (Oh) erythrocytes. Conversion to cells showing blood-group H and A specificities.

Individuals of the rare "Bombay" (Oh) blood-group phenotype lacking, due to a genetic defect, the alpha(1-2)fucosyl transferase, which is responsible for converting blood-group H precursor substances to H-specific structures. Treatment with GDP-fucose and alpha(1-2)fucosyl transferase prepared from gastric mucosa of O individuals to transform native or ficin-treated "Bombay" erythrocytes into cells phenotypically resembling O cells. The transformation was achieved, however, after prior incubation of the "Bombay" erythrocytes with neuraminidase, indicating that blood-group H precursor molecules on the surface of these cells are masked by sialyl residues. Blood-group A specificity was conferred upon neuraminidase-treated "Bombay" cells by enzymatic transfer of alpha-N-acetylgalactosamine residues, in addition to alpha-fucose residues.     

Amino acid sequence of kappa-flavitoxin: establishment of a new family of snake venom neurotoxins

The complete amino acid sequence of kappa-flavitoxin, a neurotoxin isolated from the venom of Bungarus flaviceps, has been determined by automated Edman analysis of the intact protein and of peptides derived from digests with trypsin and chymotrypsin. kappa-Flavitoxin consists of a single 66-residue polypeptide chain which is completely devoid of methionine. The amino acid sequence of kappa-flavitoxin demonstrates that although the toxin is related to the alpha-neurotoxin family, it displays a much higher degree of homology with kappa-bungarotoxin. The conserved structural features of the kappa-neurotoxins and their pharmacological profiles, which are distinct from those of all known alpha-neurotoxins, provide evidence for a new, structurally and functionally unique family of snake venom neurotoxins.     

Amino acid sequence of neurotoxin III of the scorpion Androctonus austrialis Hector.

The amino acid sequence of neurotoxin III, purified from the venom of the North African scorpion Androctonus australis Hector, has been determined by Edman degradation using a liquid-phase sequencer. Carboxypeptidase A hydrolyses confirmed not only the sequence of the five last residues but also the presence of a free alpha-carboxylic group at the C-terminus. Edman degradation was conducted on one hand with the Quadrol [N,N,N',N'-tetrakis(2-hydroxypropyl)ethylene diamine] program and S-alkylated protein before or after coupling with sulfophenylisothiocynate (the first 34 residues were thus identified), on the other hand on tryptic and chymotryptic peptides with a dimethylbenzylamine program (residues 1--23 and 31--34 were confirmed, the positions of residues 35-64 were established). Neurotoxin III was found to belong to the same group of scorpion toxins active on mammals as neurotoxin I purified from the same venom (50 homologous positions exist in the two proteins).     

NMR analysis of interaction of LqhalphaIT scorpion toxin with a peptide corresponding to the D4/S3-S4 loop of insect para voltage-gated sodium channel

Voltage-gated sodium channels (Navs) are large transmembrane proteins that initiate action potential in electrically excitable cells. This central role in the nervous system has made them a primary target for a large number of neurotoxins. Scorpion alpha-neurotoxins bind to Navs with high affinity and slow their inactivation, causing a prolonged action potential. Despite the similarity in their mode of action and three-dimensional structure, alpha-toxins exhibit great variations in selectivity toward insect and mammalian Navs, suggesting differences in the binding surfaces of the toxins and the channels. The scorpion alpha-toxin binding site, termed neurotoxin receptor site 3, has been shown to involve the extracellular S3-S4 loop in domain 4 of the alpha-subunit of voltage-gated sodium channels (D4/S3-S4). In this study, the binding site for peptides corresponding to the D4/S3-S4 loop of the para insect Nav was mapped on the highly insecticidal alpha-neurotoxin, LqhalphaIT, from the scorpion Leiurus quinquestriatus hebraeus, by following changes in the toxin amide 1H and 15N chemical shifts upon binding. This analysis suggests that the five-residue turn (residues LqK8-LqC12) of LqhalphaIT and those residues in its vicinity interact with the D4/S3-S4 loop of Nav. Residues LqR18, LqW38, and LqA39 could also form a patch contributing to the interaction with D4/S3-S4. Moreover, a new bioactive residue, LqV13, was identified as being important for Nav binding and specifically for the interaction with the D4/S3-S4 loop. The contribution of LqV13 to NaV binding was further verified by mutagenesis. Future studies involving other extracellular regions of Navs are required for further characterization of the structure of the LqhalphaIT-Navs binding site.     

Sequence-specific 1H NMR assignments and secondary structure in the sea anemone polypeptide Stichodactyla helianthus neurotoxin I

Sequence-specific assignments are reported for the 500-MHz 1H nuclear magnetic resonance (NMR) spectrum of the 48-residue polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus (Sh I). Spin systems were first identified by using two-dimensional relayed or multiple quantum filtered correlation spectroscopy, double quantum spectroscopy, and spin lock experiments. Specific resonance assignments were then obtained from nuclear Overhauser enhancement (NOE) connectivities between protons from residues adjacent in the amino acid sequence. Of a total of 265 potentially observable resonances, 248 (i.e., 94%) were assigned, arising from 39 completely and 9 partially assigned amino acid spin systems. The secondary structure of Sh I was defined on the basis of the pattern of sequential NOE connectivities, NOEs between protons on separate strands of the polypeptide backbone, and backbone amide exchange rates. Sh I contains a four-stranded antiparallel beta-sheet encompassing residues 1-5, 16-24, 30-33, and 40-46, with a beta-bulge at residues 17 and 18 and a reverse turn, probably a type II beta-turn, involving residues 27-30. No evidence of alpha-helical structure was found.     

A defensin‐like antimicrobial peptide from the venoms of spider,Ornithoctonus hainana

The defensin‐like antimicrobial peptides have been characterized from various other arthropods including insects, scorpions, and ticks. But no natural spider defensin‐like antimicrobial peptides have ever been isolated from spiders, except couple of cDNA and DNA sequences of five spider species revealed by previous genomic study. In this work, a defensin‐like antimicrobial peptide named Oh‐defensin was purified and characterized from the venoms of the spider, Ornithoctonus hainana. Oh‐defensin is composed of 52 amino acid (aa) residues including six Cys residues that possibly form three disulfide bridges. Its aa sequence is MLCKLSMFGAVLGV PACAIDCLPMGKTGGSCEGGVCGCRKLTFKILWDKKFG. By BLAST search, Oh‐defensin showed significant sequence similarity to other arthropod antimicrobial peptides of the defensin family. Oh‐defensin exerted potent antimicrobial activities against tested microorganisms including Gram‐positive bacteria, Gram‐negative bacteria, and fungi. The cDNA encoding Oh‐defensin precursor was also cloned from the cDNA library of O. hainana. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Covalent structure of botulinum neurotoxin type A: Location of sulfhydryl groups, and disulfide bridges and identification of C-termini of light and heavy chains

Botulinum neurotoxin Type A is synthesized byClostridium botulinum as a 150 kD single chain polypeptide. The posttranslational processing of the 1296 amino acid residue long gene product involves removal of the initiating methionine, formation of disulfide bridges, and limited proteolysis (nicking) by the bacterial protease(s). The mature dichain neurotoxin is made of a 50-kD light chain and a 100-kD heavy chain connected by a disulfide bridge. DNA derived amino acid sequencepredicted a total of 9 Cys residues (Binzet al., 1990,J. Biol. Chem. 265, 9153–9158; Thompsonet al., 1990,Eur. J. Biochem. 189, 73–81). Treatment of the dichain neurotoxin, dissolved in 6 M guanidine. HCl, with 4-vinylpyridine converted 5 Cys residues into S-pyridylethyl cysteine residues; but alkylation after mercaptolysis converted all 9 Cys residues in the S-pyridylethylated form. After confirming the predicted number of Cys residues by amino acid analysis, the positions of the 5 Cys residues carrying sulfhydryl groups and the 4 involved in disulfide bridges were determined by comparing the elution patterns in reversed-phase HPLC of the cyanogen bromide mixtures of the exclusively alkylated and the mercaptolyzed-alkylated neurotoxin. The chromatographically isolated components were identified by N-terminal amino acid sequence analysis. The HPLC patterns showed characteristic differences. The Cys residuespredicted in positions 133, 164, 790, 966, and 1059 were found in the sulfhydryl form; Cys 429 and 453 were found disulfide-bridged connecting the light and heavy chains, and Cys 1234 and 1279 were found in an intrachain disulfide-bridge near the C-terminus in the heavy chain. Ten amino acid residues, Thr 438-Lys 447,predicted to be present in the single chain neurotoxin were not found in the dichain neurotoxin. Nicking of single-chain neurotoxin by the protease(s) endogenous to the bacteria therefore appears to excise these 10 amino acid residues from the nicking region which leaves Lys 437 as the C-terminus of the light chain and Ala 448 as the N-terminus of the heavy chain. The N-terminal Pro 1 and C-terminal Leu 1295,predicted from the nucleotide sequence, remain conserved after nicking. Residues Pro 1-Lys 437 and Ala 448-Leu 1295 constitute the light and heavy chains, respectively. The C-termini were determined by isolation of short C-terminal peptide fragments and subsequent sequence analysis by Edman degradation. About 20% of the amino acid sequence predicted from DNA analysis was confirmed in these studies by protein-chemical methods.     

Spontaneous nicking in the nontoxic-nonhemagglutinin component of the Clostridium botulinum toxin complex

The nontoxic-nonhemagglutinin (NTNHA) component, in both isolated form and the neurotoxin (NT)/NTNHA complexed form, was prepared protease-free from toxin complexes produced by Clostridium botulinum type D strain 4947. NTNHA in both preparations was found to be spontaneously converted to the nicked NTNHA form leading to 15- and 115-kDa fragments with the excision of several amino acid residues at specific sites on SDS-PAGE during long-term incubation, while that of the NT/NTNHA/hemagglutinin complexed form remained unnicked single-chain polypeptides under the same conditions. Considering that the NTNHA preparation contained small amounts of the nicked form of NTNHA and the addition of trypsin accelerated the cleavage, it is speculated that a nicked form of NTNHA remaining after the purification and/or NTNHA itself catalyzes the cleavage of intact NTNHA.