Interaction of three-finger proteins from snake venoms and from mammalian brain with the cys-loop receptors and their models.

With the use of surface plasmon resonance (SPR) it was shown that ws-Lynx1, a water-soluble analog of the three-finger membrane-bound protein Lynx1, that modulates the activity of brain nicotinic acetylcholine receptors (nAChRs), interacts with the acetylcholine-binding protein (AChBP) with high affinity, K D = 62 nM. This result agrees with the earlier demonstrated competition of ws-Lynx1 with radioiodinated α-bungarotoxin for binding to AChBP. For the first time it was shown that ws-Lynx1 binds to GLIC, prokaryotic Cys-loop receptor (K D = 1.3 µM). On the contrary, SPR revealed that α-cobratoxin, a three-finger protein from cobra venom, does not bind to GLIC. Obtained results indicate that SPR is a promising method for analysis of topography of ws-Lynx1 binding sites using its mutants and those of AChBP and GLIC.

Complex between α-bungarotoxin and an α7 nicotinic receptor ligand-binding domain chimaera.

To identify high-affinity interactions between long-chain α-neurotoxins and nicotinic receptors, we determined the crystal structure of the complex between α-btx (α-bungarotoxin) and a pentameric ligand-binding domain constructed from the human α7 AChR (acetylcholine receptor) and AChBP (acetylcholine-binding protein). The complex buries ~2000 Ų (1 Å=0.1 nm) of surface area, within which Arg³6 and Phe³² from finger II of α-btx form a π-cation stack that aligns edge-to-face with the conserved Tyr¹84 from loop-C of α7, while Asp³° of α-btx forms a hydrogen bond with the hydroxy group of Tyr¹84. These inter-residue interactions diverge from those in a 4.2 Å structure of α-ctx (α-cobratoxin) bound to AChBP, but are similar to those in a 1.94 Å structure of α-btx bound to the monomeric α1 extracellular domain, although compared with the monomer-bound complex, the α-btx backbone exhibits a large shift relative to the protein surface. Mutational analyses show that replacing Tyr¹84 with a threonine residue abolishes high-affinity α-btx binding, whereas replacing with a phenylalanine residue maintains high affinity. Comparison of the α-btx complex with that coupled to the agonist epibatidine reveals structural rearrangements within the binding pocket and throughout each subunit. The overall findings highlight structural principles by which α-neurotoxins interact with nicotinic receptors.

A comparative study of the performance of E. coli and K. phaffii for expressing α-cobratoxin.

Three-finger toxins (3FTxs) have traditionally been obtained via venom fractionation of whole venoms from snakes. This method often yields functional toxins, but it can be difficult to obtain pure isoforms, as it is challenging to separate the many different toxins with similar physicochemical properties that generally exist in many venoms. This issue can be circumvented via the use of recombinant expression. However, achieving the correct disulfide bond formation in recombinant toxins is challenging and requires extensive optimization of expression and purification methods to enhance stability and functionality. In this study, we investigated the expression of α-cobratoxin, a well-characterized 3FTx from the monocled cobra (Naja kaouthia), in three different expression systems, namely Escherichia coli BL21 (DE3) cells with the csCyDisCo plasmid, Escherichia coli SHuffle cells, and Komagataella phaffii (formerly known as Pichia pastoris). While none of the tested systems yielded α-cobratoxin identical to the variant isolated from whole venom, the His6-tagged α-cobratoxin expressed in K. phaffii exhibited a comparable secondary structure according to circular dichroism spectra and similar binding properties to the α7 subunit of the nicotinic acetylcholine receptor. The findings presented here illustrate the advantages and limitations of the different expression systems and can help guide researchers who wish to express 3FTxs.

Dimeric α-cobratoxin X-ray structure: localization of intermolecular disulfides and possible mode of binding to nicotinic acetylcholine receptors.

In Naja kaouthia cobra venom, we have earlier discovered a covalent dimeric form of α-cobratoxin (αCT-αCT) with two intermolecular disulfides, but we could not determine their positions. Here, we report the αCT-αCT crystal structure at 1.94 Å where intermolecular disulfides are identified between Cys(3) in one protomer and Cys(20) of the second, and vice versa. All remaining intramolecular disulfides, including the additional bridge between Cys(26) and Cys(30) in the central loops II, have the same positions as in monomeric α-cobratoxin. The three-finger fold is essentially preserved in each protomer, but the arrangement of the αCT-αCT dimer differs from those of noncovalent crystallographic dimers of three-finger toxins (TFT) or from the κ-bungarotoxin solution structure. Selective reduction of Cys(26)-Cys(30) in one protomer does not affect the activity against the α7 nicotinic acetylcholine receptor (nAChR), whereas its reduction in both protomers almost prevents α7 nAChR recognition. On the contrary, reduction of one or both Cys(26)-Cys(30) disulfides in αCT-αCT considerably potentiates inhibition of the α3ß2 nAChR by the toxin. The heteromeric dimer of α-cobratoxin and cytotoxin has an activity similar to that of αCT-αCT against the α7 nAChR and is more active against α3ß2 nAChRs. Our results demonstrate that at least one Cys(26)-Cys(30) disulfide in covalent TFT dimers, similar to the monomeric TFTs, is essential for their recognition by α7 nAChR, although it is less important for interaction of covalent TFT dimers with the α3ß2 nAChR.

Identification of α-cobratoxin in equine plasma by LC-MS/MS for doping control.

Cobra venom (Naja kaouthia) contains a toxin called α-cobratoxin (α-Cbtx). This toxin is a natural protein containing 71 amino acids (MW 7821 Da) with a reported analgesic potency greater than morphine. In 2007, in USA, this substance was found in the barns of a thoroughbred trainer and since then till date, the lack of a detection of this molecule has remained a recurring problem for the horseracing industry worldwide. To solve this problem, the first method for the detection of α-cobratoxin in equine plasma has now been developed. Plasma sample (3 mL) was treated with ammonium sulfate at the isoelectric point of α-Cbtx, and the pellet was dissolved in a phosphate buffer and mixed with methanol for precipitation. The supernatant was then concentrated prior to its extraction on WCX SPE cartridges. The eluate was concentrated with two consecutive filtration steps before the trypsin digestion. The samples were analyzed using a LC-MS/MS Q Exactive instrument at 70,000 resolution on the product ions of the doubly charged precursor of the target peptide ((24)TWCDAFCSIR(33)). The method was validated (n = 18) at 5 µg/L (640 pmol/L) according to the Association of Official Racing Chemists (AORC) requirements. The lower limit of detection was 1 µg/L (130 pmol/L). The present method has made it possible for us to confirm the presence of α-Cbtx in a horse plasma sample 24 h post the administration of α-Cbtx. Thus, the present method provides the first sensitive, specific, and reliable analytical method to confirm the presence of α-Cbtx in equine plasma.

Neurotoxin II bound to acetylcholine receptors in native membranes studied by dynamic nuclear polarization NMR.

Methods enabling structural studies of membrane-integrated receptor systems without the necessity of purification provide an attractive perspective in membrane protein structural and molecular biology. This has become feasible in principle since the advent of dynamic nuclear polarization (DNP) magic-angle-spinning NMR spectroscopy, which delivers the required sensitivity. In this pilot study, we observed well-resolved solid-state NMR spectra of extensively (13)C-labeled neurotoxin II bound to the nicotinic acetylcholine receptor (nAChR) in native membranes. We show that TOTAPOL, a biradical required for DNP, is localized at membrane and protein surfaces. The concentration of active, membrane-attached biradical decreases with time, probably because of reactive components of the membrane preparation. An optimal distribution of active biradical has strong effects on the NMR data. The presence of inactive TOTAPOL in membrane-proximal situations but active biradical in the surrounding water/glycerol "glass" leads to well-resolved spectra, yet a considerable enhancement (ε = 12) is observed. The resulting spectra of a protein ligand bound to its receptor are paving the way for further DNP investigations of proteins embedded in native membrane patches.

Novel Three-Finger Neurotoxins from Naja melanoleuca Cobra Venom Interact with GABAA and Nicotinic Acetylcholine Receptors.

Cobra venoms contain three-finger toxins (TFT) including α-neurotoxins efficiently binding nicotinic acetylcholine receptors (nAChRs). As shown recently, several TFTs block GABAA receptors (GABAARs) with different efficacy, an important role of the TFTs central loop in binding to these receptors being demonstrated. We supposed that the positive charge (Arg36) in this loop of α-cobratoxin may explain its high affinity to GABAAR and here studied α-neurotoxins from African cobra N. melanoleuca venom for their ability to interact with GABAARs and nAChRs. Three α-neurotoxins, close homologues of the known N. melanoleuca long neurotoxins 1 and 2, were isolated and sequenced. Their analysis on Torpedocalifornica and α7 nAChRs, as well as on acetylcholine binding proteins and on several subtypes of GABAARs, showed that all toxins interacted with the GABAAR much weaker than with the nAChR: one neurotoxin was almost as active as α-cobratoxin, while others manifested lower activity. The earlier hypothesis about the essential role of Arg36 as the determinant of high affinity to GABAAR was not confirmed, but the results obtained suggest that the toxin loop III may contribute to the efficient interaction of some long-chain neurotoxins with GABAAR. One of isolated toxins manifested different affinity to two binding sites on Torpedo nAChR.

Peptide Inhibitors of the α-Cobratoxin-Nicotinic Acetylcholine Receptor Interaction.

Venomous snakebites cause >100 000 deaths every year, in many cases via potent depression of human neuromuscular signaling by snake α-neurotoxins. Emergency therapy still relies on antibody-based antivenom, hampered by poor access, frequent adverse reactions, and cumbersome production/purification. Combining high-throughput discovery and subsequent structure-function characterization, we present simple peptides that bind α-cobratoxin (α-Cbtx) and prevent its inhibition of nicotinic acetylcholine receptors (nAChRs) as a lead for the development of alternative antivenoms. Candidate peptides were identified by phage display and deep sequencing, and hits were characterized by electrophysiological recordings, leading to an 8-mer peptide that prevented α-Cbtx inhibition of nAChRs. We also solved the peptide:α-Cbtx cocrystal structure, revealing that the peptide, although of unique primary sequence, binds to α-Cbtx by mimicking structural features of the nAChR binding pocket. This demonstrates the potential of small peptides to neutralize lethal snake toxins in vitro, establishing a potential route to simple, synthetic, low-cost antivenoms.

Neurotoxin-specific immunoglobulins accelerate dissociation of the neurotoxin-acetylcholine receptor complex.

Toxin isolated from cobra venom and labeled with tritium was incubated with membranes rich in acetylcholine receptors. The amount of toxin bound to the receptors was determined and the kinetics of dissociation of the receptor-toxin complex was followed. Addition of an excess of horse antiserum to the venom resulted in a significant acceleration of the dissociation reaction. Similarly, a monoclonal antibody against the toxin accelerated dissociation of the receptor-toxin complex. The results indicate that specific antibody binding destabilizes the toxin-receptor complex.

Rediocides A and G as potential antitoxins against cobra venom.

Rediocides A and G, the principle components of Trigonostemon reidioides (Kurz) Craib, which is known as Lotthanong in Thai, were investigated for a detoxification mechanism against Naja kaouthia venom by in silico, in vitro, and in vivo methods. Molecular dockings of alpha-cobratoxin with rediocides A and G were performed, and the binding energies were found to be -14.17 and -14.14 kcal/mol, respectively. Rediocides bind to alpha-cobratoxin at the same location as alpha-cobratoxin binds to the nicotinic acetylcholine receptor (nAChR), i.e., at the Asp27, Phe29, Arg33, Gly34, Lys35, and Val37 residues. alpha-Cobratoxin cannot bind to nAChR, because some of its binding sites are occupied with rediocides. From in vitro SDS-PAGE, it was found that rediocides can diminish the bands of alpha-cobratoxin. In the presence of acetylcholine-binding protein (AChBP), it was apparent that rediocides can bind both alpha-cobratoxin and AChBP. From an in vivo test, it was found that injection of rediocides at 0.5 mg/kg immediately after an alpha-cobratoxin dose of three times LD(50) cannot prolong the survival time of mice. However, rediocide can prolong the survival time, if it is injected 30 min before the injection of alpha-cobratoxin. The in vitro SDS-PAGE and the in vivo results support the in silico detoxification mechanism of rediocides against cobra venom at a molecular level.

Proteomic insights into short neurotoxin-driven, highly neurotoxic venom of Philippine cobra (Naja philippinensis) and toxicity correlation of cobra envenomation in Asia.

The Philippine cobra, Naja philippinensis, is a WHO Category 1 venomous snake of medical importance responsible for fatal envenomation in the northern Philippines. To elucidate the venom proteome and pathophysiology of envenomation, N. philippinensis venom proteins were decomplexed with reverse-phase high-performance liquid chromatography, and protein fractions were subsequently digested with trypsin, followed by nano-liquid chromatography-tandem mass spectrometry analysis and data mining. Three-finger toxins (3FTX, 66.64% of total venom proteins) and phospholipases A2 (PLA2, 22.88%) constitute the main bulk of venom proteome. Other proteins are present at low abundances (<4% each); these include metalloproteinase, serine protease, cobra venom factor, cysteine-rich secretory protein, vespryn, phosphodiesterase, 5' nucleotidase and nerve growth factor. In the three-finger toxin family, the alpha-neurotoxins comprise solely short neurotoxins (SNTX, 44.55%), supporting that SNTX is the principal toxin responsible for neuromuscular paralysis and lethality reported in clinical envenomation. Cytotoxins (CTX) are the second most abundant 3FTX proteins in the venom (21.31%). The presence of CTX correlates with the venom cytotoxic effect, which is more prominent in murine cells than in human cells. From the practical standpoint, SNTX-driven neuromuscular paralysis is significant in N. philippinensis envenomation. Antivenom production and treatment should be tailored accordingly to ensure effective neutralization of SNTX. BIOLOGICAL SIGNIFICANCE: The venom proteome of Naja philippinensis, the Philippine cobra, is unravelled for the first time. Approximately half the protein bulk of the venom is made up of short neurotoxins (44.55% of the total venom proteins). As the only alpha-neurotoxins present in the venom, short neurotoxins are the causative toxins of the post-synaptic blockade and fast-onset neuromuscular paralysis in N. philippinensis envenomation. A substantial amount of cytotoxins (21.31%) was also detected in N. philippinensis venom, supporting that the venom can be cytotoxic although the effect is much weaker in human cells compared to murine cells. The finding is consistent with the low incidence of local tissue necrosis in N. philippinensis envenomation, although this does not negate the need for monitoring and care of bite wound in the patients.

Alpha-cobratoxin as a possible therapy for multiple sclerosis: a review of the literature leading to its development for this application.

The use of snake venom in the treatment of multiple sclerosis has been, at best, controversial. The anecdotal reports for snake venom's beneficial effects in this condition may be supportable now by recent scientific evidence. Cobratoxin, a neurotoxin obtained from the venom of the Thailand cobra, has demonstrated several pharmacological activities that strongly support its use in this application. By employing a chemical detoxification step, the neurotoxin can be rendered safe for administration to humans with minimal side effects. This modified neurotoxin has demonstrated neuromodulatory, antiviral, and analgesic activity, elements associated with the multiple sclerosis condition. Modified cobratoxin has demonstrated potent immunosuppressive activity in acute and chronic animal models of the disease. The drug is under investigation for use in adrenomyeloneuropathy and clinical trials in Multiple sclerosis are planned.

Venomics of Naja sputatrix, the Javan spitting cobra: A short neurotoxin-driven venom needing improved antivenom neutralization.

The venom proteome of Naja sputatrix (Javan spitting cobra) was elucidated through reverse-phase HPLC, nano-ESI-LCMS/MS and data mining. A total of 97 distinct protein forms belonging to 14 families were identified. The most abundant proteins are the three-finger toxins (3FTXs, 64.22%) and phospholipase A2 (PLA2, 31.24%), followed by nerve growth factors (1.82%), snake venom metalloproteinase (1.33%) and several proteins of lower abundance (<1%) including a variety of venom enzymes. At subproteome, the 3FTx is dominated by cytotoxins (48.08%), while short neurotoxins (7.89%) predominate over the long neurotoxins (0.48%) among other neurotoxins of lesser toxicity (muscarinic toxin-like proteins, 5.51% and weak neurotoxins, 2.26%). The major SNTX, CTX and PLA2 toxins were isolated with intravenous median lethal doses determined as 0.13, 1.06 and 0.50µg/g in mice, respectively. SABU, the Indonesia manufactured homologous tri-specific antivenom could neutralize the CTX and PLA2 fraction with moderate potency (potency=0.14-0.16mg toxin per ml antivenom). The SNTX, however, was very poorly neutralized with a potency level of 0.034mg/ml, indicating SNTX as the main limiting factor in antivenom neutralization. The finding helps elucidate the inferior efficacy of SABU reported in neutralizing N. sputatrix venom, and supports the call for antivenom improvement. BIOLOGICAL SIGNIFICANCE: The Javan spitting cobra, Naja sputatrix is by itself a unique species and should not be confused as the equatorial and the Indochinese spitting cobras. The distinction among the spitting cobras was however unclear prior to the revision of cobra systematics in the mid-90's, and results of some earlier studies are now questionable as to which species was implicated back then. The current study successfully profiled the venom proteome of authenticated N. sputatrix, and showed that the venom is made up of approximately 64% three-finger toxins (including neurotoxins and cytotoxins) and 31% phospholipases A2 by total venom proteins. The findings verified that the paralyzing components in the venom i.e. neurotoxins are predominantly the short-chain subtype (SNTX) far exceeding the long-chain subtype (LNTX) which is more abundant in the venoms of monocled cobra and Indian common cobra. The neurotoxicity of N. sputatrix venom is hence almost exclusively SNTX-driven, and effective neutralization of the SNTX is the key to early reversal of paralysis. Unfortunately, as shown through a toxin-specific assay, the immunological neutralization of the SNTX using the Indonesian antivenom (SABU) was extremely weak, implying that SABU has limited therapeutic efficacy in treating N. sputatrix envenomation clinically. From the practical standpoint, actions need to be taken at all levels from laboratory to production and policy making to ensure that the shortcoming is overcome.

Geographical venom variations of the Southeast Asian monocled cobra (Naja kaouthia): venom-induced neuromuscular depression and antivenom neutralization.

The Southeast Asian monocled cobras (Naja kaouthia) exhibit geographical variations in their venom proteomes, especially on the composition of neurotoxins. This study compared the neuromuscular depressant activity of the venoms of N. kaouthia from Malaysia (NK-M), Thailand (NK-T) and Vietnam (NK-V), and the neutralization of neurotoxicity by a monospecific antivenom. On chick biventer cervicis nerve-muscle preparation, all venoms abolished the indirect twitches, with NK-T venom being the most potent (shortest t90, time to 90% twitch inhibition), followed by NK-V and NK-M. Acetylcholine and carbachol failed to reverse the blockade, indicating irreversible/pseudo-irreversible post-synaptic neuromuscular blockade. KCl restored the twitches variably (NK-M preparation being the least responsive), consistent with different degree of muscle damage. The findings support that NK-T venom has the most abundant curarimimetic alpha-neurotoxins, while NK-M venom contains more tissue-damaging cytotoxins. Pre-incubation of tissue with N. kaouthia monovalent antivenom (NKMAV) prevented venom-induced twitch depression, with the NK-T preparation needing the largest antivenom dose. NKMAV added after the onset of neuromuscular depression could only halt the inhibitory progression but failed to restore full contraction. The findings highlight the urgency of early antivenom administration to sequester as much circulating neurotoxins as possible, thereby hastening toxin elimination from the circulation. In envenomed mice, NKMAV administered upon the first neurological sign neutralized the neurotoxic effect, with the slowest full recovery noticed in the NK-T group. This is consistent with the high abundance of neurotoxins in the NK-T venom, implying that a larger amount or repeated dosing of NKMAV may be required in NK-T envenomation.

Protective immunity against alpha-cobratoxin following a single administration of a genetic vaccine encoding a non-toxic cobratoxin variant.

Venomous snakebites result in almost 125,000 deaths per year worldwide. We present a new paradigm for the development of vaccines to protect against snakebite, using knowledge of the structure and action of specific toxins combined with a gene-based strategy to deliver a toxin gene modified to render it non-toxic while maintaining its three-dimensional structure and hence its ability to function as an immunogen. As a model for this approach, we developed a genetic vaccine to protect against alpha-cobratoxin (CTX), a potent, post-synaptic neurotoxin that is the major toxic component of the venom of Naja kaouthia, the monocellate cobra. To develop the vaccine, substitutions in the CTX cDNA were introduced at two residues critical for binding to the nicotinic acetylcholine receptor (Asp27 to Arg, Arg33 to Gly). The mutated CTX expression cassette was delivered in the context of a replication deficient adenovirus vector (AdmCTX). To assess whether expression of the mutated CTX in vivo leads to the development of protective immunity, BALB/c mice were challenged by IV administration of 2 microg of alpha-cobratoxin protein 21 or 63 days after administration of AdmCTX or Ad- Null (as a control; both, 10(9) particle units). Animals receiving AdmCTX but no alpha-cobratoxin challenge suffered no ill effects, but > or =80% of naive animals or those receiving the AdNull control vector died within 10 min from the alpha-cobratoxin challenge. In contrast, 100% of animals receiving a single dose of AdmCTX 21 or 63 days prior to alpha-cobratoxin challenge survived. The data demonstrates that an adenovirus-based vaccine can be developed to protect against lethal challenge with a potent snake venom. The effectiveness of this approach might serve as a basis to consider the development of a global public health program to protect those at risk for death by snakebite.

Nuclear magnetic resonance solution structure of the alpha-neurotoxin from the black mamba (Dendroaspis polylepis polylepis).

The three-dimensional structure in solution of the alpha-neurotoxin from the black mamba (Dendroaspis polylepis polylepis) has been determined by nuclear magnetic resonance spectroscopy. A high quality structure for this 60-residue protein was obtained from 656 NOE distance constraints and 143 dihedral angle constraints, using the distance geometry program DIANA for the structure calculation and AMBER for restrained energy minimization. For a group of 20 conformers used to represent the solution structure, the average root-mean-square deviation value calculated for the polypeptide backbone heavy atoms relative to the mean structure was 0.45 A. The protein consists of a core region from which three finger-like loops extend outwards. It includes a short, two-stranded antiparallel beta-sheet of residues 1-5 and 13-17, a three-stranded antiparallel beta-sheet involving residues 23-31, 34-42 and 51-55, and four disulfide bridges in the core region. There is also extensive non-regular hydrogen bonding between the carboxy-terminal tail of the polypeptide chain and the rest of the core region. Comparison with the crystal structure of erabutoxin-b indicates that the structure of alpha-neurotoxin is quite similar to other neurotoxin structures, but that local structural differences are seen in regions thought to be important for binding of neurotoxins to the acetylcholine receptor. For two regions of the alpha-neurotoxin structure there is evidence for an equilibrium between multiple conformations, which might be related to conformational rearrangements upon binding to the receptor. Overall, the alpha-neurotoxin presents itself as a protein with a stable core and flexible surface areas that interact with the acetylcholine receptor in such a way that high affinity binding is achieved by conformational rearrangements of the deformable regions of the neurotoxin structure.

Further evidence showing that neurotoxin-acetylcholine receptor dissociation is accelerated by monoclonal neurotoxin-specific immunoglobulin.

We have demonstrated that the dissociation of Naja nigricollis alpha-toxin from the two acetylcholine receptor sites [Weber and Changeux, Molec. Pharmac. 10, 1-14 (1974); Rousselet et al., Eur. J. Biochem. 140, 31-37 (1984)], is markedly accelerated by a monoclonal neurotoxin-specific antibody. The dissociation of the toxin occurs in a biphasic manner in the presence of a 900 molar excess of immunoglobulin (with respect to toxin concn). The progress curves are characterized by first-order kinetics. Under these conditions the maximal dissociation rate is achieved as further rate enhancement cannot be induced by exposure to an increased immunoglobulin level. In contrast when a toxin-immunoglobulin complex is incubated with a large excess of receptor, the dissociation kinetics of the complex are not enhanced. The data fit a kinetic model which implicates the existence of a transient ternary complex involving the receptor, the toxin and the antibody.

Nicotinic acetylcholine receptor α7 subunit is involved in the cobratoxin-induced antinociception in an animal model of neuropathic pain.

In this study we report that cobratoxin (CbTX), a long-chain postsynaptic α-neurotoxin isolated from the Thailand cobra, Naja naja kaouthia, has antinociceptive effect in rats with neuropathic pain. The neuropathic pain model was established in rats with partial sciatic nerve ligature (PSNL) method. The pain response was examined behaviorally with mechanical paw withdrawal and thermal paw withdrawal method. Different doses (0.56, 1.12 and 4.50 µg/kg) of CbTX were injected intrathecally. Injection of CbTX resulted in a significant dose-dependent antinociception as evidenced by increased mechanical withdrawal threshold and thermal withdrawal latency. CbTX also induces a significant dose-dependent inhibition of pain-evoked unit discharges of thalamic parafascicular neurons. Both the behavioral mechanical and thermal antinociception and the inhibition of pain-evoked discharges of neurons in thalamic parafascicular nucleus in PSNL model could be mimicked by PUN282987, selective α7 nicotinic AChR (α7 nAChR) agonist and reversed by methyllycaconitine (MLA) selective α7 nAChR antagonist. In summary, these results suggested that AChR α7 subunit was involved in the antinociceptive action of CbTX for neuropathic pain and might be the candidate target for analgesic drug design.

Motions and structural variability within toxins: implication for their use as scaffolds for protein engineering.

Animal toxins are small proteins built on the basis of a few disulfide bonded frameworks. Because of their high variability in sequence and biologic function, these proteins are now used as templates for protein engineering. Here we report the extensive characterization of the structure and dynamics of two toxin folds, the "three-finger" fold and the short alpha/beta scorpion fold found in snake and scorpion venoms, respectively. These two folds have a very different architecture; the short alpha/beta scorpion fold is highly compact, whereas the "three-finger" fold is a beta structure presenting large flexible loops. First, the crystal structure of the snake toxin alpha was solved at 1.8-A resolution. Then, long molecular dynamics simulations (10 ns) in water boxes of the snake toxin alpha and the scorpion charybdotoxin were performed, starting either from the crystal or the solution structure. For both proteins, the crystal structure is stabilized by more hydrogen bonds than the solution structure, and the trajectory starting from the X-ray structure is more stable than the trajectory started from the NMR structure. The trajectories started from the X-ray structure are in agreement with the experimental NMR and X-ray data about the protein dynamics. Both proteins exhibit fast motions with an amplitude correlated to their secondary structure. In contrast, slower motions are essentially only observed in toxin alpha. The regions submitted to rare motions during the simulations are those that exhibit millisecond time-scale motions. Lastly, the structural variations within each fold family are described. The localization and the amplitude of these variations suggest that the regions presenting large-scale motions should be those tolerant to large insertions or deletions.

Phosphorescence and ODMR study of the binding interactions of acetylcholine receptor alpha-subunit peptides with alpha-cobratoxin.

Optical detection of magnetic resonance (ODMR) and phosphorescence spectroscopy have been applied to synthetic peptides derived from the alpha-subunit of the nicotinic acetylcholine receptor of Torpedo californica and their complexes with alpha-cobratoxin (CBTX). The CBTX Trp phosphorescence is strongly quenched by the proximal disulfide linkage, while the emission wavelengths and ODMR frequencies of the 18-mer alpha 181-198 indicate a more hydrophobic Trp environment than in the 12-mer alpha 185-196. Binding to CBTX produces a subtle increase in the hydrophobicity of the Trp environment for the peptides, in qualitative agreement with a recently proposed binding model, in which a receptor Trp residue interacts strongly with a hydrophobic cleft of the toxin.