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Activating a receptor to excite a neuron Transmitting signals between nerve cells, occuring at structures known as synapses, is critical to processes such as learning and memory. Fast transmission occurs when glutamate is released from a presynaptic neuron and binds to ionotropic glutamate receptors (iGluRs) in the cell membrane of a postsynaptic neuron. The iGluR contains an ion channel that is transiently opened, to activate the postsynaptic neuron, but then closes rapidly. Chen et al. and Yelshanskaya et al. report crystal structures in a range of conformations that together provide insight into how glutamate binding causes the channel to open and how other molecules that bind to the receptor modulate this. The information could aid in the design of drugs to treat cognitive impairment or seizure disorders Science , this issue p. 1021 and p. 1070

Peptide therapeutics are acclaimed as a promising addition to the pharmaceutical arena, and they continue to attract interest due to their high potency, bioavailability, and fewer concerns with toxicity, drug to drug cross-reactions, and tissue accumulation. Around 700 species of marine snails of the genus Conus are distributed throughout tropical and subtropical waters. As different species preferentially hunt fish, worms, or molluscs they are categorized as piscivorous, vermivorous, or molluscivorous, respectively, although some cone snail species can feed on more than one prey type. These slow-moving creatures evolved into predators through incorporation of a specialized envenomation apparatus that enables them to quickly subdue their fast-moving prey. Conotoxins target a wide range of receptors and ion channels with unparalleled potency and selectivity. They have consequently become the subject of intense research in light of their immense diagnostic and therapeutic potential and are the focus of this review.

α-Conotoxins are small peptide toxins that are found in the venom of marine snails, Conus, and extremely potent antagonists of various subtypes of nicotinic acetylcholine receptors (nAChRs). nAChRs are cholinergic receptors that form ligand-gated ion channels in the plasma membranes of certain neurons and on the postsynaptic side of the neuromuscular junction. As nAChRs have an important role in regulating transmitter release, cell excitability, and neuronal integration, nAChR dysfunctions have been implicated in a variety of severe pathologies like epilepsy, myasthenic syndromes, schizophrenia, Parkinson’s and Alzheimer’s diseases. In order to expand the knowledge about cone snail toxins, we examined the venom of an unknown Conus species, Conus longurionis. This study covers the isolation of an 18-amino acid peptide from the venom of the marine snail Conus longurionis that is active on nAChRs. As far as we are aware, this is the first characterization of a conotoxin from this species. The peptide, named α-conotoxin Lo1a, was characterized by electrophysiological screening against several types of cloned nicotinic receptors expressed in Xenopus laevis oocytes. The toxin, member of the α4/7 family, blocks the response to acetylcholine in oocytes expressing α7 nAChRs, an important channel target in brain disorders, with an IC50 of 3.24 ± 0.7 µM. Also α3β4 was found to be blocked with a percentage of 40% at 10 µM. Furthermore, Lo1a shows a high selectivity for neuronal versus muscle subtype nAChRs. The three-dimensional solution structure of the α-conotoxin Lo1a was determined by NMR spectroscopy. The NMR structure reveals that the peptide backbone adopts a compact “W-shaped” conformation having two loops that are reinforced by two disulfide bonds (Cys3-Cys9 and Cys4-Cys17). Moreover, we designed two mutants, Lo1a-ΔD and Lo1a-RRR, in which an Asp is deleted or an Arg-tail replaces the Asp at the C-terminus respectively. These mutant toxins not only block α7 with a lower IC50, but also surprisingly, shift the selectivity from the neuronal subtype nAChRs towards the muscle subtype nAChRs. In summary, our findings provide insight into the assembly, structure and activity of the α-contoxin Lo1a that targets preferably α7-nAChR. Consequently, our study contributes to the discovery of new ligands to affect brain disorders including schizophrenia, Alzheimer’s disease, traumatic brain injury and ADHD. ispartof: Structure-function elucidation of a new α-conotoxin, Lo1a, from Conus longurionis vol:289 issue:14 pages:9573-9583 ispartof: location:Paris status: published

A cone snail venom peptide, μO§-conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligand-binding site. μO§-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJSSG). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) NaV1.2. A mutant channel of rNaV1.2 lacking a cysteine near the pore loop of domain II (C910L), was10(3)-fold less sensitive to GVIIJSSG than was wild-type rNaV1.2. In contrast, although rNaV1.5 was10(4)-fold less sensitive to GVIIJSSG than NaV1.2, an rNaV1.5 mutant with a cysteine in the homologous location, rNaV1.5[L869C], was10(3)-fold more sensitive than wild-type rNaV1.5. The susceptibility of rNaV1.2 to GVIIJSSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNaVβ2 or rNaVβ4, but not that of rNaVβ1 or rNaVβ3, protected rNaV1.1 to -1.7 (excluding NaV1.5) against block by GVIIJSSG. Thus, GVIIJ-related peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which NaVβ- and NaVα-subunits are present in native neurons.

Cone snails express a sophisticated arsenal of small bioactive peptides known as conopeptides or conotoxins (CTxs). Through evolutionary selection, these peptides have gained the ability to interact with a range of ion channels and receptors, such as nicotinic acetylcholine receptors (nAChRs). Here, we used reversed-phase high performance liquid chromatography (RP-HPLC) and electrospray ionization-mass spectrometry (ESI-MS) to explore the venom peptide diversity of Conus textile, a species of cone snail native to Hainan, China. One fraction of C. textile crude venom potently blocked α3β2 nAChRs. Subsequent purification, synthesis, and tandem mass spectrometric analysis demonstrated that the most active compound in this fraction was identical to α-CTx TxIA, an antagonist of α3β2 nAChRs. Then three disulfide isoforms of α-CTx TxIA were synthesized and their activities were investigated systematically for the first time. As we observed, disulfide isomerisation was particularly important for α-CTx TxIA potency. Although both globular and ribbon isomers showed similar retention times in RP-HPLC, globular TxIA potently inhibited α3β2 nAChRs with an IC50 of 5.4 nM, while ribbon TxIA had an IC50 of 430 nM. In contrast, beads isomer had little activity towards α3β2 nAChRs. Two-step oxidation synthesis produced the highest yield of α-CTx TxIA native globular isomer, while a one-step production process based on random oxidation folding was not suitable. In summary, this study demonstrated the relationship between conotoxin activity and disulfide connectivity on α-CTx TxIA.

A nano-flow high-resolution screening platform, featuring a parallel chip-based microfluidic bioassay and mass spectrometry coupled to nano-liquid chromatography, was applied to screen animal venoms for nicotinic acetylcholine receptor like (nAChR) affinity by using the acetylcholine binding protein, a mimic of the nAChR. The potential of this microfluidic platform is demonstrated by profiling the Conus textile venom proteome, consisting of over 1, 000 peptides. Within one analysis (

Marine snails of the genus Conus are a large family of predatory gastropods with an unparalleled molecular diversity of pharmacologically active compounds in their venom. Cone snail venom comprises of a rich and diverse cocktail of peptide toxins which act on a wide variety of ion channels such as voltage-gated sodium- (NaV), potassium- (KV), and calcium- (CaV) channels as well as nicotinic acetylcholine receptors (nAChRs) which are classified as ligand-gated ion channels. The mode of action of several conotoxins has been the subject of investigation, while for many others this remains unknown. This review aims to give an overview of the knowledge we have today on the molecular pharmacology of conotoxins specifically interacting with nAChRs along with the structure-function relationship data ispartof: Marine Drugs vol:12 issue:5 pages:2970-3004 ispartof: location:Switzerland status: published

Peptide de13a was previously purified from the venom of the worm-hunting cone snail Conus delessertii from the Yucatan Channel, Mexico. This peptide has eight cysteine (Cys) residues in the unique arrangement C C C CC C C C, which defines the cysteine framework XIII (“ ” represents one or more non-Cys residues). Remarkably, δ-hydroxy-lysine residues have been found only in conotoxin de13a, which also contains an unusually high proportion of hydroxylated amino acid residues. Here, we report the cDNA cloning of the complete precursor De13.1 of a related peptide, de13b, which has the same Cys framework and inter-Cys spacings as peptide de13a, and shares high protein/nucleic acid sequence identity (87%/90%) with de13a, suggesting that both peptides belong to the same conotoxin gene superfamily. Analysis of the signal peptide of precursor De13.1 reveals that this precursor belongs to a novel conotoxin gene superfamily that we chose to name gene superfamily G. Thus far superfamily G only includes two peptides, each of which contains the same, distinctive Cys framework and a high proportion of amino acid residues with hydroxylated side chains.

Marine cone snail venoms consist of large, naturally occurring combinatorial libraries of disulfide-constrained peptide neurotoxins known as conotoxins, which have profound potential in the development of analgesics. In this study, we report a synthetic combinatorial strategy that probes the hypervariable regions of conotoxin frameworks to discover novel analgesic agents by utilizing high diversity mixture-based positional-scanning synthetic combinatorial libraries (PS-SCLs). We hypothesized that the direct in vivo testing of these mixture-based combinatorial library samples during the discovery phase would facilitate the identification of novel individual compounds with desirable antinociceptive profiles while simultaneously eliminating many compounds with poor activity or liabilities of locomotion and respiration. A PS-SCL was designed based on the α-conotoxin RgIA-ΔR n-loop region and consisted of 10,648 compounds systematically arranged into 66 mixture samples. Mixtures were directly screened in vivo using the mouse 55 °C warm-water tail-withdrawal assay, which allowed deconvolution of amino acid residues at each position that confer antinociceptive activity. A second generation library of 36 individual α-conotoxin analogues was synthesized using systematic combinations of amino acids identified from PS-SCL deconvolution and further screened for antinociceptive activity. Six individual analogues exhibited comparable antinociceptive activity to that of the recognized analgesic α-conotoxin RgIA-ΔR, and were selected for further examination of antinociceptive, respiratory, and locomotor effects. Three lead compounds were identified that produced dose-dependent antinociception without significant respiratory depression or decreased locomotor activity. Our results represent a unique approach for rapidly developing novel lead α-conotoxin analogues as low-liability analgesics with promising therapeutic potential.

International audience; Cone snails produce highly complex venom comprising mostly small biologically active peptides known as conotoxins or conopeptides. Early estimates that suggested 50-200 venom peptides are produced per species have been recently increased at least 10-fold using advanced mass spectrometry. To uncover the mechanism(s) responsible for generating this impressive diversity, we used an integrated approach combining second-generation transcriptome sequencing with high sensitivity proteomics. From the venom gland transcriptome of Conus marmoreus, a total of 105 conopeptide precursor sequences from 13 gene superfamilies were identified. Over 60% of these precursors belonged to the three gene superfamilies O1, T, and M, consistent with their high levels of expression, which suggests these conotoxins play an important role in prey capture and/or defense. Seven gene superfamilies not previously identified in C. marmoreus, including five novel superfamilies, were also discovered. To confirm the expression of toxins identified at the transcript level, the injected venom of C. marmoreus was comprehensively analyzed by mass spectrometry, revealing 2710 and 3172 peptides using MALDI and ESI-MS, respectively, and 6254 peptides using an ESI-MS TripleTOF 5600 instrument. All conopeptides derived from transcriptomic sequences could be matched to masses obtained on the TripleTOF within 100 ppm accuracy, with 66 (63%) providing MS/MS coverage that unambiguously confirmed these matches. Comprehensive integration of transcriptomic and proteomic data revealed for the first time that the vast majority of the conopeptide diversity arises from a more limited set of genes through a process of variable peptide processing, which generates conopeptides with alternative cleavage sites, heterogeneous post-translational modifications, and highly variable N- and C-terminal truncations. Variable peptide processing is expected to contribute to the evolution of venoms, and explains how a limited set of ∼ 100 gene transcripts can generate thousands of conopeptides in a single species of cone snail.

Conopeptides are a diverse array of small linear and reticulated peptides that interact with high potency and selectivity with a large diversity of receptors and ion channels. They are used by cone snails for prey capture or defense. Recent advances in venom gland transcriptomic and venom peptidomic/proteomic technologies combined with bioactivity screening approaches lead to the identification of new toxins with original pharmacological profiles. Here, from transcriptomic/proteomic analyses of the Conus consors cone snail, we identified a new conopeptide called τ-CnVA, which displays the typical cysteine framework V of the T1-conotoxin superfamily. This peptide was chemically synthesized and its three-dimensional structure was solved by NMR analysis and compared to that of TxVA belonging to the same family, revealing very few common structural features apart a common orientation of the intercysteine loop. Because of the lack of a clear biological function associated with the T-conotoxin family, τ-CnVA was screened against more than fifty different ion channels and receptors, highlighting its capacity to interact selectively with the somatostatine sst3 receptor. Pharmacological and functional studies show that τ-CnVA displays a micromolar (Ki of 1.5 μM) antagonist property for the sst3 receptor, being currently the only known toxin to interact with this GPCR subfamily.

International audience; The glycopeptide CcTx, isolated from the venom of the piscivorous cone snail Conus consors, belongs to the κA-family of conopeptides. These toxins elicit excitotoxic responses in the prey by acting on voltage-gated sodium channels. The structure of CcTx, a first in the κA-family, has been determined by high-resolution NMR spectroscopy together with the analysis of its O-glycan at Ser7. A new type of glycopeptide O-glycan core structure, here registered as core type 9, containing two terminal L-galactose units {α-L-Galp-(1→4)-α-D-GlcpNAc-(1→6)-[α-L-Galp-(1→2)-β-D-Galp-(1→3)-]α-D-GalpNAc-(1→O)}, is highlighted. A sequence comparison to other putative members of the κA-family suggests that O-linked glycosylation might be more common than previously thought. This observation alone underlines the requirement for more careful and in-depth investigations into this type of post-translational modification in conotoxins.

In natural proteins and peptides, amino acids exist almost invariably as l-isomers. There are, however, several examples of naturally-occurring peptides containing d-amino acids. In this study we investigated the role of a naturally-occurring d-amino acid in a small peptide identified in the transcriptome of a marine cone snail. This peptide belongs to a family of peptides known as contryphans, all of which contain a single d-amino acid residue. The solution structure of this peptide was solved by NMR, but further investigations with molecular dynamics simulations suggest that its solution behaviour may be more dynamic than suggested by the NMR ensemble. Functional tests in mice uncovered a novel bioactivity, a depressive phenotype that contrasts with the hyperactive phenotypes typically induced by contryphans. Trp3 is important for bioactivity, but this role is independent of the chirality at this position. The d-chirality of Trp3 in this peptide was found to be protective against enzymatic degradation. Analysis by NMR and molecular dynamics simulations indicated an interaction of Trp3 with lipid membranes, suggesting the possibility of a membrane-mediated mechanism of action for this peptide.

Microorganisms produce a large number of medically relevant small molecules through unique biosynthetic mechanisms. These small molecules have been widely used to develop drugs to treat a range of infectious diseases, cancers and other pathologies. In their natural environment microorganisms use these small molecules for signalling or defence. As microorganisms have adapted to different environments, they have greatly expanded their chemical profile and thus the investigation of microorganisms from new environments has often led to the discovery of novel chemistry. Microbes often exist in association with higher eukaryotic hosts and are therefore exposed to unique environments and thus produce unique chemistry. The host organism can benefit by utilising microbial metabolites for their own defence against infections, predators, and for chemical signalling. Interestingly, some venomous animals such as puffer fish and the blue ringed octopus utilise toxins produced by associated microbes to defend against predators. However this chemistry remains largely unexplored for the vast majority of venomous animals. In this study, we investigated metabolites from microbes associated with venomous animals including spiders, centipedes, cone snails, scorpions and wasps. We cultivated microbes from 15 venomous animals to produce a library of ~360 strains. The microbe library was cultured under a range of conditions and the resultant solvent extracts were subjected to chemical, biological and genomic studies. This PhD thesis focuses on metabolites produced by three wasp-associated fungi, Aspergillus sp. (CMB-W031), Talaromyces sp. (CMB- W045) and Quambalaria sp. (CMB-W001) and two cone snail-derived bacteria, Streptomyces sp. (CMB-CS038 and CMB-CS150) and Bacillus sp. (CMB-CS100). Chapter 1 Reviews the current status of drug discovery and symbiotic microbial strains involved in producing bioactive metabolites Chapter 2 Describes our approach to venomous animal microbial cultivation and microbe library biological and chemical profiling Chapter 3 Describes the identification and structure elucidation of a new nitrated diketopiperazine, waspergillamide A from a wasp-derived fungi Aspergillus sp. (CMB-W031) Chapter 4 Describes the identification and structure elucidation of new siderophores and azaphilones from a wasp-derived fungi Talaromyces sp. (CMB-W045) ii Chapter 5 Describes the chemical investigation of several cone snail-derived Streptomyces sp. (CMB-CS038, CMB-CS150, CMB-CS138, CMB-CS143 and CMB-CS145) and their common biosynthetic capacity. Chapter 6 Describes the identification and characterization of a pheromone from the wasp-derived fungi Quambalaria sp. (CMB-W001) Chapter 7 Describes the identification of a novel biosynthetic gene cluster in the cone snail-derived Bacillus sp. (CMB-CS100), through in silico approaches Chapter 8 Conclusions and outlook Our studies showed that venomous animals are a rich source of fungi and bacteria with the capacity to produce diverse metabolites. Chemical and biological screening on microbes demonstrated their ability to synthesise bioactive components. The isolation of new metabolites from wasp-derived fungi reveals the unexplored potential of venomous animal associated microbes as a source of new chemistry. The discovery of an insect pheromone from a wasp-associated fungi and antifungal compounds from cone snail bacteria hints at their use as signalling molecules and as anti-infective agents respectively by their host organisms. To explore otherwise silent biosynthetic pathways, we sequenced and analysed the genome of a cone snail bacterium and identified a novel lantipeptide gene cluster whose expression in traditional culture conditions was validated by proteomics. Our results indicate this approach can also be applied to other isolates in combination with chemical approaches to discover and characterise new metabolites. In summary, this thesis highlights the potential of venomous animal-associated microbes as sources of new bioactive metabolites.

Background The venom of predatory marine cone snails mainly contains a diverse array of unique bioactive peptides commonly referred to as conopeptides or conotoxins. These peptides have proven to be valuable pharmacological probes and potential drugs because of their high specificity and affinity to important ion channels, receptors and transporters of the nervous system. Most previous studies have focused specifically on the conopeptides from piscivorous and molluscivorous cone snails, but little attention has been devoted to the dominant vermivorous species. Results The vermivorous Chinese tubular cone snail, Conus betulinus, is the dominant Conus species inhabiting the South China Sea. The transcriptomes of venom ducts and venom bulbs from a variety of specimens of this species were sequenced using both next-generation sequencing and traditional Sanger sequencing technologies, resulting in the identification of a total of 215 distinct conopeptides. Among these, 183 were novel conopeptides, including nine new superfamilies. It appeared that most of the identified conopeptides were synthesized in the venom duct, while a handful of conopeptides were identified only in the venom bulb and at very low levels. Conclusions We identified 215 unique putative conopeptide transcripts from the combination of five transcriptomes and one EST sequencing dataset. Variation in conopeptides from different specimens of C. betulinus was observed, which suggested the presence of intraspecific variability in toxin production at the genetic level. These novel conopeptides provide a potentially fertile resource for the development of new pharmaceuticals, and a pathway for the discovery of new conotoxins. Electronic supplementary material The online version of this article (doi:10.1186/s13742-016-0122-9) contains supplementary material, which is available to authorized users.

A novel conotoxin, named as PiVIIA, was isolated from the venom of Conus princeps, a marine predatory cone snail collected in the Pacific Southern Coast of Mexico. Chymotryptic digest of the S-alkylated peptide in combination with liquid chromatography coupled to tandem mass spectrometry, were used to define the sequencing of this peptide. Eleven N-terminal amino acids were verified by automated Edman degradation. PiVIIA is a 25-mer peptide (CDAOTHYCTNYWγCCSGYCγHSHCW) with six cysteine residues forming three disulphide bonds, a hydroxyproline (O) and two gamma carboxyglutamic acid (γ) residues. Based on the arrangement of six Cys residues (C-C-CC-C-C), this conotoxin might belong to the O2-superfamily. Moreover, PiVIIA has a conserved motif (-γCCS-) that characterizes γ-conotoxins from molluscivorous Conus. Peptide PiVIIA has 45% sequence identity with γ-PnVIIA-the prototype of this family. Biological activity of PiVIIA was assessed by voltage-clamp recording in rat dorsal root ganglion neurons. Perfusion of PiVIIA in the µM range produces a significant increase in the Ca(2+) currents, without significantly modifying the Na⁺, K⁺ or proton-gated acid sensing ionic currents. These results indicate that PiVIIA is a new conotoxin whose activity deserves further studies to define its potential use as a positive modulator of neuronal activity.

Venoms are widely employed by numerous animals across disparate lineages for predation and defense. Among them, the deadly carnivorous cone snails are reputed for the potency of their venoms comprising small neurotoxic peptides known as conotoxins. Though a majority of cone snails prey on worms, some species prey on fish and other mollusks despite being slow movers. This remarkable prey diversification contributes to their evolutionary success. The origins of these dietary shifts have historically been explained based on the synergistic pharmacology of toxin classes. However, the recent discovery that some mollusk- and fish-hunting snails inject distinct defensive and predatory venoms has led to an alternative hypothesis where defense plays a pivotal role in the evolution of conotoxins and cone snails. This chapter provides an overview of cone snails and highlights recent advances in our understanding of conotoxin evolution.

Over 200 components with molecular mass ranging mainly from 400 to 4000 Da were characterized from the venom of the vermivorous cone snail Conus fulgetrum that inhabit Egyptian Red Sea. One major component having a molecular mass of 2946 Da was purified by HPLC, and its primary structure was determined by a combination of Edman degradation and MS/MS analysis.

International audience; Predatory marine snails of the genus Conus use venom containing a complex mixture of bioactive peptides to subdue their prey. Here we report on a comprehensive analysis of the protein content of injectable venom from Conus consors, an indo-pacific fish-hunting cone snail. By matching MS/MS data against an extensive set of venom gland transcriptomic mRNA sequences, we identified 105 components out of ~400 molecular masses detected in the venom. Among them, we described new conotoxins belonging to the A, M- and O1-superfamilies as well as a novel superfamily of disulphide free conopeptides. A high proportion of the deduced sequences (36%) corresponded to propeptide regions of the A- and M-superfamilies, raising the question of their putative role in injectable venom. Enzymatic digestion of higher molecular mass components allowed the identification of new conkunitzins (~7 kDa) and two proteins in the 25 and 50 kDa molecular mass ranges respectively characterised as actinoporin-like and hyaluronidase-like protein. These results provide the most exhaustive and accurate proteomic overview of an injectable cone snail venom to date, and delineate the major protein families present in the delivered venom. This study demonstrates the feasibility of this analytical approach and paves the way for transcriptomics-assisted strategies in drug discovery.

To date, cone snail toxins ("conotoxins") are of great interest in the pursuit of novel subtype-selective modulators of voltage-gated sodium channels (Na(v)s). Na(v)s participate in a wide range of electrophysiological processes. Consequently, their malfunctioning has been associated with numerous diseases. The development of subtype-selective modulators of Na(v)s remains highly important in the treatment of such disorders. In current research, a series of novel, synthetic, and bioactive compounds were designed based on two naturally occurring μ-conotoxins that target Na(v)s. The initial designed peptide contains solely 13 amino acids and was therefore named "Mini peptide." It was derived from the μ-conotoxins KIIIA and BuIIIC. Based on this Mini peptide, 10 analogues were subsequently developed, comprising 12-16 amino acids with two disulfide bridges. Following appropriate folding and mass verification, blocking effects on Na(v)s were investigated. The most promising compound established an IC(50) of 34.1 ± 0.01 nm (R2-Midi on Na(v)1.2). An NMR structure of one of our most promising compounds was determined. Surprisingly, this structure does not reveal an α-helix. We prove that it is possible to design small peptides based on known pharmacophores of μ-conotoxins without losing their potency and selectivity. These data can provide crucial material for further development of conotoxin-based therapeutics. ispartof: Journal of Biological Chemistry vol:287 issue:37 pages:31382-31392 ispartof: location:United States status: published

Milked venom from cone snails represent a novel biological resource with a proven track record for drug discovery. To strengthen this correlation, we undertook a chromatographic and mass spectrometric study of individual milked venoms from Conus purpurascens. Milked venoms demonstrate extensive peptide differentiation amongst individual specimens and during captivity. Individual snails were found to lack a consistent set of described conopeptides, but instead demonstrated the ability to change venom expression, composition and post-translational modification incorporation; all variations contribute to an increase in chemical diversity and prey targeting strategies. Quantitative amino acid analysis revealed that milked venom peptides are expressed at ranges up to 3.51-121.01 μM within single milked venom samples. This provides for a 6.37-20,965 fold-excess of toxin to induce apparent IC₅₀ for individual conopeptides identified in this study. Comparative molecular mass analysis of duct venom, milked venom and radula tooth extracts from single C. purpurascens specimens demonstrated a level of peptide continuity. Numerous highly abundant and unique conopeptides remain to be characterized. This study strengthens the notion that approaches in conopeptide drug lead discovery programs will potentially benefit from a greater understanding of the toxinological nature of the milked venoms of Conus.

Cone snail venoms are a rich source of peptides, many of which are potent and selective modulators of ion channels and receptors. Here we report the isolation and characterization of two novel conotoxins from the venom of Conus imperialis. These two toxins contain a novel cysteine framework, C-C-C-CC-C, which has not been found in other conotoxins described to date. We name it framework XXIII and designate the two toxins im23a and im23b; cDNAs of these toxins exhibit a novel signal peptide sequence, which defines a new K-superfamily. The disulfide connectivity of im23a has been mapped by chemical mapping of partially reduced intermediates and by NMR structure calculations, both of which establish a I-II, III-IV, V-VI pattern of disulfide bridges. This pattern was also confirmed by synthesis of im23a with orthogonal protection of individual cysteine residues. The solution structure of im23a reveals that im23a adopts a novel helical hairpin fold. A cluster of acidic residues on the surface of the molecule is able to bind calcium. The biological activity of the native and recombinant peptides was tested by injection into mice intracranially and intravenously to assess the effects on the central and peripheral nervous systems, respectively. Intracranial injection of im23a or im23b into mice induced excitatory symptoms; however, the biological target of these new toxins has yet to be identified.

International audience; Conopeptides are a diverse group of recently evolved venom peptides used for prey capture and/or defense. Each species of cone snails produces in excess of 1000 conopeptides, with those pharmacologically characterized (≈ 0.1%) targeting a diverse range of membrane proteins typically with high potency and specificity. The majority of conopeptides inhibit voltage- or ligand-gated ion channels, providing valuable research tools for the dissection of the role played by specific ion channels in excitable cells. It is noteworthy that many of these targets are found to be expressed in pain pathways, with several conopeptides having entered the clinic as potential treatments for pain [e.g., pyroglutamate1-MrIA (Xen2174)] and one now marketed for intrathecal treatment of severe pain [ziconotide (Prialt)]. This review discusses the diversity, pharmacology, structure-activity relationships, and therapeutic potential of cone snail venom peptide families acting at voltage-gated ion channels (ω-, μ-, μO-, δ-, ι-, and κ-conotoxins), ligand-gated ion channels (α-conotoxins, σ-conotoxin, ikot-ikot, and conantokins), G-protein-coupled receptors (ρ-conopeptides, conopressins, and contulakins), and neurotransmitter transporters (χ-conopeptides), with expanded discussion on the clinical potential of sodium and calcium channel inhibitors and α-conotoxins. Expanding the discovery of new bioactives using proteomic/transcriptomic approaches combined with high-throughput platforms and better defining conopeptide structure-activity relationships using relevant membrane protein crystal structures are expected to grow the already significant impact conopeptides have had as both research probes and leads to new therapies.

The venom peptides (i.e., conotoxins or conopeptides) that species in the genus Conus collectively produce are remarkably diverse, estimated to be around 50,000 to 140,000, but the pace of discovery and characterization of these peptides have been rather slow. To date, only a minor fraction have been identified and studied. However, the advent of next-generation DNA sequencing technologies has opened up opportunities for expediting the exploration of this diversity. The whole transcriptome of a venom duct from the vermivorous marine snail C. pulicarius was sequenced using the 454 sequencing platform. Analysis of the data set resulted in the identification of over eighty unique putative conopeptide sequences, the highest number discovered so far from a Conus venom duct transcriptome. More importantly, majority of the sequences were potentially novel, many with unexpected structural features, hinting at the vastness of the diversity of Conus venom peptides that remains to be explored. The sequences represented at least 14 major superfamilies/types (disulfide- and non-disulfide-rich), indicating the structural and functional diversity of conotoxins in the venom of C. pulicarius . In addition, the contryphans were surprisingly more diverse than what is currently known. Comparative analysis of the O-superfamily sequences also revealed insights into the complexity of the processes that drive the evolution and diversification of conotoxins.

International audience; Cone snail venoms are considered an untapped reservoir of extremely diverse peptides, named conopeptides, displaying a wide array of pharmacological activities. We report here for the first time, the presence of high molecular weight compounds that participate in the envenomation cocktail used by these marine snails. Using a combination of proteomic and transcriptomic approaches, we identified glycosyl hydrolase proteins, of the hyaluronidase type (Hyal), from the dissected and injectable venoms ("injectable venom" stands for the venom variety obtained by milking of the snails. This is in contrast to the "dissected venom", which was obtained from dissected snails by extraction of the venom glands) of a fish-hunting cone snail, Conus consors (Pionoconus clade). The major Hyal isoform, Conohyal-Cn1, is expressed as a mixture of numerous glycosylated proteins in the 50 kDa molecular mass range, as observed in 2D gel and mass spectrometry analyses. Further proteomic analysis and venom duct mRNA sequencing allowed full sequence determination. Additionally, unambiguous segment location of at least three glycosylation sites could be determined, with glycans corresponding to multiple hexose (Hex) and N-acetylhexosamine (HexNAc) moieties. With respect to other known Hyals, Conohyal-Cn1 clearly belongs to the hydrolase-type of Hyals, with strictly conserved consensus catalytic donor and positioning residues. Potent biological activity of the native Conohyals could be confirmed in degrading hyaluronic acid. A similar Hyal sequence was also found in the venom duct transcriptome of C. adamsonii (Textilia clade), implying a possible widespread recruitment of this enzyme family in fish-hunting cone snail venoms. These results provide the first detailed Hyal sequence characterized from a cone snail venom, and to a larger extent in the Mollusca phylum, thus extending our knowledge on this protein family and its evolutionary selection in marine snail venoms.

I used histological sections and 3D reconstructions to document development through metamorphosis of the foregut and proboscis in the conoidean neogastropod Conus lividus. A goal was to determine how highly derived features of the post-metamorphic feeding system of this gastropod predator develop without interfering with larval structures for microherbivory. A second goal was to compare foregut development in this conoidean with previous observations on foregut development in the buccinoidean neogastropod Nassarius mendicus. These two neogastropods both have a feeding larval stage, but they show major differences in post-metamorphic foregut morphology. Basic events in development of the proboscis and proboscis sheath in C. lividus and N. mendicus were similar. However, the elongate buccal tube of C. lividus forms during metamorphosis as a composite of apical epidermal tissue that grows inward and ventral foregut tissue that extends outward. The larval mouth is not carried through metamorphosis. Comparative observations on foregut development in caenogastropods, which now include data on C. lividus, suggest that the foregut incorporates dorsal and ventral modules having different ontogenetic and functional fates. This developmental modularity may have facilitated evolutionary diversification of the post-metamorphic foregut. Foregut diversification in predatory gastropods may have been further fast-tracked by developmental uncoupling of larval and post-metamorphic mouths.

The Irukandji syndrome is caused by the sting of some small jellyfish species. The syndrome has severe life-threatening consequences. The exacerbating pain and cardiovascular symptoms (tachycardia and hypertension) are hard to control in many cases. We suggest a way to experiment a new possible therapy with an FDA approved analgesic, ziconotide, a synthetic derivative from a marine cone snail ( Conus magus ) venom component, which is administrated intravenously. The proposed experimental plasma concentration of ziconotide for rats is in the range of 0–6 μg ml −1 . Based on a molecular biological scenario of the venom action mechanism at cellular level, we suggest that the proposed method should be functional in re-establishing the normal cardiovascular parameters of the experimental animals and concomitantly it should abolish the severe pain caused by envenomation. We expect that positive experimental results in agreement with our theory will lead to the possibility of a new therapy for the Irukandji syndrome and possibly for other envenomations with similar ethyology.

Venomous animals produce a diverse range of peptides and small molecules that are of both therapeutic and pharmacologic value. One such animal, the cone snail, produces peptides known as conotoxins, which may be of interest to those studying the mammalian immune system. Conotoxins are a family of venom peptides that display extraordinary diversity and often exquisite specificity for membrane protein targets, especially voltage and ligand activated ion channels. Conopeptides are proving to be important pharmacological tools to probe human physiology, with some showing promise as therapeutics for conditions such as neuropathic pain. The potential of these peptides to interact and modulate the human immune system has not been investigated despite literature suggesting that conotoxins could be valuable research tools and potential therapeutics in the area of immunology. Known pharmacological targets of conopeptides expressed by immunocompetent cells include voltage-gated potassium channel (Kv), voltage-gated calcium channel (Cav), nicotinic and acetylcholine receptors. In addition, the 5-HT3, GABAB and NMDA receptors that are not considered classic immunomodulators may play a secondary role in modulating immune responses. This review highlights venom peptides with the potential to act at immunological targets within the mammalian immune system.

Predatory marine cone snails (genus Conus) utilize complex venoms mainly composed of small peptide toxins that target voltage- and ligand-gated ion channels in their prey. Although the venoms of a number of cone snail species have been intensively profiled and functionally characterized, nothing is known about the initiation of venom expression at an early developmental stage. Here, we report on the expression of venom mRNA in embryos of Conus victoriae and the identification of novel α- and O-conotoxin sequences. Embryonic toxin mRNA expression is initiated well before differentiation of the venom gland, the organ of venom biosynthesis. Structural and functional studies revealed that the embryonic α-conotoxins exhibit the same basic three-dimensional structure as the most abundant adult toxin but significantly differ in their neurological targets. Based on these findings, we postulate that the venom repertoire of cone snails undergoes ontogenetic changes most likely reflecting differences in the biotic interactions of these animals with their prey, predators, or competitors. To our knowledge, this is the first study to show toxin mRNA transcripts in embryos, a finding that extends our understanding of the early onset of venom expression in animals and may suggest alternative functions of peptide toxins during development.

The oceans are a source of combinatorial library of unique natural products, 'not found in the terrestrial environment'. Marine invertebrates such as sponges, molluscs, bryozoans, tunicates (Urochordata) and their associated microorganisms are the major representatives of promising bioactive compounds. Among these, the predatory molluscan cone snails have evolved with highly structured small and complex array of peptides (more than 50,000) linked to their prey capture and defence. These peptides have become a valuable source of neuro pharmacological targets as many of them selectively modulate ion channels and transporters. A group of scientists from United States, Europe, Australia, Israel and China have been characterized drugs for neuropathic pain and pharmacological targets from the peptides of a few cone snail species. Several are now in Clinical and preclinical development. Less than 1% of the cono peptides are pharmacologically characterized. India has a diversity of 20-30% of total cone snail species distributed worldwide. A group of Indian Scientists have made promising drug discovery programs from Conus peptides. This review will focus on the Conus peptides from Indian cone snails species, their neuro pharmacological targets and future directions.

Crustacean cardioactive peptide (CCAP) and related peptides are multifunctional regulatory neurohormones found in invertebrates. We isolated a CCAP-related peptide (conoCAP-a, for cone snail CardioActive Peptide) and cloned the cDNA of its precursor from venom of Conus villepinii. The precursor of conoCAP-a encodes for two additional CCAP-like peptides: conoCAP-b and conoCAP-c. This multi-peptide precursor organization is analogous to recently predicted molluscan CCAP-like preprohormones, and suggests a mechanism for the generation of biological diversification without gene amplification. While arthropod CCAP is a cardio-accelerator, we found that conoCAP-a decreases the heart frequency in Drosophila larvae, demonstrating that conoCAP-a and CCAP have opposite effects. Intravenous injection of conoCAP-a in rats caused decreased heart frequency and blood pressure in contrast to the injection of CCAP, which did not elicit any cardiac effect. Perfusion of rat ventricular cardiac myocytes with conoCAP-a decreased systolic calcium, indicating that conoCAP-a cardiac negative inotropic effects might be mediated via impairment of intracellular calcium trafficking. The contrasting cardiac effects of conoCAP-a and CCAP indicate that molluscan CCAP-like peptides have functions that differ from those of their arthropod counterparts. Molluscan CCAP-like peptides sequences, while homologous, differ between taxa and have unique sequences within a species. This relates to the functional hypervariability of these peptides as structure activity relationship studies demonstrate that single amino acids variations strongly affect cardiac activity. The discovery of conoCAPs in cone snail venom emphasizes the significance of their gene plasticity to have mutations as an adaptive evolution in terms of structure, cellular site of expression, and physiological functions.

Cone snail venoms have yielded pharmacologically active natural products of exceptional scientific interest. However, cone snails are a small minority of venomous molluscan biodiversity, the vast majority being tiny venomous morphospecies in the family Turridae. A novel method called lumun-lumun opens access to these micromolluscs and their venoms. Old fishing nets are anchored to the sea bottom for a period of 1-6months and marine biotas rich in small molluscs are established. In a single lumun-lumun community, we found a remarkable gastropod biodiversity (155 morphospecies). Venomous predators belonging to the superfamily Conoidea (36 morphospecies) were the largest group, the majority being micromolluscs in the family Turridae. We carried out an initial analysis of the most abundant of the turrid morphospecies recovered, Clathurella (Lienardia) cincta (Dunker, 1871). In contrast to all cDNA clones characterized from cone snail venom ducts, one of the C. cincta clones identified encoded two different peptide precursors presumably translated from a single mRNA. The prospect of easily accessing so many different morphospecies of venomous marine snails raises intriguing toxinological possibilities: the 36 conoidean morphospecies in this one net alone have the potential to yield thousands of novel pharmacologically active compounds.

The cone snail Conus pulicarius from the Philippines provides a specific habitat for actinomycetes and other bacteria. A phenotypic screen using primary cultures of mouse dorsal root ganglion neurons revealed that one C. pulicarius associate, Streptomyces sp. CP32, produces a series of natural products that enhance or diminish whole-cell Ca(2+) flux. These compounds include known thiazoline compounds and a series of new derivatives, pulicatins A-E (6-10). Individual compounds were shown to bind to a series of human receptors, with selective binding to the human serotonin 5-HT(2B) receptor. Here, we report the structure elucidation of the new compounds and results of the neurological assays.

Conus species are characterized by their hyperdiverse toxins, encoded by a few gene superfamilies. Our phylogenies of the genus, based on mitochondrial genes, confirm previous results that C. californicus is highly divergent from all other species. Genetic and biochemical analysis of their venom peptides comprise the fifteen most abundant conopeptides and over 50 mature cDNA transcripts from the venom duct. Although C. californicus venom retains many of the general properties of other Conus species, they share only half of the toxin gene superfamilies found in other Conus species. Thus, in these two lineages, approximately half of the rapidly diversifying gene superfamilies originated after an early Tertiary split. Such results demonstrate that, unlike endogenously acting gene families, these genes are likely to be significantly more restricted in their phylogenetic distribution. In concordance with the evolutionary duistance of C. californicus from other species, there are aspects of prey-capture behavior and prey preferences of this species that diverges significantly from all other Conus.

Marine cone snails are a large family of gastropods that have evolved highly potent venoms for predation and defense. The cone snail venom has exceptional molecular diversity in neuropharmacologically active compounds, targeting a range of receptors, ion channels, and transporters. These conotoxins have helped to dissect the structure and function of many of these therapeutically significant targets in the central and peripheral nervous systems, as well as unravelling the complex cellular mechanisms modulated by these receptors and ion channels. This review provides an overview of α-conotoxins targeting neuronal nicotinic acetylcholine receptors. The structure and activity of both classical and non-classical α-conotoxins are discussed, along with their contributions towards understanding nicotinic acetylcholine receptor (nAChR) structure and function.

In our present work, seven conotoxins and conopeptides were cloned from four cone snail species based on the M-superfamily signal peptides. Among them, two conopeptides, Vt3.1 and Vt3.2, showed unusual sequence characteristics. Both of them contained two cysteines that are separated by just one non-cysteine residue. In vitro, the chemically synthesized Vt3.1 formed dimers with different intermolecular disulfide linkages. Only the dimer with crossed disulfides showed bioactivity when injected into the intraventricular region of mice brains. Therefore, Vt3.1 and Vt3.2 represent a new group of conopeptides that form disulfide-crosslinked dimers in vitro and probably in vivo.

SUMMARYCone snails use an extensile, tubular proboscis as a conduit to deliver a potent cocktail of bioactive venom peptides into their prey. Previous studies have focused mainly on understanding the venom's role in prey capture but successful prey capture requires both rapid physiological and biomechanical mechanisms. Conus catus, a fish-hunting species, uses a high-speed hydraulic mechanism to inject its hollow, spear-like radular tooth into prey. We take an integrated approach to investigating the biomechanics of this process by coupling kinematic studies with morphological analyses. Taking advantage of the opaque venom and translucent proboscis of a mollusc-hunting juvenile cone snail, Conus pennaceus, we have determined that a high-speed prey capture mechanism is not unique to cone species that hunt fish prey. Two morphological structures were found to play crucial roles in this process. A constriction of the lumen near the tip of the proboscis, composed of tall epithelial cells densely packed with microfilaments, impedes forward movement of the radular tooth prior to its propulsion. Proximal to the constriction, a muscular sphincter was found to regulate venom flow and pressurization in the proboscis. In C. pennaceus, the rapid appearance and flushing of venom within the proboscis during prey capture suggests a mechanism involving the delivery of a discrete quantity of venom. The interplay between these elements provides a unique and effective biomechanical injection system for the fast-acting cone snail venom peptides.

Cone snails have evolved an assortment of venom peptides as an evolutionary strategy for rapid prey immobilization and defence. Earlier studies estimated approximately 100 conopeptides per species. In this study we optimized liquid chromatography and electrospray ionization mass spectrometry for the detection of conopeptides in crude venom to characterize conopeptides present in the venom of individual specimens of Conus textile, C. imperialis and C. marmoreus. Using this approach, we have expanded the predicted number of venom peptides 10-fold to an estimate of 1000-1900 conopeptides per species. Our investigation has also revealed a surprisingly high level of intra-species variation that distinguishes cone snails from other venomous species including spiders and scorpions. Given this inherent diversity and variability, more sensitive bioassays and sequencing techniques will be required to fully explore conotoxin bioactivity.

Described herein is a general approach to identify novel compounds using the biodiversity of a megadiverse group of animals; specifically, the phylogenetic lineage of the venomous gastropods that belong to the genus Conus (“cone snails”). Cone snail biodiversity was exploited to identify three new μ-conotoxins, BuIIIA, BuIIIB and BuIIIC, encoded by the fish-hunting species Conus bullatus . BuIIIA, BuIIIB and BuIIIC are strikingly divergent in their amino acid composition compared to previous μ-conotoxins known to target the voltage-gated Na channel skeletal muscle subtype Na v 1.4. Our preliminary results indicate that BuIIIB and BuIIIC are potent inhibitors of Na v 1.4 (average block ∼96%, at a 1 μM concentration of peptide), displaying a very slow off-rate not seen in previously characterized μ-conotoxins that block Na v 1.4. In addition, the three new C. bullatus μ-conopeptides help to define a new branch of the M-superfamily of conotoxins, namely M-5. The exogene strategy used to discover these Na channel-inhibiting peptides was based on both understanding the phylogeny of Conus , as well as the molecular genetics of venom μ-conotoxin peptides previously shown to generally target voltage-gated Na channels. The discovery of BuIIIA, BuIIIB and BuIIIC Na channel blockers expands the diversity of ligands useful in determining the structure–activity relationship of voltage-gated sodium channels.

To date, studies conducted on cone snail venoms have attributed the origins of this complex mixture of neuroactive peptides entirely to gene expression by the secretory cells lining the lumen of the venom duct. However, specialized tissues such as the salivary glands also secrete their contents into the anterior gut and could potentially contribute some venom components injected into target animals; evidence supporting this possibility is reported here. Sequence analysis of a cDNA library created from a salivary gland of Conus pulicarius revealed the expression of two transcripts whose predicted gene products, after post-translational processing, strikingly resemble mature conopeptides belonging to the alpha-conotoxin family. These two transcripts, like alpha-conotoxin transcripts, putatively encode mature peptides containing the conserved A-superfamily cysteine pattern (CC-C-C) but the highly conserved A-superfamily signal sequences were not present. Analysis of A-superfamily members expressed in the venom duct of the same C. pulicarius specimens revealed three putative alpha-conotoxin sequences; the salivary gland transcripts were not found in the venom duct cDNA library, suggesting that these alpha-conotoxins are salivary gland specific. Therefore, expression of conotoxin-like gene products by the salivary gland could potentially add to the complexity of Conus venoms.

As part of continuing studies of the venom components present in Conus austini (syn.: Conus cancellatus), a vermivorous cone snail collected in the western Gulf of Mexico, Mexico, two major peptides, as14a and as14b, were purified and characterized. Their amino acid sequences were determined by automatic Edman sequencing after reduction and alkylation. Their molecular masses, established by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, confirmed the chemical analyses and indicated that as14a and as14b have free C-termini. Each peptide contains 4-Cys residues arranged in a pattern (C-C-C-C, framework 14). The primary structure of as14a is GGVGRCIYNCMNSGGGLNFIQCKTMCY (experimental monoisotopic mass 2883.92 Da; calculated monoisotopic mass 2884.20 Da), whereas that of as14b is RWDVDQCIYYCLNGVVGYSYTECQTMCT (experimental monoisotopic mass 3308.63 Da; calculated monoisotopic mass 3308.34 Da). Both purified peptides elicited scratching and grooming activity in mice, and as14b also caused body and rear limb extension and tail curling immediately upon injection. The high sequence similarity of peptide as14a with peptide vil14a from the vermivorous C. villepinii suggests that the former might block K+ channels.

A novel peptide, conorfamide-Sr2 (CNF-Sr2), was purified from the venom extract of Conus spurius , collected in the Caribbean Sea off the Yucatan Peninsula. Its primary structure was determined by automated Edman degradation and amino acid analysis, and confirmed by electrospray ionization mass spectrometry. Conorfamide-Sr2 contains 12 amino acids and no Cys residues, and it is only the second FMRFamide-related peptide isolated from a venom. Its primary structure GPMγDPLγIIRI-nh 2 , (γ, gamma-carboxyglutamate; -nh 2 , amidated C-terminus; calculated monoisotopic mass, 1468.72 Da; experimental monoisotopic mass, 1468.70 Da) shows two features that are unusual among FMRFamide-related peptides (FaRPs, also known as RFamide peptides), namely the novel presence of gamma-carboxyglutamate, and a rather uncommon C-terminal residue, Ile. CNF-Sr2 exhibits paralytic activity in the limpet Patella opea and causes hyperactivity in the freshwater snail Pomacea paludosa and in the mouse. The sequence similarities of CNF-Sr2 with FaRPs from marine and freshwater mollusks and mice might explain its biological effects in these organisms. It also resembles FaRPs from polychaetes (the prey of C. spurius ), which suggests a natural biological role. Based on these similarities, CNF-Sr2 might interact with receptors of these three distinct types of FaRPs, G-protein-coupled receptors, Na + channels activated by FMRFamide (FaNaCs), and acid-sensing ion channels (ASICs). The biological activities of CNF-Sr2 in mollusks and mice make it a potential tool to study molecular targets in these and other organisms.

The M-superfamily, one of eight major conotoxin superfamilies found in the venom of the cone snail, contains a Cys framework with disulfide-linked loops labeled 1, 2, and 3 (-CC (1) C (2) C (3) CC-). M-Superfamily conotoxins can be divided into the m-1, -2, -3, and -4 branches, based upon the number of residues located in the third Cys loop between the fourth and fifth Cys residues. Here we provide a three-dimensional solution structure for the m-1 conotoxin tx3a found in the venom of Conus textile. The 15-amino acid peptide, CCSWDVCDHPSCTCC, has disulfide bonds between Cys (1) and Cys (14), Cys (2) and Cys (12), and Cys (7) and Cys (15) typical of the C1-C5, C2-C4, and C3-C6 connectivity pattern seen in m-1 branch peptides. The tertiary structure of tx3a was determined by two-dimensional (1)H NMR in combination with the combined assignment and dynamics algorithm for nuclear magnetic resonance (NMR) applications CYANA program. Input for structure calculations consisted of 62 inter- and intraproton, five phi angle, and four hydrogen bond constraints. The root-mean-square deviation values for the 20 final structures are 0.32 +/- 0.07 and 0.84 +/- 0.11 A for the backbone and heavy atoms, respectively. Surprisingly, the structure of tx3a has a "triple-turn" motif seen in the m-2 branch conotoxin mr3a, which is absent in mr3e, the only other member of the m-1 branch of the M-superfamily whose structure is known. Interestingly, injection of tx3a into mice elicits an excitatory response similar to that of the m-2 branch peptide mr3a, even though the conotoxins have different disulfide connectivity patterns.