Novel excitatory Conus peptides define a new conotoxin superfamily

A new class of Conus peptides, the I-superfamily of conotoxins, has been characterized using biochemical, electrophysiological and molecular genetic methods. Peptides in this superfamily have a novel pattern of eight Cys residues. Five peptides that elicited excitatory symptomatology, r11a, r11b, r11c, r11d and r11e, were purified from Conus radiatus venom; four were tested on amphibian peripheral axons and shown to elicit repetitive action potentials, consistent with being members of the 'lightning-strike cabal' of toxins that effect instant immobilization of fish prey. A parallel analysis of Conus cDNA clones revealed a new class of conotoxin genes that was particularly enriched (with 18 identified paralogues) in a Conus radiatus venom duct library; several C. radiatus clones encoded the excitatory peptides directly characterized from venom. The remarkable diversity of related I-superfamily peptides within a single Conus species is unprecedented. When combined with the excitatory effects observed on peripheral circuitry, this unexpected diversity suggests a corresponding molecular complexity of the targeted signaling components in peripheral axons; the I-conotoxin superfamily should provide a rich lode of pharmacological tools for dissecting and understanding these. Thus, the I-superfamily conotoxins promise to provide a significant new technology platform for dissecting the molecular components of axons.     

I-conotoxins in vermivorous species of the West Atlantic: peptide sr11a from Conus spurius

Peptide sr11a was purified from the venom of Conus spurius, a vermivorous cone snail collected in the Yucatan Channel, in the Western Atlantic. Its primary structure was determined by automatic Edman degradation after reduction and alkylation. Its molecular mass, as determined by MALDI-TOF mass spectrometry (average mass 3650.77 Da), confirmed the chemical data (calculated average mass, 3651.13 Da). The sequence of peptide sr11a (CRTEGMSCgamma gamma NQQCCWRSCCRGECEAPCRFGP&; gamma, gamma-carboxy-Glu; &, amidated C-terminus) shows eight Cys residues arranged in the pattern that defines the I-superfamily of conotoxins. Peptide sr11a contains two gamma-carboxy-Glu residues, a post-translational modification that has been found in other I-conotoxins from species that live in the West Pacific: r11e from the piscivorous Conus radiatus, and kappa-BtX from the vermivorous Conus betulinus. Peptide sr11a is the eighth I-conotoxin isolated from a Conus venom and the first I-conotoxin from a species from the Western Atlantic. Peptide sr11a produced stiffening of body, limbs and tail when injected intracranially into mice.     

The A-superfamily of conotoxins: structural and functional divergence

The generation of functional novelty in proteins encoded by a gene superfamily is seldom well documented. In this report, we define the A-conotoxin superfamily, which is widely expressed in venoms of the predatory cone snails (Conus), and show how gene products that diverge considerably in structure and function have arisen within the same superfamily. A cDNA clone encoding alpha-conotoxin GI, the first conotoxin characterized, provided initial data that identified the A-superfamily. Conotoxin precursors in the A-superfamily were identified from six Conus species: most (11/16) encoded alpha-conotoxins, but some (5/16) belong to a family of excitatory peptides, the kappaA-conotoxins that target voltage-gated ion channels. alpha-Conotoxins are two-disulfide-bridged nicotinic antagonists, 13-19 amino acids in length; kappaA-conotoxins are larger (31-36 amino acids) with three disulfide bridges. Purification and biochemical characterization of one peptide, kappaA-conotoxin MIVA is reported; five of the other predicted conotoxins were previously venom-purified. A comparative analysis of conotoxins purified from venom, and their precursors reveal novel post-translational processing, as well as mutational events leading to polymorphism. Patterns of sequence divergence and Cys codon usage define the major superfamily branches and suggest how these separate branches arose.     

A helical conotoxin from Conus imperialis has a novel cysteine framework and defines a new superfamily

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.     

Substrate specificity of plant UDP-dependent glycosyltransferases predicted from crystal structures and homology modeling

Plant family 1 UDP-dependent glycosyltransferases (UGTs) catalyze the glycosylation of a plethora of bioactive natural products. In Arabidopsis thaliana, 120 UGT encoding genes have been identified. The crystal-based 3D structures of four plant UGTs have recently been published. Despite low sequence conservation, the UGTs show a highly conserved secondary and tertiary structure. The sugar acceptor and sugar donor substrates of UGTs are accommodated in the cleft formed between the N- and C-terminal domains. Several regions of the primary sequence contribute to the formation of the substrate binding pocket including structurally conserved domains as well as loop regions differing both with respect to their amino acid sequence and sequence length. In this review we provide a detailed analysis of the available plant UGT crystal structures to reveal structural features determining substrate specificity. The high 3D structural conservation of the plant UGTs render homology modeling an attractive tool for structure elucidation. The accuracy and utility of UGT structures obtained by homology modeling are discussed and quantitative assessments of model quality are performed by modeling of a plant UGT for which the 3D crystal structure is known. We conclude that homology modeling offers a high degree of accuracy. Shortcomings in homology modeling are also apparent with modeling of loop regions remaining as a particularly difficult task.     

Identification of six novel T-1 conotoxins from Conus pulicarius by molecular cloning

Cone snails are a group of ancient marine gastropods with highly sophisticated defense and prey strategies using conotoxins in their venom. Conotoxins are a diverse array of small peptides, mostly with multiple disulfide bridges. Using a 3' RACE approach, we identified six novel peptides from the venom ducts of a worm-hunting cone snail Conus pulicarius. These peptides are named Pu5.1-Pu5.6 as their primary structures show the typical pattern of T-1 conotoxin family, a large and diverse group of peptides widely distributed in venom ducts of all major feeding types of Conus. Except for the conserved signal peptide sequences in the precursors and unique arrangement of Cys residues (CC-CC) in mature domains, the six novel T-1 conotoxins show remarkable sequence diversity in their pro and mature regions and are, thus, likely to be functionally diversified. Here, we present a simple and fast strategy of gaining novel disulfide-rich conotoxins via molecular cloning and our detailed sequence analysis will pave the way for the future functional characterization of toxin-receptor interaction.     

A novel M-superfamily conotoxin with a unique motif from Conus vexillum

Cone snails are tropical marine mollusks that envenomate prey with a complex mixture of neuropharmacologically active compounds for the purpose of feeding and defence, each evolved to act in a highly specific manner on different parts of the nervous system. Here, we report the peptide purification, molecular cloning, chemical synthesis, and functional characterization of a structurally unique toxin isolated from the venom of Conus vexillum. The novel peptide, designated Vx2, was composed of 21 amino acid residues cross-linked by 3 disulfide bonds (WIDPSHYCCCGGGCTDDCVNC). Intriguingly, its mature peptide sequence shows low level of similarity with other identified conotoxins, and its unique motif (-CCCGGGC-) was not reported in other Conus peptides. However, its signal peptide sequence shares high similarity with those of the M-superfamily conotoxins. Hence, Vx2 could be classified into a new family of the M-superfamily.     

Molecular cloning, expression and characterization of protein disulfide isomerase from Conus marmoreus

The oxidative folding of disulfide-rich conotoxins is essential for their biological functions. In vivo, disulfide bond formation is mainly catalyzed by protein disulfide isomerase. To elucidate the physiologic roles of protein disulfide isomerase in the folding of conotoxins, we have cloned a novel full-length protein disulfide isomerase from Conus marmoreus. Its ORF encodes a 500 amino acid protein that shares sequence homology with protein disulfide isomerases from other species, and 70% homology with human protein disulfide isomerase. Enzymatic analyses of recombinant C. marmoreus protein disulfide isomerase showed that it shared functional similarities with human protein disulfide isomerase. Using conotoxins tx3a and sTx3.1 as substrate, we analyzed the oxidase and isomerase activities of the C. marmoreus protein disulfide isomerase and found that it was much more efficient than glutathione in catalyzing oxidative folding and disulfide isomerization of conotoxins. We further demonstrated that macromolecular crowding had little effect on the protein disulfide isomerase-catalyzed oxidative folding and disulfide isomerization of conotoxins. On the basis of these data, we propose that the C. marmoreus protein disulfide isomerase plays a key role during in vivo folding of conotoxins.     

Characterization of a T-superfamily conotoxin TxVC from Conus textile that selectively targets neuronal nAChR subtypes

T-superfamily conotoxins have a typical cysteine pattern of “CC–CC”, and are known to mainly target calcium or sodium ion channels. Recently, we screened the targets of a series of T-superfamily conotoxins and found that a new T-superfamily conotoxin TxVC (KPCCSIHDNSCCGL-NH₂) from the venom of Conus textile. It selectively targeted the neuronal nicotinic acetylcholine receptor (nAChR) subtypes α4β2 and α3β2, with IC₅₀ values of 343.4 and 1047.2 nM, respectively, but did not exhibit obvious pharmacological effects on voltage-gated potassium, sodium or calcium channel in DRG cells, the BK channels expressed in HEK293 cells, or the Kv channels in LβT2 cells. The changes in the inhibitory activities of its Ala mutants, the NMR structure, and molecular simulation results based on other conotoxins targeting nAChR α4β2, all demonstrated that the residues Ile⁶ and Leu¹⁴ were the main hydrophobic pharmacophores. To our best knowledge, this is the first T-superfamily conotoxin that inhibits neuronal nAChRs and possesses high binding affinity to α4β2. This finding will expand the knowledge of the targets of T-superfamily conotoxins and the motif information could help the design of new nAChR inhibitors.     

Solution structure of an M-1 conotoxin with a novel disulfide linkage

The M-superfamily of conotoxins has a typical Cys framework (-CC-C-C-CC-), and is one of the eight major superfamilies found in the venom of the cone snail. Depending on the number of residues located in the last Cys loop (between Cys4 and Cys5), the M-superfamily family can be divided into four branches, namely M-1, -2, -3 and -4. Recently, two M-1 branch conotoxins (mr3e and tx3a) have been reported to possess a new disulfide bond arrangement between Cys1 and Cys5, Cys2 and Cys4, and Cys3 and Cys6, which is different from those seen in the M-2 and M-4 branches. Here we report the 3D structure of mr3e determined by 2D (1)H NMR in aqueous solution. Twenty converged structures of this peptide were obtained on the basis of 190 distance constraints obtained from NOE connectivities, as well as six varphi dihedral angle, three hydrogen bond, and three disulfide bond constraints. The rmsd values about the averaged coordinates of the backbone atoms were 0.43 +/- 0.19 A. Although mr3e has the same Cys arrangement as M-2 and M-4 conotoxins, it adopts a distinctive backbone conformation with the overall molecule resembling a 'flying bird'. Thus, different disulfide linkages may be employed by conotoxins with the same Cys framework to result in a more diversified backbone scaffold.     

Functional properties of the epithelial Ca2+ channel, ECaC.

ECaC is the first member of a new subfamily of Ca2+ channels embedded in the large TRPC family that includes numerous channel proteins. The channel has been proposed as the main gatekeeper of transcellular Ca2+ transport in kidney and intestine. The functional characterization of this channel is evolving rapidly and may have far reaching consequences for other channels of the TRPC family. The goal of this mini-review is to summarize the major functional and structural characteristics of ECaC, including (i) its proposed functional role, (ii) its channel structure and expression pattern, (iii) its main electrophysiological characteristics and (iv) its regulation.     

The three-dimensional solution structure of mini-M conotoxin BtIIIA reveals a disconnection between disulfide connectivity and peptide fold

Conotoxins are bioactive peptides from the venoms of marine snails and have been divided into several superfamilies based on homologies in their precursor sequences. The M-superfamily conotoxins can be further divided into five branches based on the number of residues in the third loop of the peptide sequence. Recently two M-1 branch conotoxins (tx3a and mr3e) with a C1–C5, C2–C4, C3–C6 disulfide connectivity and one M-2 branch conotoxin (mr3a) with a C1–C6, C2–C4, C3–C5 disulfide connectivity were described. Here we report the disulfide connectivity, chemical synthesis and the three-dimensional NMR structure of the novel 14-residue conotoxin BtIIIA, extracted from the venom of Conus betulinus. It has the same disulfide connectivity as mr3a, which puts it in the M-2 branch conotoxins but has a distinctly different structure from other M-2 branch conotoxins. 105 NOE distance restraints and seven dihedral angle restraints were used for the structure calculations. The three-dimensional structure was determined with CYANA based on torsion angle dynamics and refinement in a water solvent box was carried out with CNS. Fifty structures were calculated and the 20 lowest energy structures superimposed with a RMSD of 0.49 ± 0.16 Å. Even though it has the M-2 branch disulfide connectivity, BtIIIA was found to have a ‘flying bird’ backbone motif depiction that is found in the M-1 branch conotoxin mr3e. This study shows that conotoxins with the same cysteine framework can have different disulfide connectivities and different peptide folds.     

The structure of a novel insect peptide explains its Ca2+ channel blocking and antifungal activities

Diapause-specific peptide (DSP), derived from the leaf beetle, inhibits Ca2+ channels and has antifungal activity. DSP acts on chromaffin cells of the adrenal medulla in a fashion similar to that of omega-conotoxin GVIA, a well-known neurotoxic peptide, and blocks N-type voltage-dependent Ca2+ channels. However, the amino acid sequence of DSP has little homology with any other known Ca2+ channel blockers or antifungal peptides. In this paper, we analyzed the solution structure of DSP by using two-dimensional 1H nuclear magnetic resonance and determined the pairing of half-cystine residues forming disulfide bonds. The arrangement of the three disulfide bridges in DSP was distinct from that of other antifungal peptides and conotoxins. The overall structure of DSP is compact due in part to the three disulfide bridges and, interestingly, is very similar to those of the insect- and plant-derived antifungal peptides. On the other hand, the disulfide arrangement and the three-dimensional structure of DSP and GVIA are not similar. Nevertheless, some surface residues of DSP superimpose on the key functional residues of GVIA. This homologous distribution of hydrophobic and charged side chains may result in the functional similarity between DSP and GVIA. Thus, we propose here that the three-dimensional structure of DSP can explain its dual function as a Ca2+ channel blocker and antifungal peptide.     

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

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

Purification and structural characterization of a D-amino acid-containing conopeptide, conomarphin, from Conus marmoreus

Cone snails, a group of gastropod animals that inhabit tropical seas, are capable of producing a mixture of peptide neurotoxins, namely conotoxins, for defense and predation. Conotoxins are mainly disulfide-rich short peptides that act on different ion channels, neurotransmitter receptors, or transporters in the nervous system. They exhibit highly diverse compositions, structures, and biological functions. In this work, a novel Cys-free 15-residue conopeptide from Conus marmoreus was purified and designated as conomarphin. Conomarphin is unique because of its D-configuration Phe at the third residue from the C-terminus, which was identified using HPLC by comparing native conomarphin fragments and the corresponding synthetic peptides cleaved by different proteases. Surprisingly, the cDNA-encoded precursor of conomarphin was found to share the conserved signal peptide with other M-superfamily conotoxins, clearly indicating that conomarphin should belong to the M-superfamily, although conomarphin shares no homology with other six-Cys-containing M-superfamily conotoxins. Furthermore, NMR spectroscopy experiments established that conomarphin adopts a well-defined structure in solution, with a tight loop in the middle of the peptide and a short 3(10)-helix at the C-terminus. By contrast, no loop in L-Phe13-conomarphin was found, which suggests that D-Phe13 is essential for the structure of conomarphin. In conclusion, conomarphin may represent a new conotoxin family, whose biological activity remains to be identified.     

Molecular evolution of ion channels: Amino acid sequences and 3D structures

Comparative analysis of structure and function of macromolecules, such as proteins, is an integral part of modern evolutionary biology. The first and critical step in understanding evolution of homologous proteins is their amino acid sequence alignment. However, standard algorithms fail to provide unambiguous sequence alignment for proteins of poor homology. More reliable results can be provided by comparing experimental 3D structures obtained at atomic resolution with the aid of X-ray structural analysis. If such structures are lacking, homology modeling is used which considers indirect experimental data on functional roles of individual amino acid residues. An important problem is that sequence alignment, which reflects genetic modifications, not necessarily corresponds to functional homology, which depends on 3D structures critical for natural selection. Since the alignment techniques relying only on the analysis of primary structures carry no information on the functional properties of proteins, the inclusion of 3D structures into consideration is of utmost importance. Here we consider several ion channels as examples to demonstrate that alignment of their 3D structures can significantly improve sequence alignment obtained by traditional methods.     

Identification of a novel class of conotoxins defined as V-conotoxins with a unique cysteine pattern and signal peptide sequence

Cone snails are predatory gastropod mollusks distributed in all tropical marine habitats with a highly sophisticated defense strategy using small peptides in their venoms. Here, we report the discovery and initial characterization of the V-superfamily conotoxins. A novel conotoxin vi15a was purified from the venom of a worm-hunting species Conus virgo. The sequence of vi15a was determined to have a unique arrangement of cysteine residues (C-C-CC-C-C-C-C), which defines the new V-superfamily conotoxins. The cDNA of vi15a was cloned with RACE method. Its unique signal peptide sequence led to the cloning of another V-superfamily conotoxin, Vt15.1, from Conus vitulinus. These results, as well as the existence of Lt15.1 from Conus litteratus and ca15a from Conus caracteristicus with the same cysteine pattern, suggest that V-superfamily might be a large and diverse group of peptides widely distributed in different Conus species. Like other eight Cys-containing toxins, V-superfamily conotoxins might also adopt an “ICK+1” disulfide bond connectivity. The identification of this novel class of conotoxins will certainly improve our understanding of the structure diversity of disulfide rich toxins.     

Isolation and characterization of a novel conus peptide with apparent antinociceptive activity

Cone snails are tropical marine mollusks that envenomate prey with a complex mixture of neuropharmacologically active compounds. We report the discovery and biochemical characterization of a structurally unique peptide isolated from the venom of Conus marmoreus. The new peptide, mr10a, potently increased withdrawal latency in a hot plate assay (a test of analgesia) at intrathecal doses that do not produce motor impairment as measured by rotarod test. The sequence of mr10a is NGVCCGYKLCHOC, where O is 4-trans-hydroxyproline. This sequence is highly divergent from all other known conotoxins. Analysis of a cDNA clone encoding the toxin, however, indicates that it is a member of the recently described T-superfamily. Total chemical synthesis of the three possible disulfide arrangements of mr10a was achieved, and elution studies indicate that the native form has a disulfide connectivity of Cys1-Cys4 and Cys2-Cys3. This disulfide linkage is unprecedented among conotoxins and defines a new family of Conus peptides.