Solution structure of Ptu1, a toxin from the assassin bug Peirates turpis that blocks the voltage-sensitive calcium channel N-type.

Ptu1 is a toxin from the assassin bug Peirates turpis which has been demonstrated to bind reversibly the N-type calcium channels and to have lower affinity than the omega-conotoxin MVIIA. We have determined the solution structure of Ptu1 by use of conventional two-dimensional NMR techniques followed by distance-geometry and molecular dynamics. The calculated structure of Ptu1 belongs to the inhibitory cystin knot structural family (ICK) that consists of a compact disulfide-bonded core from which four loops emerge. Analysis of the 25 converged solutions indicates that the molecular structure of Ptu1 contains a 2-stranded antiparallel beta-sheet (residues 24-27 and 31-34) as the only secondary structure. The loop 2 that has been described to be critical for the binding of the toxin on the channel is similar in Ptu1 and MVIIA. In this loop, the critical residue, Tyr13, in MVIIA is retrieved in Ptu1 as Phe13, but the presence of an acidic residue (Asp16) in Ptu1 could disturb the binding of Ptu1 on the channel and could explain the lower affinity of Ptu1 toward the N-type calcium channel compared to the one of MVIIA. Analysis of the electrostatic charge's repartition gives some insights about the importance of the basic residues, which could interact with acidic residues of the channel and then provide a stabilization of the toxin on the channel.     

Modular toxin from the lynx spider Oxyopes takobius: Structure of spiderine domains in solution and membrane‐mimicking environment

We have recently demonstrated that a common phenomenon in evolution of spider venom composition is the emergence of so‐called modular toxins consisting of two domains, each corresponding to a “usual” single‐domain toxin. In this article, we describe the structure of two domains that build up a modular toxin named spiderine or OtTx1a from the venom of Oxyopes takobius. Both domains were investigated by solution NMR in water and detergent micelles used to mimic membrane environment. The N‐terminal spiderine domain OtTx1a‐AMP (41 amino acid residues) contains no cysteines. It is disordered in aqueous solution but in micelles, it assumes a stable amphiphilic structure consisting of two α‐helices separated by a flexible linker. On the contrary, the C‐terminal domain OtTx1a‐ICK (59 residues) is a disulfide‐rich polypeptide reticulated by five S–S bridges. It presents a stable structure in water and its core is the inhibitor cystine knot (ICK) or knottin motif that is common among single‐domain neurotoxins. OtTx1a‐ICK structure is the first knottin with five disulfide bridges and it represents a good reference for the whole oxytoxin family. The affinity of both domains to membranes was measured with NMR using titration by liposome suspensions. In agreement with biological tests, OtTx1a‐AMP was found to show high membrane affinity explaining its potent antimicrobial properties.     

Solution structure of two insect-specific spider toxins and their pharmacological interaction with the insect voltage-gated Na+ channel

Delta-paluIT1 and delta-paluIT2 are toxins purified from the venom of the spider Paracoelotes luctuosus. Similar in sequence to mu-agatoxins from Agelenopsis aperta, their pharmacological target is the voltage-gated insect sodium channel, of which they alter the inactivation properties in a way similar to alpha-scorpion toxins, but they bind on site 4 in a way similar to beta-scorpion toxins. We determined the solution structure of the two toxins by use of two-dimensional nuclear magnetic resonance (NMR) techniques followed by distance geometry and molecular dynamics. The structures of delta-paluIT1 and delta-paluIT2 belong to the inhibitory cystine knot structural family, i.e. a compact disulfide-bonded core from which four loops emerge. Delta-paluIT1 and delta-paluIT2 contain respectively two- and three-stranded anti-parallel beta-sheets as unique secondary structure. We compare the structure and the electrostatic anisotropy of those peptides to other sodium and calcium channel toxins, analyze the topological juxtaposition of key functional residues, and conclude that the recognition of insect voltage-gated sodium channels by these toxins involves the beta-sheet, in addition to loops I and IV. Besides the position of culprit residues on the molecular surface, difference in dipolar moment orientation is another determinant of receptor binding and biological activity differences. We also demonstrate by electrophysiological experiments on the cloned insect voltage-gated sodium channel, para, heterologuously co-expressed with the tipE subunit in Xenopus laevis oocytes, that delta-paluIT1 and delta-paluIT2 procure an increase of Na+ current. delta-PaluIT1-OH seems to have less effect when the same concentrations are used.     

Structure and sodium channel activity of an excitatory I1-superfamily conotoxin

Conotoxin iota-RXIA, from the fish-hunting species Conus radiatus, is a member of the recently characterized I1-superfamily, which contains eight cysteine residues arranged in a -C-C-CC-CC-C-C- pattern. iota-RXIA (formerly designated r11a) is one of three characterized I1 peptides in which the third last residue is posttranslationally isomerized to the d configuration. Naturally occurring iota-RXIA with d-Phe44 is significantly more active as an excitotoxin than the l-Phe analogue both in vitro and in vivo. We have determined the solution structures of both forms by NMR spectroscopy, the first for an I1-superfamily member. The disulfide connectivities were determined from structure calculations and confirmed chemically as 5-19, 12-22, 18-27, and 21-38, suggesting that iota-RXIA has an ICK structural motif with one additional disulfide (21-38). Indeed, apart from the first few residues, the structure is well defined up to around residue 35 and does adopt an ICK structure. The C-terminal region, including Phe44, is disordered. Comparison of the d-Phe44 and l-Phe44 forms indicates that the switch from one enantiomer to the other has very little effect on the structure, even though it is clearly important for receptor interaction based on activity data. Finally, we identify the target of iota-RXIA as a voltage-gated sodium channel; iota-RXIA is an agonist, shifting the voltage dependence of activation of mouse NaV1.6 expressed in Xenopus oocytes to more hyperpolarized potentials. Thus, there is a convergence of structure and function in iota-RXIA, as its disulfide pairing and structure resemble those of funnel web spider toxins that also target sodium channels.     

Novel families of toxin-like peptides in insects and mammals: a computational approach

Most animal toxins are short proteins that appear in venom and vary in sequence, structure and function. A common characteristic of many such toxins is their apparent structural stability. Sporadic instances of endogenous toxin-like proteins that function in non-venom context have been reported. We have utilized machine learning methodology, based on sequence-derived features and guided by the notion of structural stability, in order to conduct a large-scale search for toxin and toxin-like proteins. Application of the method to insect and mammalian sequences revealed novel families of toxin-like proteins. One of these proteins shows significant similarity to ion channel inhibitors that are expressed in cone snail and assassin bug venom, and is surprisingly expressed in the bee brain. A toxicity assay in which the protein was injected to fish induced a strong yet reversible paralytic effect. We suggest that the protein may function as an endogenous modulator of voltage-gated Ca(2+) channels. Additionally, we have identified a novel mammalian cluster of toxin-like proteins that are expressed in the testis. We suggest that these proteins might be involved in regulation of nicotinic acetylcholine receptors that affect the acrosome reaction and sperm motility. Finally, we highlight a possible evolutionary link between venom toxins and antibacterial proteins. We expect our methodology to enhance the discovery of additional novel protein families.     

A new family of small (4kDa) neurotoxins from the venoms of spiders of the genus Phoneutria

A family of 4kDa neurotoxic peptides was purified from venoms of Phoneutria spiders. All have six cysteine residues, and low similarity with other neurotoxins. Three toxins caused moderate inhibition of L-type Ca(2+) channels. The structure of toxin PRTx27C3 was modeled and compared with toxin ADO1. The importance of four residues is suggested.     

Structure-function studies of omega-atracotoxin, a potent antagonist of insect voltage-gated calcium channels.

The omega-atracotoxins are a family of 36 to 37-residue peptide neurotoxins that block insect but not mammalian voltage-gated calcium channels. The high phylogenetic specificity of these toxins recommends them as lead compounds for targeting insects that have developed resistance to chemical pesticides. We have begun to examine structure-function relationships in the omega-atracotoxins in order to explore the molecular basis of their activity and phylogenetic specificity. By probing the venom of the Blue Mountains funnel-web spider, Hadronyche versuta, for insecticidal toxins with masses close to that of omega-atracotoxin-Hv1a (omega-ACTX-Hv1a), we have isolated and sequenced five additional omega-atracotoxins. Five of the six omega-atracotoxins isolated from the venom of H. versuta (omega-ACTX-Hv1a to -Hv1e) differ from one another by only 1-3 residues and have similar insecticidal potencies. In contrast, omega-ACTX-Hv1f differs from the other toxins by up to 10 residues and it has markedly reduced insecticidal potency, thus providing information on key functional residues. The new atracotoxin sequences have revealed that the three N-terminal residues are highly conserved. Despite the fact that these residues are structurally disordered in solution we show here, by a series of N-terminal truncations, that they contribute significantly to insecticidal potency. However, loss of activity does not correlate with deletion of highly conserved residues, which leads us to propose that the disposition of the N-terminal charge, rather than the chemical properties of the N-terminal residues themselves, may be critical for the activity of omega-atracotoxin on insect calcium channels.     

Polypeptide neurotoxins from spider venoms.

Spider venoms contain a variety of toxic components. The polypeptide toxins are divided into low and high molecular mass types. Small polypeptide toxins interacting with cation channels display spatial structure homology. They can affect the functioning of calcium, sodium, or potassium channels. A family of high molecular mass toxic proteins was found in the venom of the spider genus Latrodectus. These neurotoxins, latrotoxins, cause a massive transmitter release from a diversity of nerve endings. The latrotoxins are proteins of about 1000 amino acid residues and share a high level of structure identity. The structural and functional properties of spider polypeptide toxins are reviewed in this paper.     

Cloning,purification and biochemical characterization of dipetarudin,a new chimeric thrombin inhibitor

The development of thrombin inhibitors could provide invaluable progress for antithrombotic therapy. In this paper, we report the cloning, purification and biochemical characterization of dipetarudin, a chimeric thrombin inhibitor composed of the N-terminal head structure of dipetalogastin II, the strongest inhibitor from the assassin bug Dipetalogaster maximus, and the exosite 1 blocking segment of hirudin, connected through a five glycine linker. The cloning of dipetarudin was performed by a simple method which had not been used previously to clone chimeras. Biochemical characterization of dipetarudin revealed that it is a slow, tight-binding inhibitor with a molecular mass (M(r)=7560) and a thrombin inhibitory activity (K(i)=446 fM) comparable to r-hirudin.     

Recombinant production and solution structure of PcTx1, the specific peptide inhibitor of ASIC1a proton-gated cation channels

Acid-sensing ion channels (ASICs) are thought to be important ion channels, particularly for the perception of pain. Some of them may also contribute to synaptic plasticity, learning, and memory. Psalmotoxin 1 (PcTx1), the first potent and specific blocker of the ASIC1a proton-sensing channel, has been successfully expressed in the Drosophila melanogaster S2 cell recombinant expression system used here for the first time to produce a spider toxin. The recombinant toxin was identical in all respects to the native peptide, and its three-dimensional structure in solution was determined by means of (1)H 2D NMR spectroscopy. Surface characteristics of PcTx1 provide insights on key structural elements involved in the binding of PcTx1 to ASIC1a channels. They appear to be localized in the beta-sheet and the beta-turn linking the strands, as indicated by electrostatic anisotropy calculations, surface charge distribution, and the presence of residues known to be implicated in channel recognition by other inhibitor cystine knot (ICK) toxins.     

Solution structure of PcFK1, a spider peptide active against Plasmodium falciparum

Psalmopeotoxin I (PcFK1) is a 33-amino-acid residue peptide isolated from the venom of the tarantula Psalmopoeus cambridgei. It has been recently shown to possess strong antiplasmodial activity against the intra-erythrocyte stage of Plasmodium falciparum in vitro. Although the molecular target for PcFK1 is not yet determined, this peptide does not lyse erythrocytes, is not cytotoxic to nucleated mammalian cells, and does not inhibit neuromuscular function. We investigated the structural properties of PcFK1 to help understand the unique mechanism of action of this peptide and to enhance its utility as a lead compound for rational development of new antimalarial drugs. In this paper, we have determined the three-dimensional solution structure by (1)H two-dimensional NMR means of recombinant PcFK1, which is shown to belong to the ICK structural superfamily with structural determinants common to several neurotoxins acting as ion channels effectors.     

Chemical disulfide mapping identifies an inhibitor cystine knot in the agouti signaling protein

The agouti signaling protein (ASIP) and its homolog, the agouti-related protein (AgRP), act as inverse agonists that control, respectively, pigmentation and metabolic function in mammals. NMR investigations find that the C-terminal domains of these proteins adopt a fold consistent with an inhibitor cystine knot (ICK), previously identified in invertebrate toxins. Although these structural studies suggest that ASIP and AgRP define a new mammalian protein fold class, the results with ASIP are inconclusive. Here, we apply direct chemical mapping to determine the complete set of disulfide linkages in ASIP. The results demonstrate unequivocally that ASIP adopts the ICK fold and thereby supports a recent evolution structure function analysis, which proposes that ASIP and AgRP arose from a common antagonist ligand.     

ICK1, a cyclin-dependent protein kinase inhibitor from Arabidopsis thaliana interacts with both Cdc2a and CycD3, and its expression is induced by abscisic acid

Cyclin-dependent kinase (CDK) inhibitor genes encode low molecular weight proteins which have important functions in cell cycle regulation, development and perhaps also in tumorigenesis. The first plant CDK inhibitor gene ICK1 was recently identified from Arabidopsis thaliana . Although the C-terminal domain of ICK1 contained an important consensus sequence with the mammalian CDK inhibitor p27^Kip1, the remainder of the deduced ICK1 sequence showed little similarity to any known CDK inhibitors. In vitro assays showed that recombinant ICK1 exhibited unique kinase inhibitory properties. In the present study we characterized ICK1 in terms of its gene structure, its interaction with both A. thaliana Cdc2a and CycD3, and its induction by the plant growth regulator, abscisic acid (ABA). ICK1 was expressed at a relatively low level in the tissues surveyed. However, ICK1 was induced by ABA, and along with ICK1 induction there was a decrease in Cdc2-like histone H1 kinase activity. These results suggest a molecular mechanism by which plant cell division might be inhibited by ABA. ICK1 clones were also identified from independent yeast two-hybrid screens using the CycD3 construct. The implication that ICK1 protein could interact with both Cdc2a and CycD3 was confirmed by in vitro binding assays. Furthermore, deletion analysis indicated that different regions of ICK1 are required for the interactions with Cdc2a and CycD3. These results provide a mechanistic basis for understanding the role of CDK inhibitors in cell cycle regulation in plant cells.     

Solution structure of BmKTX, a K+ blocker toxin from the Chinese scorpion Buthus Martensi

BmKTX is a toxin recently purified from the venom of Buthus Martensi, which belongs to the kaliotoxin family. We have determined its solution structure by use of conventional two-dimensional NMR techniques followed by distance-geometry and energy minimization. The calculated structure is composed of a short alpha-helix (residues 14 to 20) connected by a tight turn to a two-stranded antiparallel beta-sheet (sequences 25-27 and 32-34). The beta-turn connecting these strands belongs to type I. The N-terminal segment (sequence 1 to 8) runs parallel to the beta-sheet although it cannot be considered as a third strand. Comparison of the conformation of BmKTX and toxins of the kaliotoxin family clearly demonstrates that they are highly related. Therefore, analysis of the residues belonging to the interacting surface of those toxins allows us to propose a functional map of BmKTX slightly different from the one of KTX and AgTX2, which may explain the variations in affinities of these toxins towards the Kv1.3 channels.     

A short motif in Arabidopsis CDK inhibitor ICK1 decreases the protein level,probably through a ubiquitin‐independent mechanism

The ICK/KRP family of cyclin‐dependent kinase (CDK) inhibitors modulates the activity of plant CDKs through protein binding. Previous work has shown that changing the levels of ICK/KRP proteins by overexpression or downregulation affects cell proliferation and plant growth, and also that the ubiquitin proteasome system is involved in degradation of ICK/KRPs. We show in this study that the region encompassing amino acids 21 to 40 is critical for ICK1 levels in both Arabidopsis and yeast. To determine how degradation of ICK1 is controlled, we analyzed the accumulation of hemagglutinin (HA) epitope‐tagged ICK1 proteins in yeast mutants defective for two ubiquitin E3 ligases. The highest level of HA‐ICK1 protein was observed when both the N‐terminal 1–40 sequence was removed and the SCF (SKP1–Cullin1–F‐box complex) function disrupted, suggesting the involvement of both SCF‐dependent and SCF‐independent mechanisms in the degradation of ICK1 in yeast. A short motif consisting of residues 21–30 is sufficient to render green fluorescent protein (GFP) unstable in plants and had a similar effect in plants regardless of whether it was fused to the N‐terminus or C‐terminus of GFP. Furthermore, results from a yeast ubiquitin receptor mutant rpn10Δ indicate that protein ubiquitination is not critical in the degradation of GFP‐ICK1^1–40 in yeast. These results thus identify a protein‐destabilizing sequence motif that does not contain a typical ubiquitination residue, suggesting that it probably functions through an SCF‐independent mechanism.     

The impact of the fourth disulfide bridge in scorpion toxins of the alpha-KTx6 subfamily

Animal toxins are highly reticulated and structured polypeptides that adopt a limited number of folds. In scorpion species, the most represented fold is the alpha/beta scaffold in which an helical structure is connected to an antiparallel beta-sheet by two disulfide bridges. The intimate relationship existing between peptide reticulation and folding remains poorly understood. Here, we investigated the role of disulfide bridging on the 3D structure of HsTx1, a scorpion toxin potently active on Kv1.1 and Kv1.3 channels. This toxin folds along the classical alpha/beta scaffold but belongs to a unique family of short-chain, four disulfide-bridged toxins. Removal of the fourth disulfide bridge of HsTx1 does not affect its helical structure, whereas its two-stranded beta-sheet is altered from a twisted to a nontwisted configuration. This structural change in HsTx1 is accompanied by a marked decrease in Kv1.1 and Kv1.3 current blockage, and by alterations in the toxin to channel molecular contacts. In contrast, a similar removal of the fourth disulfide bridge of Pi1, another scorpion toxin from the same structural family, has no impact on its 3D structure, pharmacology, or channel interaction. These data highlight the importance of disulfide bridging in reaching the correct bioactive conformation of some toxins.     

High-resolution NMR structure of the chemically-synthesized melanocortin receptor binding domain AGRP(87-132) of the agouti-related protein.

The agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin receptors MC3R and MC4R found in the hypothalamus and exhibits potent orexigenic (appetite-stimulating) activity. The cysteine-rich C-terminal domain of this protein, corresponding to AGRP(87-132), contains five disulfide bonds and exhibits receptor binding affinity and antagonism equivalent to that of the full-length protein. The three-dimensional structure of this domain has been determined by 1H NMR at 800 MHz. The first 34 residues of AGRP(87-132) are well-ordered and contain a three-stranded antiparallel beta sheet, where the last two strands form a beta hairpin. The relative spatial positioning of the disulfide cross-links demonstrates that the ordered region of AGRP(87-132) adopts the inhibitor cystine knot (ICK) fold previously identified for numerous invertebrate toxins. Interestingly, this may be the first example of a mammalian protein assigned to the ICK superfamily. The hairpin's turn region presents a triplet of residues (Arg-Phe-Phe) known to be essential for melanocortin receptor binding. The structure also suggests that AGRP possesses an additional melanocortin-receptor contact region within a loop formed by the first 16 residues of its C-terminal domain. This specific region shows little sequence homology to the corresponding region of the agouti protein, which is an MC1R antagonist involved in pigmentation. Consideration of these sequence differences, along with recent experiments on mutant and chimeric melanocortin receptors, allows us to postulate that this loop in the first 16 residues of its C-terminal domain confers AGRP's distinct selectivity for MC3R and MC4R.     

Omega-agatoxins: novel calcium channel antagonists of two subtypes from funnel web spider (Agelenopsis aperta) venom

A new series of polypeptide presynaptic antagonists ("omega-agatoxins") was purified from venom of the funnel web spider Agelenopsis aperta. Physiological data indicate that all of these peptides are antagonists of voltage-sensitive calcium channels. Although all three omega-agatoxins (Aga) described here (omega-Aga-IA, omega-Aga-IB, and omega-Aga-IIA) block insect neuromuscular transmission presynaptically, biochemical data permit their subclassification as Type I and Type II toxins. Type I toxins (omega-Aga-IA and -IB) are 7.5 kDa, have closely related amino acid sequences, and exhibit characteristic tryptophan-like UV absorbance spectra. Complete Edman sequencing of omega-Aga-IA reveals it to be a 66-amino acid polypeptide containing 9 cysteines and 5 tryptophan residues. omega-Aga-IIA, a Type II toxin, is 11 kDa, shows limited amino acid sequence similarity to the Type I toxins, and exhibits mixed tryptophan- and tyrosine-like absorbance. Nanomolar concentrations of omega-Aga-IIA inhibit the specific binding of 125I-labeled omega-conotoxin GVIA to chick synaptosomal membranes while omega-Aga-IA and -IB have no effect under identical conditions. The omega-agatoxins thus are defined as two subtypes of neuronal calcium channel toxins with different structural characteristics and calcium channel binding specificities.     

Solution structures of the cis- and trans-Pro30 isomers of a novel 38-residue toxin from the venom of Hadronyche Infensa sp. that contains a cystine-knot motif within its four disulfide bonds

The primary sequence and three-dimensional structure of a novel peptide toxin isolated from the Australian funnel-web spider Hadronyche infensa sp. is reported. ACTX-Hi:OB4219 contains 38 amino acids, including eight-cysteine residues that form four disulfide bonds. The connectivities of these disulfide bonds were previously unknown but have been unambiguously determined in this study. Three of these disulfide bonds are arranged in an inhibitor cystine-knot (ICK) motif, which is observed in a range of other disulfide-rich peptide toxins. The motif incorporates an embedded ring in the structure formed by two of the disulfides and their connecting backbone segments penetrated by a third disulfide bond. Using NMR spectroscopy, we determined that despite the isolation of a single native homologous product by RP-HPLC, ACTX-Hi:OB4219 possesses two equally populated conformers in solution. These two conformers were determined to arise from cis/trans isomerization of the bond preceding Pro30. Full assignment of the NMR spectra for both conformers allowed for the calculation of their structures, revealing the presence of a triple-stranded antiparallel beta sheet consistent with the inhibitor cystine-knot (ICK) motif.