Inhibitor Cystine Knot Research Articles

Dissecting a role of evolutionary‐conserved but noncritical disulfide bridges in cysteine‐rich peptides using ω‐conotoxin GVIA and its selenocysteine analogs

Conotoxins comprise a large group of peptidic neurotoxins that use diverse disulfide-rich scaffolds. Each scaffold is determined by an evolutionarily conserved pattern of cysteine residues. Although many structure-activity relationship studies confirm the functional and structural importance of disulfide crosslinks, there is growing evidence that not all disulfide bridges are critical in maintaining activities of conotoxins. To answer the fundamental biological question of what the role of noncritical disulfide bridges is, we investigated function and folding of disulfide-depleted analogs of ω-conotoxin GVIA (GVIA) that belongs to an inhibitory cystine knot motif family and blocks N-type calcium channels. Removal of a noncritical Cys1-Cys16 disulfide bridge in GVIA or its selenopeptide analog had, as predicted, rather minimal effects on the inhibitory activity on calcium channels, as well as on in vivo activity following intracranial administration. However, the disulfide-depleted GVIA exhibited significantly lower folding yields for forming the remaining two native disulfide bridges. The disulfide-depleted selenoconotoxin GVIA analog also folded with significantly lower yields, suggesting that the functionally noncritical disulfide pair plays an important cooperative role in forming the native disulfide scaffold. Taken together, our results suggest that distinct disulfide bridges may be evolutionarily preserved by the oxidative folding or/and stabilization of the bioactive conformation of a disulfide-rich scaffold.

Agouti-Related Protein Segments Outside of the Receptor Binding Core Are Required for Enhanced Short- and Long-term Feeding Stimulation

The agouti-related protein (AgRP) plays a central role in energy balance by reducing signaling through the hypothalamic melanocortin receptors (McRs) 3 and 4, in turn stimulating feeding and decreasing energy expenditure. Mature AgRP(83-132), produced by endoproteolytic processing, contains a central region that folds as an inhibitor cystine knot (ICK) stabilized by a network of disulfide bonds; this domain alone carries the molecular features for high affinity McR binding and inverse agonism. Outside of the ICK domain are two polypeptide segments, an N-terminal extension and a C-terminal loop, both completely conserved but of unknown function. Here we examine the physiological roles of these non-ICK segments by developing a panel of modified AgRPs that were administered to rats through intracerebroventricular (ICV) injection. Analysis of food consumption demonstrates that basic (positively charged) residues are essential for potent short- and long-term AgRP stimulated feeding. Moreover, we demonstrate an approximate linear relationship between protein charge density and 24 h food intake. Next, we developed artificial AgRP(83-132) analogues with increased positive charge and found that these species were substantially more potent than wild type. A single dose of one protein, designated AgRP-4K, results in enhanced feeding for well over a week and weight gain that is nearly double that of AgRP(83-132). These studies suggest new strategies for the development of potent orexigenic species and may serve as leads for the development of therapeutics for treating wasting conditions such as cachexia.

New Mode of Action for a Knottin Protein Bioinsecticide: PEA ALBUMIN 1 SUBUNIT b (PA1b) IS THE FIRST PEPTIDIC INHIBITOR OF V-ATPase

PA1b (for pea albumin 1 subunit b) is a plant bioinsecticide lethal to several pests that are important in agriculture or human health. PA1b belongs to the inhibitory cystine knot family or knottin family. Originating from a plant (the garden pea) commonly eaten by humans without any known toxic or allergic effects, PA1b is a candidate for transgenic applications and is one of the most promising biopesticides for pest control. Using whole-cell patch-clamp techniques on Sf9 PA1b-sensitive lepidopteran insect cells, we discovered that PA1b reversibly blocked ramp membrane currents in a dose-dependent manner (EC(50) = 0.52 μM). PA1b had the same effect as bafilomycin, a specific inhibitor of the vacuolar proton pump (V-type H(+)-ATPase), and the PA1b-sensitive current depended on the internal proton concentration. Biochemical assays on purified V-ATPase from the lepidopteran model Manduca sexta showed that PA1b inhibited the V(1)V(0)-type H(+)-ATPase holoenzyme activity (IC(50) ∼ 70 nM) by interacting with the membrane-bound V(0) part of the V-ATPase. V-ATPase is a complex protein that has been studied increasingly because of its numerous physiological roles. In the midgut of insects, V-ATPase activity is essential for energizing nutrient absorption, and the results reported in this work explain the entomotoxic properties of PA1b. Targeting V-ATPase is a promising means of combating insect pests, and PA1b represents the first peptidic V-ATPase inhibitor. The search for V-ATPase inhibitors is currently of great importance because it has been demonstrated that V-ATPase plays a role in so many physiological processes.

Solution structure of a short-chain insecticidal toxin LaIT1 from the venom of scorpion Liocheles australasiae

The solution structure of an insecticidal toxin LaIT1, a 36-residue peptide with a unique amino-acid sequence and two disulfide bonds, isolated from the venom of the scorpion Liocheles australasiae was determined by heteronuclear NMR spectroscopy. Structural similarity search showed that LaIT1 exhibits an inhibitory cystine knot (ICK)-like fold, which usually contains three or more disulfide bonds. Mutational analysis has revealed that two Arg residues of LaIT1, Arg13 and Arg15, play significant roles in insecticidal activity.

Unique scorpion toxin with a putative ancestral fold provides insight into evolution of the inhibitor cystine knot motif.

The three-disulfide inhibitor cystine knot (ICK) motif is a fold common to venom peptides from spiders, scorpions, and aquatic cone snails. Over a decade ago it was proposed that the ICK motif is an elaboration of an ancestral two-disulfide fold coined the disulfide-directed β-hairpin (DDH). Here we report the isolation, characterization, and structure of a novel toxin [U(1)-liotoxin-Lw1a (U(1)-LITX-Lw1a)] from the venom of the scorpion Liocheles waigiensis that is the first example of a native peptide that adopts the DDH fold. U(1)-LITX-Lw1a not only represents the discovery of a missing link in venom protein evolution, it is the first member of a fourth structural fold to be adopted by scorpion-venom peptides. Additionally, we show that U(1)-LITX-Lw1a has potent insecticidal activity across a broad range of insect pest species, thereby providing a unique structural scaffold for bioinsecticide development.

A Novel Family of Insect-Selective Peptide Neurotoxins Targeting Insect Large-Conductance Calcium-Activated K+ Channels Isolated from the Venom of the Theraphosid Spider Eucratoscelus constrictus

Spider venoms are actively being investigated as sources of novel insecticidal agents for biopesticide engineering. After screening 37 theraphosid spider venoms, a family of three new "short-loop" inhibitory cystine knot insecticidal toxins (κ-TRTX-Ec2a, κ-TRTX-Ec2b, and κ-TRTX-Ec2c) were isolated and characterized from the venom of the African tarantula Eucratoscelus constrictus. Whole-cell patch-clamp recordings from cockroach dorsal unpaired median neurons revealed that, despite significant sequence homology with other theraphosid toxins, these 29-residue peptides lacked activity on insect voltage-activated sodium and calcium channels. It is noteworthy that κ-TRTX-Ec2 toxins were all found to be high-affinity blockers of insect large-conductance calcium-activated K(+) (BK(Ca)) channel currents with IC(50) values of 3 to 25 nM. In addition, κ-TRTX-Ec2a caused the inhibition of insect delayed-rectifier K(+) currents, but only at significantly higher concentrations. κ-TRTX-Ec2a and κ-TRTX-Ec2b demonstrated insect-selective effects, whereas the homologous κ-TRTX-Ec2c also resulted in neurotoxic signs in mice when injected intracerebroventricularly. Unlike other theraphosid toxins, κ-TRTX-Ec2 toxins induce a voltage-independent channel block, and therefore, we propose that these toxins interact with the turret and/or loop region of the external entrance to the channel and do not project deeply into the pore of the channel. Furthermore, κ-TRTX-Ec2a and κ-TRTX-Ec2b differ from other theraphotoxins at the C terminus and positions 5 to 6, suggesting that these regions of the peptide contribute to the phyla selectivity and are involved in targeting BK(Ca) channels. This study therefore establishes these toxins as tools for studying the role of BK(Ca) channels in insects and lead compounds for the development of novel insecticides.

Screening Spider Toxin Libraries for TRP Channel Inhibitors

Pain caused by activation of pain-sensing peripheral neurons (“nociceptors”) is a major source of human suffering and economic loss. A variety of stimulus- and voltage-gated ion channels in peripheral nociceptors play essential roles in the transduction and transmission of normal and pathological pain signals in human patients, and thus are important targets for pharmacological intervention. Around one hundred inhibitory cystine knot (ICK) peptide toxins were selected from a database of published and unpublished spider toxins. The tethered-toxin (“t-toxin”) method for the heterologous expression of ICK ion channel toxins in membrane tethered form directly in a channel-expressing target cell was applied. Pools of cRNA of t-toxins (∼6 per pool) were co-expressed with ion channels in Xenopus oocytes and the currents were measured by two-electrode voltage clamp 2-4 days after injection. One pool was found to cause ∼50 % suppression of the TRPA1 current induced by 100 μM mustard oil, and subscreening of this pool revealed a single t-toxin responsible for this effect. When co-expressed with TRPV1, this t-toxin suppressed the inward current induced by 20 μM capsaicin ∼80%. The effect of this t-toxin on other ion channels was also investigated. It completely blocked depolarization-activated inward currents of Para, a Drosophila voltage-gated sodium channel. When co-expressed with Nav1.2, it reduced peak currents by 60%. Interestingly, however, it did not affect currents flowing through Kir4.1, an inwardly rectifying potassium channel. These results suggest that this relatively promiscuous toxin acts on a structural feature common to ion channels with six transmembrane domains (Trp channels and voltage-gated channels), but lacking in those that only possess the two pore-spanning transmembrane domains (inward rectifier).

Optimizing structural modeling for a specific protein scaffold: knottins or inhibitor cystine knots

BackgroundKnottins are small, diverse and stable proteins with important drug design potential. They can be classified in 30 families which cover a wide range of sequences (1621 sequenced), three-dimensional structures (155 solved) and functions (> 10). Inter knottin similarity lies mainly between 15% and 40% sequence identity and 1.5 to 4.5 Å backbone deviations although they all share a tightly knotted disulfide core. This important variability is likely to arise from the highly diverse loops which connect the successive knotted cysteines. The prediction of structural models for all knottin sequences would open new directions for the analysis of interaction sites and to provide a better understanding of the structural and functional organization of proteins sharing this scaffold.ResultsWe have designed an automated modeling procedure for predicting the three-dimensionnal structure of knottins. The different steps of the homology modeling pipeline were carefully optimized relatively to a test set of knottins with known structures: template selection and alignment, extraction of structural constraints and model building, model evaluation and refinement. After optimization, the accuracy of predicted models was shown to lie between 1.50 and 1.96 Å from native structures at 50% and 10% maximum sequence identity levels, respectively. These average model deviations represent an improvement varying between 0.74 and 1.17 Å over a basic homology modeling derived from a unique template. A database of 1621 structural models for all known knottin sequences was generated and is freely accessible from our web server at http://knottin.cbs.cnrs.fr. Models can also be interactively constructed from any knottin sequence using the structure prediction module Knoter1D3D available from our protein analysis toolkit PAT at http://pat.cbs.cnrs.fr.ConclusionsThis work explores different directions for a systematic homology modeling of a diverse family of protein sequences. In particular, we have shown that the accuracy of the models constructed at a low level of sequence identity can be improved by 1) a careful optimization of the modeling procedure, 2) the combination of multiple structural templates and 3) the use of conserved structural features as modeling restraints.

D-Maurocalcine, a Pharmacologically Inert Efficient Cell-penetrating Peptide Analogue

Maurocalcine has been the first demonstrated animal toxin acting as a cell-penetrating peptide. Although it possesses competitive advantages, its use as a cell-penetrating peptide (CPP) requires that analogues be developed that lack its characteristic pharmacological activity on ryanodine-sensitive calcium channels without affecting its cell-penetrating and vector efficiencies. Here, we present the synthesis, three-dimensional (1)H NMR structure, and activity of D-maurocalcine. We demonstrate that it possesses all of the desired features for an excellent CPP: preserved structure, lack of pharmacological action, conserved vector properties, and absence of cell toxicity. This is the first report of a folded/oxidized animal toxin in its D-diastereomer conformation for use as a CPP. The protease resistance of this new peptide analogue, combined with its efficient cell penetration at concentrations devoid of cell toxicity, suggests that D-maurocalcine should be an excellent vector for in vivo applications.

Novel Class of Spider Toxin: ACTIVE PRINCIPLE FROM THE YELLOW SAC SPIDER CHEIRACANTHIUM PUNCTORIUM VENOM IS A UNIQUE TWO-DOMAIN POLYPEPTIDE

Venom of the yellow sac spider Cheiracanthium punctorium (Miturgidae) was found unique in terms of molecular composition. Its principal toxic component CpTx 1 (15.1 kDa) was purified, and its full amino acid sequence (134 residues) was established by protein chemistry and mass spectrometry techniques. CpTx 1 represents a novel class of spider toxin with modular architecture. It consists of two different yet homologous domains (modules) each containing a putative inhibitor cystine knot motif, characteristic of the widespread single domain spider neurotoxins. Venom gland cDNA sequencing provided precursor protein (prepropeptide) structures of three CpTx 1 isoforms (a-c) that differ by single residue substitutions. The toxin possesses potent insecticidal (paralytic and lethal), cytotoxic, and membrane-damaging activities. In both fly and frog neuromuscular preparations, it causes stable and irreversible depolarization of muscle fibers leading to contracture. This effect appears to be receptor-independent and is inhibited by high concentrations of divalent cations. CpTx 1 lyses cell membranes, as visualized by confocal microscopy, and destabilizes artificial membranes in a manner reminiscent of other membrane-active peptides by causing numerous defects of variable conductance and leading to bilayer rupture. The newly discovered class of modular polypeptides enhances our knowledge of the toxin universe.

Structure and Orientation of a Voltage-Sensor Toxin in Lipid Membranes

Amphipathic protein toxins from tarantula venom inhibit voltage-activated potassium (Kv) channels by binding to a critical helix-turn-helix motif termed the voltage sensor paddle. Although these toxins partition into membranes to bind the paddle motif, their structure and orientation within the membrane are unknown. We investigated the interaction of a tarantula toxin named SGTx with membranes using both fluorescence and NMR spectroscopy. Depth-dependent fluorescence-quenching experiments with brominated lipids suggest that Trp30 in SGTx is positioned ∼9 Å from the center of the bilayer. NMR spectra reveal that the inhibitor cystine knot structure of the toxin does not radically change upon membrane partitioning. Transferred cross-saturation NMR experiments indicate that the toxin's hydrophobic protrusion contacts the hydrophobic core of the membrane, whereas most surrounding polar residues remain at interfacial regions of the bilayer. The inferred orientation of the toxin reveals a twofold symmetry in the arrangement of basic and hydrophobic residues, a feature that is conserved among tarantula toxins. These results have important implications for regions of the toxin involved in recognizing membranes and voltage-sensor paddles, and for the mechanisms by which tarantula toxins alter the activity of different types of ion channels.

Antimicrobial peptide-like genes in Nasonia vitripennis: a genomic perspective

BackgroundAntimicrobial peptides (AMPs) are an essential component of innate immunity which can rapidly respond to diverse microbial pathogens. Insects, as a rich source of AMPs, attract great attention of scientists in both understanding of the basic biology of the immune system and searching molecular templates for anti-infective drug design. Despite a large number of AMPs have been identified from different insect species, little information in terms of these peptides is available from parasitic insects.ResultsBy using integrated computational approaches to systemically mining the Hymenopteran parasitic wasp Nasonia vitripennis genome, we establish the first AMP repertoire whose members exhibit extensive sequence and structural diversity and can be distinguished into multiple molecular types, including insect and fungal defensin-like peptides (DLPs) with the cysteine-stabilized α-helical and β-sheet (CSαβ) fold; Pro- or Gly-rich abaecins and hymenoptaecins; horseshoe crab tachystatin-type AMPs with the inhibitor cystine knot (ICK) fold; and a linear α-helical peptide. Inducible expression pattern of seven N. vitripennis AMP genes were verified, and two representative peptides were synthesized and functionally identified to be antibacterial. In comparison with Apis mellifera (Hymenoptera) and several non-Hymenopteran model insects, N. vitripennis has evolved a complex antimicrobial immune system with more genes and larger protein precursors. Three classical strategies that are likely responsible for the complexity increase have been recognized: 1) Gene duplication; 2) Exon duplication; and 3) Exon-shuffling.ConclusionThe present study established the N. vitripennis peptidome associated with antimicrobial immunity by using a combined computational and experimental strategy. As the first AMP repertoire of a parasitic wasp, our results offer a basic platform for further studying the immunological and evolutionary significances of these newly discovered AMP-like genes in this class of insects.

Site-Specific Effects of Diselenide Bridges on the Oxidative Folding of a Cystine Knot Peptide, ω-Selenoconotoxin GVIA

Structural and functional studies of small, disulfide-rich peptides depend on their efficient chemical synthesis and folding. A large group of peptides derived from animals and plants contains the Cys pattern C-C-CC-C-C that forms the inhibitory cystine knot (ICK) or knottin motif. Here we report the effect of site-specific incorporation of pairs of selenocysteine residues on oxidative folding and the functional activity of omega-conotoxin GVIA, a well-characterized ICK-motif peptidic antagonist of voltage-gated calcium channels. Three selenoconotoxin GVIA analogues were chemically synthesized; all three folded significantly faster in the glutathione-based buffer compared to wild-type GVIA. One analogue, GVIA[C8U,C19U], exhibited significantly higher folding yields. A recently described NMR-based method was used for mapping the disulfide connectivities in the three selenoconotoxin analogues. The diselenide-directed oxidative folding of selenoconotoxins was predominantly driven by amino acid residue loop sizes formed by the resulting diselenide and disulfide cross-links. Both in vivo and in vitro activities of the analogues were assessed; the block of N-type calcium channels was comparable among the analogues and wild-type GVIA, suggesting that the diselenide replacement did not affect the bioactive conformation. Thus, diselenide substitution may facilitate oxidative folding of pharmacologically diverse ICK peptides. The diselenide replacement has been successfully applied to a growing number of bioactive peptides, including alpha-, mu-, and omega-conotoxins, suggesting that the integrated oxidative folding of selenopeptides described here may prove to be a general approach for efficient synthesis of diverse classes of disulfide-rich peptides.

Synthesis, biophysical, and biological studies of wild-type and mutant psalmopeotoxins—Anti-malarial cysteine knot peptides from Psalmopoeus cambridgei

Psalmopeotoxin I and II (PcFK1 and PcFK2), an anti-malarial peptide first extracted from Psalmopoeus cambridgei was synthesized and characterized. Both peptides belong to the Inhibitor Cystine Knot (ICK) superfamily, containing three disulfide bridges. The six cysteine residues are conserved similar to other members of the ICK superfamily, suggesting their critical role for either folding or function. In this study, the peptides were synthesized using Fmoc solid-phase peptide synthesis (SPPS). The three disulfide bonds of were constructed by regioselective and random oxidative approaches. The resulting disulfide bond patterns were verified by the HPLC-MS analysis of intact peptides and by the disulfide bond mapping using tryptic digestion. Implications of the disulfide bonds on the biophysical and biological properties of PcFKs were studied using three disulfide mutants in which a particular pair of cysteines was replaced with two isosteric serine residues. Structures and biophysical characteristics of all variants were studied using far-UV CD and fluorescence spectroscopy. Biological activities of all variants were evaluated using antiplasmodial assay against the K1 multi-drug-resistant strain of P. falciparum. The experimental results showed that the three disulfide bridges could not be correctly synthesized by the random oxidative strategy. Structural and biophysical analyses revealed that all variants had similar structures to the twisted β-sheet. However, the studies of disulfide bond removal indicated that each disulfide bond had different effects on both biophysical and biological activities of PcFKs. Correlation of biophysical parameters and biological activities showed that both PcFKs may have different mechanisms of actions for antiplasmodial activity.

Determination with Matrix-Assisted Laser Desorption/Ionization Tandem Time-of-Flight Mass Spectrometry of the Extensive Disulfide Bonding in Tarantula Venom Peptide Psalmopeotoxin I

Psalmopeotoxin I (PcFK1) is a 33-residue peptide isolated from the venom of the tarantula Psalmopoeus cambridgei. This peptide specifically inhibits the intra-erythrocyte stage of Plasmodium falciparum in vitro. It contains six cysteine residues forming three disulfide bridges and belongs to the superfamily of natural peptides containing the inhibitor cystine knot (ICK) fold. We produced the wild-type and mutated forms of the recombinant peptide to examine the mechanism of action of PcFK1. The purified toxins were consistently produced as two isobaric peptides (r-PcFK1-1 and r-PcFK1-2) with different retention properties but identical anti-plasmodial -biological activity. Comparison of (15)N-NMR heteronuclear single quantum correlation spectra revealed that although rPcFK1-1 was highly structured, rPcFK1-2 does not have a stable three-dimensional structure. We used high-energy collision-induced fragmentation of the peptides with a matrix-assisted laser desorption/ionization tandem time-of- flight mass spectrometer to further investigate the structure of the native peptides in its natural form and produced in E. coli. The fragmentation spectra of the native peptides were very complex due to the occurrence in the spectrum of ions resulting from (1) cross-linking of fragments through a disulfide bridge and (2) asymmetric fragmentations of the disulfide bridges and (3) multiple neutral losses. The tandem mass spectrometry fragmentation pattern of r-PcFK1-1 was similar to that of the natural peptide isolated from crude venom, but r-PcFK1-2 had a clearly distinct fragmentation pattern, more closely resembling the fragmentation spectra of reduced and alkylated peptides. Observed ions could be attributed to specific fragments by comparing spectra between the wild-type and selected variants with point mutations (Y11W, R20T, Y26W, K28V). The disulfide connections in r-PcFK1-2 differed from those of the native peptide and showed a rare disulfide bridge between vicinal cysteine residues. The r-PcFK1_(R20T) variant showed a very limited fragmentation pattern when analyzed in positive mode but displayed much more fragmentation in negative mode pointing out the importance of the R20 residue in the fragmentation of PcFK1. Using the reductive matrix 1,5-diaminonaphtalene promoted strongly in source decay fragmentation of the peptides in MS mode. Our findings illustrated the critical role of the electronic environment around the central Cys(18)-Cys(19) doublet in PcFK1 in internal fragmentation of the peptide.

S3b Amino Acid Substitutions and Ancillary Subunits Alter the Affinity of Heteropoda venatoria Toxin 2 for Kv4.3

Heteropoda venatoria toxin 2 (HpTx2) is an inhibitor cystine knot (ICK)-gating modifier toxin that selectively inhibits Kv4 channels. To characterize the molecular determinants of interaction, we performed alanine scanning of the Kv4.3 S3b region. HpTx2-Kv4.3 interaction had an apparent K(d) value of 2.3 microM. Two alanine mutants in Kv4.3 increased K(d) values to 6.4 microM for V276A and 25 microM for L275A. Simultaneous mutation of both amino acids to alanine nearly eliminated toxin interaction. Unlike Hanatoxin and other well characterized ICK toxins, HpTx2 binding does not require a charged amino acid for interaction. To determine whether the identity of the S3b binding site amino acids altered HpTx2 specificity, we constructed Kv4.3 [LV275IF]. This mutation decreased the K(d) value to 0.54 microM, suggesting that the hydrophobic character of the putative binding site is the most important property for interaction with HpTx2. One mutant, N280A, caused stronger interaction of HpTx2 with Kv4.3; the K(d) value for Kv4.3 [N280A] was 0.26 microM. To understand Kv4.3-based transient outward currents in native tissues, we tested the affinity of HpTx2 for Kv4.3 coexpressed with KChIP2b. The toxin's K(d) value for Kv4.3 + KChIP2b was 0.95 microM. KChIP2b stabilizes the closed state of Kv4.3, suggesting that the increased toxin affinity is due to increased stabilization of the closed state. These data show that HpTx2 binding to Kv4.3 has aspects in common with other ICK gating modifier toxins but that the interventions that increase toxin affinity suggest flexibility toward channel binding that belies its unusual specificity for Kv4 channels.

Imperatoxin A, A Calcin Toxin From Pandinus Imperator Scorpions, Ablates Calcium Sparks In Permeabilized Cells

Imperatoxin A (IpTxa) is a 33 amino acid peptide toxin from the African scorpion Pandinus imperator. Its definitive structural and functional characteristics designate it as a member of the calcin family, a unique group of peptide toxins, which to date includes Hemicalcin (HCa), Hadrucalcin (HdCa), IpTxa, Maurocalcin (MCa), and Opicalcins 1 & 2 (Opi 1 & 2). Calcins are unified by 1) a compact, globular structure containing an inhibitor cystine knot, 2) positive net charge, 3) the ability to activate ryanodine receptors (RyR) with high affinity and selectivity, and 4) presumed or confirmed cell-penetrating capabilities. Previous studies have demonstrated the ability of MCa to carry a fluorescent cargo to the interior of intact cells, and we have used a similar method to confirm that IpTxa is also a cell-penetrator. In addition, we have demonstrated IpTxa's ability to transiently enhance calcium release from the sarcoplasmic reticulum (SR) of intact cells. In the current study, we used confocal microscopy to explore the effects of IpTxa on saponin-permeabilized cells. Perfusion of 10nM IpTxa on cells resulted in a dramatic reduction of sparks to as low as 1% of control within two minutes. This effect was even more pronounced and rapid in the presence of 50nM toxin, with complete ablation of sparks in less than 1 minute. At the same time, mean fluorescence is often increased in our scans relative to control, although rapid application of caffeine reveals that SR calcium content is retained. Our results suggest that IpTxa depletes calcium from the SR, a hypothesis that would explain not only the ablation of sparks, but also the transient nature of enhanced calcium release in intact cells observed upon perfusion with IpTxa.

A folded and functional synthetic PA1b: an interlocked entomotoxic miniprotein.

PA1b (Pea Albumin 1, subunit b) is a hydrophobic, 37-amino acid miniprotein isolated from pea seeds (Pivum sativum), crosslinked by three interlocked disulfide bridges, signature of the ICK (inhibitory cystine-knot) family. It acts as an entomotoxic factor against major insect pests in stored crops and vegetables, making it a promising bioinsecticide. Here we report an efficient and simple protocol for the production of large quantities of highly pure, biologically active synthetic PA1b. The features of PA1b oxidative refolding revealed the off-pathway products and competitive aggregation processes. The efficiency of the oxidative folding can be significantly improved by using hydrophobic alcoholic cosolvents and decreasing the temperature. The homogeneity of the synthetic oxidized PA1b was established by reversed-phase HPLC. The correct pairing of the three disulfide bridges, as well as the three-dimensional structure of synthetic PA1b was assessed by NMR. Synthetic PA1b binds to microsomal proteins from Sitophilus oryzae with a Kd of 8 nM, a figure quite similar to that determined for PA1b extracted from its natural source. Moreover, the synthetic miniprotein was as potent as the extracted one towards the sensitive strains of weevils. Our findings will open the way to the production of PA1b analogues by chemical means to an in-depth understanding of the PA1b mechanism of action.

Knottin cyclization: impact on structure and dynamics

BackgroundPresent in various species, the knottins (also referred to as inhibitor cystine knots) constitute a group of extremely stable miniproteins with a plethora of biological activities. Owing to their small size and their high stability, knottins are considered as excellent leads or scaffolds in drug design. Two knottin families contain macrocyclic compounds, namely the cyclotides and the squash inhibitors. The cyclotide family nearly exclusively contains head-to-tail cyclized members. On the other hand, the squash family predominantly contains linear members. Head-to-tail cyclization is intuitively expected to improve bioactivities by increasing stability and lowering flexibility as well as sensitivity to proteolytic attack.ResultsIn this paper, we report data on solution structure, thermal stability, and flexibility as inferred from NMR experiments and molecular dynamics simulations of a linear squash inhibitor EETI-II, a circular squash inhibitor MCoTI-II, and a linear analog lin-MCoTI. Strikingly, the head-to-tail linker in cyclic MCoTI-II is by far the most flexible region of all three compounds. Moreover, we show that cyclic and linear squash inhibitors do not display large differences in structure or flexibility in standard conditions, raising the question as to why few squash inhibitors have evolved into cyclic compounds. The simulations revealed however that the cyclization increases resistance to high temperatures by limiting structure unfolding.ConclusionIn this work, we show that, in contrast to what could have been intuitively expected, cyclization of squash inhibitors does not provide clear stability or flexibility modification. Overall, our results suggest that, for squash inhibitors in standard conditions, the circularization impact might come from incorporation of an additional loop sequence, that can contribute to the miniprotein specificity and affinity, rather than from an increase in conformational rigidity or protein stability. Unfolding simulations showed however that cyclization is a stabilizing factor in strongly denaturing conditions. This information should be useful if one wants to use the squash inhibitor scaffold in drug design.

Animal Toxins Acting on Voltage-Gated Potassium Channels

Animal venoms are rich natural sources of bioactive compounds, including peptide toxins acting on the various types of ion channels, i.e. K(+), Na(+), Cl(-) and Ca(2+). Among K+ channel-acting toxins, those selective for voltage-gated K(+) (Kv) channels are widely represented and have been isolated from the venoms of numerous animal species, such as scorpions, sea anemones, snakes, marine cone snails and spiders. The toxins characterized hitherto contain between 22 and 60 amino acid residues, and are cross-linked by two to four disulfide bridges. Depending on their types of fold, toxins can be classified in eight structural categories, which showed a combination of beta-strands, helices, or a mixture of both. The main architectural motifs thereof are referred to as alpha/beta scaffold and inhibitor cystine knot (ICK). A detailed analysis of toxin structures and pharmacological selectivities indicates that toxins exhibiting a similar type of fold can exert their action on several subtypes of Kv channels, whereas a particular Kv channel can be targeted by toxins that possess unrelated folds. Therefore, it appears that the ability of structurally divergent toxins to interact with a particular Kv channel relies onto a similar spatial distribution of amino acid residues that are key to the toxin-channel interaction (rather than the type of toxin fold). The diversity of Kv channel blockers and their therapeutic value in the potential treatment of a number of specific human diseases, especially autoimmune disorders, inflammatory neuropathies and cancer, are reviewed.