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Mutations in SCN4A gene encoding Na v 1.4 channel α-subunit, are known to cause neuromuscular disorders such as myotonia or paralysis. Here, we study the effect of two amino acid replacements, K1302Q and G1306E, in the DIII-IV loop of the channel, corresponding to mutations found in patients with myotonia. We combine clinical, electrophysiological, and molecular modeling data to provide a holistic picture of the molecular mechanisms operating in mutant channels and eventually leading to pathology. We analyze the existing clinical data for patients with the K1302Q substitution, which was reported for adults with or without myotonia phenotypes, and report two new unrelated patients with the G1306E substitution, who presented with severe neonatal episodic laryngospasm and childhood-onset myotonia. We provide a functional analysis of the mutant channels by expressing Na v 1.4 α-subunit in Xenopus oocytes in combination with β1 subunit and recording sodium currents using two-electrode voltage clamp. The K1302Q variant exhibits abnormal voltage dependence of steady-state fast inactivation, being the likely cause of pathology. K1302Q does not lead to decelerated fast inactivation, unlike several other myotonic mutations such as G1306E. For both mutants, we observe increased window currents corresponding to a larger population of channels available for activation. To elaborate the structural rationale for our experimental data, we explore the contacts involving K/Q1302 and E1306 in the AlphaFold2 model of wild-type Na v 1.4 and Monte Carlo-minimized models of mutant channels. Our data provide the missing evidence to support the classification of K1302Q variant as likely pathogenic and may be used by clinicians., (© 2024 Federation of American Societies for Experimental Biology.)

BeKm-1 is a peptide toxin from scorpion venom that blocks the pore of the potassium channel hERG (K v 11.1) in the human heart. Although individual protein structures have been resolved, the structure of the complex between hERG and BeKm-1 is unknown. Here, we used molecular dynamics and ensemble docking, guided by previous double-mutant cycle analysis data, to obtain an in silico model of the hERG-BeKm-1 complex. Adding to the previous mutagenesis study of BeKm-1, our model uncovers the key role of residue Arg20, which forms three interactions (a salt bridge and hydrogen bonds) with the channel vestibule simultaneously. Replacement of this residue even by lysine weakens the interactions significantly. In accordance, the recombinantly produced BeKm-1 R20K mutant exhibited dramatically decreased activity on hERG. Our model may be useful for future drug design attempts., (© 2024 Federation of European Biochemical Societies.)

Oligoarginine peptides, known mostly for their cell-penetrating properties, are also inhibitors of the nicotinic acetylcholine receptors (nAChRs). Since octa-arginine (R8) inhibits α9α10 nAChR and suppresses neuropathic pain, we checked if other polycationic compounds containing amino and/or guanidino groups could be effective and tested the activity of the disulfide-fixed "cyclo"R8, a series of biogenic polyamines (putrescine, spermidine, and spermine), C -methylated spermine analogs, agmatine and its analogs, as well as acylpolyamine argiotoxin-636 from spider venom. Their inhibitory potency on muscle-type, α7 and α9α10 nAChRs was determined using radioligand analysis, electrophysiology, and calcium imaging. "Cyclo"R8 showed similar activity to that of R8 against α9α10 nAChR (IC 50 ≈ 60 nM). Biogenic polyamines as well as agmatine and its analogs displayed low activity on muscle-type Torpedo californica , as well as α7 and α9α10 nAChRs, which increased with chain length, the most active being spermine and its C -methylated derivatives having IC 50 of about 30 μM against muscle-type T. californica nAChR. Argiotoxin-636, which contains a polyamine backbone and terminal guanidino group, also weakly inhibited T. californica nAChR (IC 50 ≈ 15 μM), but it revealed high potency against rat α9α10 nAChR (IC 50 ≈ 200 nM). We conclude that oligoarginines and similar polycationic compounds effectively inhibiting α9α10 nAChR may serve as a basis for the development of analgesics to reduce neuropathic pain., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ojomoko, Kryukova, Egorova, Salikhov, Epifanova, Denisova, Khomutov, Sukhov, Vassilevski, Khomutov, Tsetlin and Shelukhina.)

Here, we introduce the third release of Kalium database (http://kaliumdb.org/), a manually curated comprehensive depository that accumulates data on polypeptide ligands of potassium channels. The major goal of this amplitudinous update is to summarize findings for natural polypeptide ligands of K + channels, as well as data for the artificial derivatives of these substances obtained over the decades of exploration. We manually analyzed more than 700 original manuscripts and systematized the information on mutagenesis, production of radio- and fluorescently labeled derivatives, and the molecular pharmacology of K + channel ligands. As a result, data on more than 1200 substances were processed and added enriching the database content fivefold. We also included the electrophysiological data obtained on the understudied and neglected K + channels including the heteromeric and concatenated channels. We associated target channels in Kalium with corresponding entries in the official database of the International Union of Basic and Clinical Pharmacology. Kalium was supplemented with an adaptive Statistics page, where users are able to obtain actual data output. Several other improvements were introduced, such as a color code to distinguish the range of ligand activity concentrations and advanced tools for filtration and sorting. Kalium is a fully open-access database, crosslinked to other databases of interest. It can be utilized as a convenient resource containing ample up-to-date information about polypeptide ligands of K + channels., (© 2023 The Protein Society.)

Animal venoms are a promising source of potential bioinsecticides. To find hits with pronounced oral insect toxicity, we screened 82 venoms using Achroia grisella (Lepidoptera) and Tenebrio molitor (Coleoptera) larvae, and adult Drosophila melanogaster (Diptera). We also injected the most potent venoms in adult D. melanogaster to compare their efficiency in different routes of administration. 18 venoms from spiders and snakes show high oral toxicity and can be further exploited to isolate new insecticides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Scorpion α-toxins (α-NaTx) inhibiting the inactivation of voltage-gated sodium channels (Na v ) are a well-studied family of small proteins. We previously showed that the structure of α-NaTx specificity module responsible for selective Na v binding is governed by an interplay between the nest and niche protein motifs. Here, we report the solution structure of the toxin Lqq4 from the venom of the scorpion Leiurus quinquestriatus. Unexpectedly, we find that this toxin presents an ensemble of long-lived structurally distinct states. We unequivocally assign these states to the alternative configurations (cis-trans isomers) of two peptide bonds: V56-P57 and C17-G18; neither of the cis isomers has been described in α-NaTx so far. We argue that the native conformational space of α-NaTx is wider than assumed previously., (© 2023 Federation of European Biochemical Societies.)

Previous studies have identified some key amino acid residues in scorpion toxins blocking potassium channels. In particular, the most numerous toxins belonging to the α-KTx family and affecting voltage-gated potassium channels (K V ) present a conserved K-C-X-N motif in the C-terminal half of their sequence. Here, we show that the X position of this motif is almost always occupied by either methionine or isoleucine. We compare the activity of three pairs of peptides that differ just by this residue on a panel of K V 1 channels and find that toxins bearing methionine affect preferentially K V 1.1 and 1.6 isoforms. The refined K-C-M/I-N motif stands out as the principal structural element of α-KTx conferring high affinity and selectivity to K V channels., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Among voltage-gated potassium channel (K V ) isoforms, K V 1.6 is one of the most widespread in the nervous system. However, there are little data concerning its physiological significance, in part due to the scarcity of specific ligands. The known high-affinity ligands of K V 1.6 lack selectivity, and conversely, its selective ligands show low affinity. Here, we present a designer peptide with both high affinity and selectivity to K V 1.6. Previously, we have demonstrated that K V isoform-selective peptides can be constructed based on the simplistic α-hairpinin scaffold, and we obtained a number of artificial Tk-hefu peptides showing selective blockage of K V 1.3 in the submicromolar range. We have now proposed amino acid substitutions to enhance their activity. As a result, we have been able to produce Tk-hefu-11 that shows an EC 50 of ≈70 nM against K V 1.3. Quite surprisingly, Tk-hefu-11 turns out to block K V 1.6 with even higher potency, presenting an EC 50 of ≈10 nM. Furthermore, we have solved the peptide structure and used molecular dynamics to investigate the determinants of selective interactions between artificial α-hairpinins and K V channels to explain the dramatic increase in K V 1.6 affinity. Since K V 1.3 is not highly expressed in the nervous system, we hope that Tk-hefu-11 will be useful in studies of K V 1.6 and its functions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Apamin is often cited as one of the few substances selectively acting on small-conductance Ca 2+ -activated potassium channels (K Ca 2). However, published pharmacological and structural data remain controversial. Here, we investigated the molecular pharmacology of apamin by two-electrode voltage-clamp in Xenopus laevis oocytes and patch-clamp in HEK293, COS7, and CHO cells expressing the studied ion channels, as well as in isolated rat brain neurons. The microtitre broth dilution method was used for antimicrobial activity screening. The spatial structure of apamin in aqueous solution was determined by NMR spectroscopy. We tested apamin against 42 ion channels (K Ca , K V , Na V , nAChR, ASIC, and others) and confirmed its unique selectivity to K Ca 2 channels. No antimicrobial activity was detected for apamin against Gram-positive or Gram-negative bacteria. The NMR solution structure of apamin was deposited in the Protein Data Bank. The results presented here demonstrate that apamin is a selective nanomolar or even subnanomolar-affinity K Ca 2 inhibitor with no significant effects on other molecular targets. The spatial structure as well as ample functional data provided here support the use of apamin as a K Ca 2-selective pharmacological tool and as a template for drug design., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kuzmenkov, Peigneur, Nasburg, Mineev, Nikolaev, Pinheiro-Junior, Arseniev, Wulff, Tytgat and Vassilevski.)

Fluorescence can be exploited to monitor intermolecular interactions in real time and at a resolution up to a single molecule. It is a method of choice to study ligand-receptor interactions. However, at least one of the interacting molecules should possess good fluorescence characteristics, which can be achieved by the introduction of a fluorescent label. Gene constructs with green fluorescent protein (GFP) are widely used to follow the expression of the respective fusion proteins and monitor their function. Recently, a small synthetic analogue of GFP chromophore ( p -HOBDI-BF 2 ) was successfully used for tagging DNA molecules, so we decided to test its applicability as a potential fluorescent label for proteins and peptides. This was done on α-cobratoxin (α-CbTx), a three-finger protein used as a molecular marker of muscle-type, neuronal α7 and α9/α10 nicotinic acetylcholine receptors (nAChRs), as well as on azemiopsin, a linear peptide neurotoxin selectively inhibiting muscle-type nAChRs. An activated N-hydroxysuccinimide ester of p -HOBDI-BF 2 was prepared and utilized for toxin labeling. For comparison we used a recombinant α-CbTx fused with a full-length GFP prepared by expression of a chimeric gene. The structure of modified toxins was confirmed by mass spectrometry and their activity was characterized by competition with iodinated α-bungarotoxin in radioligand assay with respective receptor preparations, as well as by thermophoresis. With the tested protein and peptide neurotoxins, introduction of the synthetic GFP chromophore induced considerably lower decrease in their affinity for the receptors as compared with full-length GFP attachment. The obtained fluorescent derivatives were used for nAChR visualization in tissue slices and cell cultures., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kasheverov, Kuzmenkov, Kudryavtsev, Chudetskiy, Shelukhina, Barykin, Ivanov, Siniavin, Ziganshin, Baranov, Tsetlin, Vassilevski and Utkin.)

The α-Hairpinins are a family of plant defense peptides with a common fold presenting two short α-helices stabilized by two invariant S-S-bridges. We have shown previously that substitution of just two amino acid residues in a wheat α-hairpinin Tk-AMP-X2 leads to Tk-hefu-2 that features specific affinity to voltage-gated potassium channels K V 1.3. Here, we utilize a combined molecular modeling approach based on molecular dynamics simulations and protein surface topography technique to improve the affinity of Tk-hefu-2 to K V 1.3 while preserving its specificity. An important advance of this work compared with our previous studies is transition from the analysis of various physicochemical properties of an isolated toxin molecule to its consideration in complex with its target, a membrane-bound ion channel. As a result, a panel of computationally designed Tk-hefu-2 derivatives was synthesized and tested against K V 1.3. The most active mutant Tk-hefu-10 showed a half-maximal inhibitory concentration of ∼150 nM being >10 times more active than Tk-hefu-2 and >200 times more active than the original Tk-hefu. We conclude that α-hairpinins provide an attractive disulfide-stabilized scaffold for the rational design of ion channel inhibitors. Furthermore, the success rate can be considerably increased by the proposed "target-based" iterative strategy of molecular design., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Old world scorpions produce an abundance of toxins called α-NaTx, which interfere with the fast inactivation of voltage-gated sodium channels. Their selectivity to channels of mammals or insects depends on a part of toxin named the specificity module. We report here the spatial structure of a major and broadly active toxin MeuNaTxα-1 from the venom of Mesobuthus eupeus. Notably, its specificity module is markedly different from other α-NaTx with known 3D structure. Close inspection shows that its conformation is a result of an interplay between protein motifs such as the nest and niche, which eventually shape α-NaTx structural diversity., (© 2021 Wiley Periodicals LLC.)

Voltage-gated sodium channels (NaV) have a modular architecture and contain five membrane domains. The central pore domain is responsible for ion conduction and contains a selectivity filter, while the four peripheral voltage-sensing domains (VSD-I/IV) are responsible for activation and rapid inactivation of the channel. "Gating modifier" toxins from arthropod venoms interact with VSDs, influencing the activation and/or inactivation of the channel, and may serve as prototypes of new drugs for the treatment of various channelopathies and pain syndromes. The toxin-binding sites located on VSD-I, II and IV of mammalian NaV channels have been previously described. In this work, using the example of the Hm-3 toxin from the crab spider Heriaeus melloteei , we showed the presence of a toxin-binding site on VSD-III of the human skeletal muscle NaV1.4 channel. A developed cell-free protein synthesis system provided milligram quantities of isolated (separated from the channel) VSD-III and its 15N-labeled analogue. The interactions between VSD-III and Hm-3 were studied by NMR spectroscopy in the membrane-like environment of DPC/LDAO (1 : 1) micelles. Hm-3 has a relatively high affinity to VSD-III (dissociation constant of the complex Kd ~6 μM), comparable to the affinity to VSD‑I and exceeding the affinity to VSD-II. Within the complex, the positively charged Lys25 and Lys28 residues of the toxin probably interact with the S1-S2 extracellular loop of VSD-III. The Hm-3 molecule also contacts the lipid bilayer surrounding the channel., (Copyright ® 2021 National Research University Higher School of Economics.)

Widow spiders are among the few spider species worldwide that can cause serious envenoming in humans. The clinical syndrome resulting from Latrodectus spp. envenoming is called latrodectism and characterized by pain (local or regional) associated with diaphoresis and nonspecific systemic effects. The syndrome is caused by α-latrotoxin, a ~130 kDa neurotoxin that induces massive neurotransmitter release. Due to this function, α-latrotoxin has played a fundamental role as a tool in the study of neuroexocytosis. Nevertheless, some questions concerning its mode of action remain unresolved today. The diagnosis of latrodectism is purely clinical, combined with the patient's history of spider bite, as no analytical assays exist to detect widow spider venom. By utilizing antibody phage display technology, we here report the discovery of the first recombinant human monoclonal immunoglobulin G antibody (TPL0020_02_G9) that binds α-latrotoxin from the Mediterranean black widow spider ( Latrodectus tredecimguttatus ) and show neutralization efficacy ex vivo . Such antibody can be used as an affinity reagent for research and diagnostic purposes, providing researchers with a novel tool for more sophisticated experimentation and analysis. Moreover, it may also find therapeutic application in future., (Copyright © 2020 Føns, Ledsgaard, Nikolaev, Vassilevski, Sørensen, Chevalier, Fiebig and Laustsen.)

Voltage-gated potassium channels (K V s) perform vital physiological functions and are targets in different disorders ranging from ataxia and arrhythmia to autoimmune diseases. An important issue is the search for and production of selective ligands of these channels. Peptide toxins found in scorpion venom named KTx excel in both potency and selectivity with respect to some potassium channel isoforms, which may present only minute differences in their structure. Despite several decades of research the molecular determinants of KTx selectivity are still poorly understood. Here we analyze MeKTx13-3 (Kalium ID: α-KTx 3.19) from the lesser Asian scorpion Mesobuthus eupeus , a high-affinity K V 1.1 blocker (IC 50 ~2 nM); it also affects K V 1.2 (IC 50 ~100 nM), 1.3 (~10 nM) and 1.6 (~60 nM). By constructing computer models of its complex with K V 1.1-1.3 channels we identify specific contacts between the toxin and the three isoforms. We then perform mutagenesis to disturb the identified contacts with K V 1.1 and 1.2 and produce recombinant MeKTx13-3_AAAR, which differs by four amino acid residues from the parent toxin. As predicted by the modeling, this derivative shows decreased activity on K V 1.1 (IC 50 ~550 nM) and 1.2 (~200 nM). It also has diminished activity on K V 1.6 (~1500 nM) but preserves K V 1.3 affinity as measured using the voltage-clamp technique on mammalian channels expressed in Xenopus oocytes. In effect, we convert a selective K V 1.1 ligand into a new specific K V 1.3 ligand. MeKTx13-3 and its derivatives are attractive tools to study the structure-function relationship in potassium channel blockers., (Copyright © 2020 Gigolaev, Kuzmenkov, Peigneur, Tabakmakher, Pinheiro-Junior, Chugunov, Efremov, Tytgat and Vassilevski.)

Tk-hefu is an artificial peptide designed based on the α-hairpinin scaffold, which selectively blocks voltage-gated potassium channels K v 1.3. Here we present its spatial structure resolved by NMR spectroscopy and analyze its interaction with channels using computer modeling. We apply protein surface topography to suggest mutations and increase Tk-hefu affinity to the K v 1.3 channel isoform. We redesign the functional surface of Tk-hefu to better match the respective surface of the channel pore vestibule. The resulting peptide Tk-hefu-2 retains K v 1.3 selectivity and displays ∼15 times greater activity compared with Tk-hefu. We verify the mode of Tk-hefu-2 binding to the channel outer vestibule experimentally by site-directed mutagenesis. We argue that scaffold engineering aided by protein surface topography represents a reliable tool for design and optimization of specific ion channel ligands., (© 2019 Berkut et al.)

Neurotoxins are among the main components of scorpion and snake venoms. Scorpion neurotoxins affect voltage-gated ion channels, while most snake neurotoxins target ligand-gated ion channels, mainly nicotinic acetylcholine receptors (nAChRs). We report that scorpion venoms inhibit α-bungarotoxin binding to both muscle-type nAChR from Torpedo californica and neuronal human α7 nAChR. Toxins inhibiting nAChRs were identified as OSK-1 (α-KTx family) from Orthochirus scrobiculosus and HelaTx1 (κ-KTx family) from Heterometrus laoticus, both being blockers of voltage-gated potassium channels. With an IC 50 of 1.6 μm, OSK1 inhibits acetylcholine-induced current through mouse muscle-type nAChR heterologously expressed in Xenopus oocytes. Other well-characterized scorpion toxins from these families also bind to Torpedo nAChR with micromolar affinities. Our results indicate that scorpion neurotoxins present target promiscuity., (© 2019 Federation of European Biochemical Societies.)

Voltage-gated sodium (Na V ) channels are essential for the normal functioning of cardiovascular, muscular, and nervous systems. These channels have modular organization; the central pore domain allows current flow and provides ion selectivity, whereas four peripherally located voltage-sensing domains (VSDs-I/IV) are needed for voltage-dependent gating. Mutations in the S4 voltage-sensing segments of VSDs in the skeletal muscle channel Na V 1.4 trigger leak (gating pore) currents and cause hypokalemic and normokalemic periodic paralyses. Previously, we have shown that the gating modifier toxin Hm-3 from the crab spider Heriaeus melloteei binds to the S3-S4 extracellular loop in VSD-I of Na V 1.4 channel and inhibits gating pore currents through the channel with mutations in VSD-I. Here, we report that Hm-3 also inhibits gating pore currents through the same channel with the R675G mutation in VSD-II. To investigate the molecular basis of Hm-3 interaction with VSD-II, we produced the corresponding 554-696 fragment of Na V 1.4 in a continuous exchange cell-free expression system based on the Escherichia coli S30 extract. We then performed a combined nuclear magnetic resonance (NMR) and electron paramagnetic resonance spectroscopy study of isolated VSD-II in zwitterionic dodecylphosphocholine/lauryldimethylamine-N-oxide or dodecylphosphocholine micelles. To speed up the assignment of backbone resonances, five selectively 13 C, 15 N-labeled VSD-II samples were produced in accordance with specially calculated combinatorial scheme. This labeling approach provides assignment for ∼50% of the backbone. Obtained NMR and electron paramagnetic resonance data revealed correct secondary structure, quasi-native VSD-II fold, and enhanced ps-ns timescale dynamics in the micelle-solubilized domain. We modeled the structure of the VSD-II/Hm-3 complex by protein-protein docking involving binding surfaces mapped by NMR. Hm-3 binds to VSDs I and II using different modes. In VSD-II, the protruding ß-hairpin of Hm-3 interacts with the S1-S2 extracellular loop, and the complex is stabilized by ionic interactions between the positively charged toxin residue K24 and the negatively charged channel residues E604 or D607. We suggest that Hm-3 binding to these charged groups inhibits voltage sensor transition to the activated state and blocks the depolarization-activated gating pore currents. Our results indicate that spider toxins represent a useful hit for periodic paralyses therapy development and may have multiple structurally different binding sites within one Na V molecule.

Potassium channels are the most diverse group of ion channels in humans. They take vital parts in numerous physiological processes and their malfunction gives rise to a range of pathologies. In addition to small molecules, there is a wide selection of several hundred polypeptide ligands binding to potassium channels, the majority of which have been isolated from animal venoms. Until recently, only scorpion toxins received focused attention being systematically assembled in the manually curated Kalium database, but there is a diversity of well-characterized potassium channel ligands originating from other sources. To address this issue, here we present the updated and improved Kalium 2.0 that covers virtually all known polypeptide ligands of potassium channels and reviews all available pharmacological data. In addition to an expansion, we have introduced several new features to the database including posttranslational modification annotation, indication of ligand mode of action, BLAST search, and possibility of data export.

An effective bacterial system for the production of β-toxin Ts1, the main component of the Brazilian scorpion Tityus serrulatus venom, was developed. Recombinant toxin and its 15 N-labeled analogue were obtained via direct expression of synthetic gene in Escherichia coli with subsequent folding from the inclusion bodies. According to NMR spectroscopy data, the recombinant toxin is structured in an aqueous solution and contains a significant fraction of β-structure. The formation of a stable disulfide-bond isomer of Ts1, having a disordered structure, has also been observed during folding. Recombinant Ts1 blocks Na + current through Na V 1.5 channels without affecting the processes of activation and inactivation. At the same time, the effect upon Na V 1.4 channels is associated with a shift of the activation curve towards more negative membrane potentials.

Scorpion venom is an unmatched source of selective high-affinity ligands of potassium channels. There is a high demand for such compounds to identify and manipulate the activity of particular channel isoforms. The objective of this study was to obtain and characterize a specific ligand of voltage-gated potassium channel K V 1.2. As a result, we report the remarkable selectivity of the peptide MeKTx11-1 (α-KTx 1.16) from Mesobuthus eupeus scorpion venom to this channel isoform. MeKTx11-1 is a high-affinity blocker of K V 1.2 (IC 50 ∼0.2 nM), while its activity against K V 1.1, K V 1.3, and K V 1.6 is 10 000, 330 and 45 000 fold lower, respectively, as measured using the voltage-clamp technique on mammalian channels expressed in Xenopus oocytes. Two substitutions, G9V and P37S, convert MeKTx11-1 to its natural analog MeKTx11-3 (α-KTx 1.17) having 15 times lower activity and reduced selectivity to K V 1.2. We produced MeKTx11-1 and MeKTx11-3 as well as their mutants MeKTx11-1(G9V) and MeKTx11-1(P37S) recombinantly and demonstrated that point mutations provide an intermediate effect on selectivity. Key structural elements that explain MeKTx11-1 specificity were identified by molecular modeling of the toxin-channel complexes. Confirming our molecular modeling predictions, site-directed transfer of these elements from the pore region of K V 1.2 to K V 1.3 resulted in the enhanced sensitivity of mutant K V 1.3 channels to MeKTx11-1. We conclude that MeKTx11-1 may be used as a selective tool in neurobiology., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Sodium channel alpha-toxins from scorpion venom (α-NaTx) inhibit the inactivation of voltage-gated sodium channels. We used solution NMR to investigate the structure of BeM9 toxin from Mesobuthus eupeus scorpion, a prototype α-NaTx classified as an "α-like" toxin due to its wide spectrum of activity on insect and mammalian channels. We identified a new motif that we named "arginine hand," whereby arginine side chain forms several hydrogen bonds with main chain atoms. The arginine hand was found in the "specificity module," a part of the molecule that dictates toxin selectivity; and just single arginine-to-lysine point mutation drastically changed BeM9 selectivity profile., (© 2018 Wiley Periodicals, Inc.)

AMPA receptors mediate fast excitatory neurotransmission and are critical for CNS development and function. Calcium-permeable subsets of AMPA receptors are strongly implicated in acute and chronic neurological disorders. However, despite the clinical importance, the therapeutic landscape for specifically targeting them, and not the calcium-impermeable AMPA receptors, remains largely undeveloped. To address this problem, we used cryo-electron microscopy and electrophysiology to investigate the mechanisms by which small-molecule blockers selectively inhibit ion channel conductance in calcium-permeable AMPA receptors. We determined the structures of calcium-permeable GluA2 AMPA receptor complexes with the auxiliary subunit stargazin bound to channel blockers, including the orb weaver spider toxin AgTx-636, the spider toxin analog NASPM, and the adamantane derivative IEM-1460. Our structures provide insights into the architecture of the blocker binding site and the mechanism of trapping, which are critical for development of small molecules that specifically target calcium-permeable AMPA receptors., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Animal toxins are traditional and indispensible molecular tools that find application in different fields of biochemistry, neurobiology and pharmacology. These compounds possess several outstanding properties such as high affinity and selectivity with respect to particular molecular targets, most importantly ion channels and neuroreceptors, and stability. In addition to using toxins per se, a wide variety of labelled modifications have been obtained including radioactive and fluorescent derivatives. Here, we discuss the major types of labelled toxins, methods of their production and principal possibilities of application ranging from receptor localization and visualization to development of screening systems and diagnostic tools, and drug discovery., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Gating pore currents through the voltage-sensing domains (VSDs) of the skeletal muscle voltage-gated sodium channel Na V 1.4 underlie hypokalemic periodic paralysis (HypoPP) type 2. Gating modifier toxins target ion channels by modifying the function of the VSDs. We tested the hypothesis that these toxins could function as blockers of the pathogenic gating pore currents. We report that a crab spider toxin Hm-3 from Heriaeus melloteei can inhibit gating pore currents due to mutations affecting the second arginine residue in the S4 helix of VSD-I that we have found in patients with HypoPP and describe here. NMR studies show that Hm-3 partitions into micelles through a hydrophobic cluster formed by aromatic residues and reveal complex formation with VSD-I through electrostatic and hydrophobic interactions with the S3b helix and the S3-S4 extracellular loop. Our data identify VSD-I as a specific binding site for neurotoxins on sodium channels. Gating modifier toxins may constitute useful hits for the treatment of HypoPP., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

We report isolation, sequencing, and electrophysiological characterization of OSK3 (α-KTx 8.8 in Kalium and Uniprot databases), a potassium channel blocker from the scorpion Orthochirus scrobiculosus venom. Using the voltage clamp technique, OSK3 was tested on a wide panel of 11 voltage-gated potassium channels expressed in Xenopus oocytes, and was found to potently inhibit Kv1.2 and Kv1.3 with IC 50 values of ~331nM and ~503nM, respectively. OdK1 produced by the scorpion Odontobuthus doriae differs by just two C-terminal residues from OSK3, but shows marked preference to Kv1.2. Based on the charybdotoxin-potassium channel complex crystal structure, a model was built to explain the role of the variable residues in OdK1 and OSK3 selectivity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (© 2016 The Protein Society.)

Traditionally, arachnid venoms are known to contain two particularly important groups of peptide toxins. One is disulfide-rich neurotoxins with a predominance of β-structure that specifically target protein receptors in neurons or muscle cells. The other is linear cationic cytotoxins that form amphiphilic α-helices and exhibit rather non-specific membrane-damaging activity. In the present paper, we describe the first 3D structure of a modular arachnid toxin, purotoxin-2 (PT2) from the wolf spider Alopecosa marikovskyi (Lycosidae), studied by NMR spectroscopy. PT2 is composed of an N-terminal inhibitor cystine knot (ICK, or knottin) β-structural domain and a C-terminal linear cationic domain. In aqueous solution, the C-terminal fragment is hyper-flexible, whereas the knottin domain is very rigid. In membrane-mimicking environment, the C-terminal domain assumes a stable amphipathic α-helix. This helix effectively tethers the toxin to membranes and serves as a membrane-access and membrane-anchoring device. Sequence analysis reveals that the knottin + α-helix architecture is quite widespread among arachnid toxins, and PT2 is therefore the founding member of a large family of polypeptides with similar structure motifs. Toxins from this family target different membrane receptors such as P2X in the case of PT2 and calcium channels, but their mechanism of action through membrane access may be strikingly similar., (© 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Ion channels play a central role in a host of physiological and pathological processes and are the second largest target for existing drugs. There is an increasing need for reliable tools to detect and visualize particular ion channels, but existing solutions suffer from a number of limitations such as high price, poor specificity, and complicated protocols. As an alternative, we produced recombinant chimeric constructs (FP-Tx) consisting of fluorescent proteins (FP) fused with potassium channel toxins from scorpion venom (Tx). In particular, we used two FP, eGFP and TagRFP, and two Tx, OSK1 and AgTx2, to create eGFP-OSK1 and RFP-AgTx2. We show that these chimeras largely retain the high affinity of natural toxins and display selectivity to particular ion channel subtypes. FP-Tx are displaced by other potassium channel blockers and can be used as an imaging tool in ion channel ligand screening setups. We believe FP-Tx chimeras represent a new efficient molecular tool for neurobiology.

In the present study, we show that venom of the ant spider Lachesana tarabaevi is unique in terms of molecular composition and toxicity. Whereas venom of most spiders studied is rich in disulfide-containing neurotoxic peptides, L. tarabaevi relies on the production of linear (no disulfide bridges) cytolytic polypeptides. We performed full-scale peptidomic examination of L. tarabaevi venom supported by cDNA library analysis. As a result, we identified several dozen components, and a majority (∼80% of total venom protein) exhibited membrane-active properties. In total, 33 membrane-interacting polypeptides (length of 18-79 amino acid residues) comprise five major groups: repetitive polypeptide elements (Rpe), latarcins (Ltc), met-lysines (MLys), cyto-insectotoxins (CIT) and latartoxins (LtTx). Rpe are short (18 residues) amphiphilic molecules that are encoded by the same genes as antimicrobial peptides Ltc 4a and 4b. Isolation of Rpe confirms the validity of the iPQM (inverted processing quadruplet motif) proposed to mark the cleavage sites in spider toxin precursors that are processed into several mature chains. MLys (51 residues) present 'idealized' amphiphilicity when modelled in a helical wheel projection with sharply demarcated sectors of hydrophobic, cationic and anionic residues. Four families of CIT (61-79 residues) are the primary weapon of the spider, accounting for its venom toxicity. Toxins from the CIT 1 and 2 families have a modular structure consisting of two shorter Ltc-like peptides. We demonstrate that in CIT 1a, these two parts act in synergy when they are covalently linked. This finding supports the assumption that CIT have evolved through the joining of two shorter membrane-active peptides into one larger molecule., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

Kalium (http://kaliumdb.org/) is a manually curated database that accumulates data on potassium channel toxins purified from scorpion venom (KTx). This database is an open-access resource, and provides easy access to pages of other databases of interest, such as UniProt, PDB, NCBI Taxonomy Browser, and PubMed. General achievements of Kalium are a strict and easy regulation of KTx classification based on the unified nomenclature supported by researchers in the field, removal of peptides with partial sequence and entries supported by transcriptomic information only, classification of β-family toxins, and addition of a novel λ-family. Molecules presented in the database can be processed by the Clustal Omega server using a one-click option. Molecular masses of mature peptides are calculated and available activity data are compiled for all KTx. We believe that Kalium is not only of high interest to professional toxinologists, but also of general utility to the scientific community.Database URL:http://kaliumdb.org/., (© The Author(s) 2016. Published by Oxford University Press.)

Arthropod venoms feature the presence of cytolytic peptides believed to act synergetically with neurotoxins to paralyze prey or deter aggressors. Many of them are linear, i.e., lack disulfide bonds. When isolated from the venom, or obtained by other means, these peptides exhibit common properties. They are cationic; being mostly disordered in aqueous solution, assume amphiphilic α-helical structure in contact with lipid membranes; and exhibit general cytotoxicity, including antifungal, antimicrobial, hemolytic, and anticancer activities. To suit the pharmacological needs, the activity spectrum of these peptides should be modified by rational engineering. As an example, we provide a detailed review on latarcins (Ltc), linear cytolytic peptides from Lachesana tarabaevi spider venom. Diverse experimental and computational techniques were used to investigate the spatial structure of Ltc in membrane-mimicking environments and their effects on model lipid bilayers. The antibacterial activity of Ltc was studied against a panel of Gram-negative and Gram-positive bacteria. In addition, the action of Ltc on erythrocytes and cancer cells was investigated in detail with confocal laser scanning microscopy. In the present review, we give a critical account of the progress in the research of Ltc. We explore the relationship between Ltc structure and their biological activity and derive molecular characteristics, which can be used for optimization of other linear peptides. Current applications of Ltc and prospective use of similar membrane-active peptides are outlined.

Potassium (K+) channels are a widespread superfamily of integral membrane proteins that mediate selective transport of K+ ions through the cell membrane. They have been found in all living organisms from bacteria to higher multicellular animals, including humans. Not surprisingly, K+ channels bind ligands of different nature, such as metal ions, low molecular mass compounds, venom-derived peptides, and antibodies. Functionally these substances can be K+ channel pore blockers or modulators. Representatives of the first group occlude the channel pore, like a cork in a bottle, while the second group of ligands alters the operation of channels without physically blocking the ion current. A rich source of K+ channel ligands is venom of different animals: snakes, sea anemones, cone snails, bees, spiders, and scorpions. More than a half of the known K+ channel ligands of polypeptide nature are scorpion toxins (KTx), all of which are pore blockers. These compounds have become an indispensable molecular tool for the study of K+ channel structure and function. A recent special interest is the possibility of toxin application as drugs to treat diseases involving K+ channels or related to their dysfunction (channelopathies).

The lesser Asian scorpion Mesobuthus eupeus (Buthidae) is one of the most widely spread and dispersed species of the Mesobuthus genus, and its venom is actively studied. Nevertheless, a considerable amount of active compounds is still under-investigated due to the high complexity of this venom. Here, we report a comprehensive analysis of putative potassium channel toxins (KTxs) from the cDNA library of M. eupeus venom glands, and we compare the deduced KTx structures with peptides purified from the venom. For the transcriptome analysis, we used conventional tools as well as a search for structural motifs characteristic of scorpion venom components in the form of regular expressions. We found 59 candidate KTxs distributed in 30 subfamilies and presenting the cysteine-stabilized α/β and inhibitor cystine knot types of fold. M. eupeus venom was then separated to individual components by multistage chromatography. A facile fluorescent system based on the expression of the KcsA-Kv1.1 hybrid channels in Escherichia coli and utilization of a labeled scorpion toxin was elaborated and applied to follow Kv1.1 pore binding activity during venom separation. As a result, eight high affinity Kv1.1 channel blockers were identified, including five novel peptides, which extend the panel of potential pharmacologically important Kv1 ligands. Activity of the new peptides against rat Kv1.1 channel was confirmed (IC50 in the range of 1-780 nm) by the two-electrode voltage clamp technique using a standard Xenopus oocyte system. Our integrated approach is of general utility and efficiency to mine natural venoms for KTxs., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

We present a structural and functional study of a sodium channel activation inhibitor from crab spider venom. Hm-3 is an insecticidal peptide toxin consisting of 35 amino acid residues from the spider Heriaeus melloteei (Thomisidae). We produced Hm-3 recombinantly in Escherichia coli and determined its structure by NMR spectroscopy. Typical for spider toxins, Hm-3 was found to adopt the so-called "inhibitor cystine knot" or "knottin" fold stabilized by three disulfide bonds. Its molecule is amphiphilic with a hydrophobic ridge on the surface enriched in aromatic residues and surrounded by positive charges. Correspondingly, Hm-3 binds to both neutral and negatively charged lipid vesicles. Electrophysiological studies showed that at a concentration of 1 μm Hm-3 effectively inhibited a number of mammalian and insect sodium channels. Importantly, Hm-3 shifted the dependence of channel activation to more positive voltages. Moreover, the inhibition was voltage-dependent, and strong depolarizing prepulses attenuated Hm-3 activity. The toxin is therefore concluded to represent the first sodium channel gating modifier from an araneomorph spider and features a "membrane access" mechanism of action. Its amino acid sequence and position of the hydrophobic cluster are notably different from other known gating modifiers from spider venom, all of which are described from mygalomorph species. We hypothesize parallel evolution of inhibitor cystine knot toxins from Araneomorphae and Mygalomorphae suborders., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Venoms from three poneromorph ant species (Paraponera clavata, Ectatomma quadridens and Ectatomma tuberculatum) were investigated for the growth inhibition of Gram-positive and Gram-negative bacteria. It was shown that the venom of E. quadridens and its peptide fraction in particular possess marked antibacterial action. Three linear antimicrobial peptides sharing low similarity to the well-known ponericin peptides were isolated from this ant venom by means of size-exclusion and reversed-phase chromatography. The peptides showed antimicrobial activity at low micromolar concentrations. Their primary structure was established by direct Edman sequencing in combination with mass spectrometry. The most active peptide designated ponericin-Q42 was chemically synthesized. Its secondary structure was investigated in aqueous and membrane-mimicking environment, and the peptide was shown to be partially helical already in water, which is unusual for short linear peptides. Analysis of its activity on different bacterial strains, human erythrocytes and chronic myelogenous leukemia K562 cells revealed that the peptide shows broad spectrum cytolytic activity at micromolar and submicromolar concentrations. Ponericin-Q42 also possesses weak toxic activity on flesh fly larvae with LD50 of ∼105 μg/g., (Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.)

The multilayered plant immune system relies on rapid recognition of pathogen-associated molecular patterns followed by activation of defense-related genes, resulting in the reinforcement of plant cell walls and the production of antimicrobial compounds. To suppress plant defense, fungi secrete effectors, including a recently discovered Zn-metalloproteinase from Fusarium verticillioides, named fungalysin Fv-cmp. This proteinase cleaves class IV chitinases, which are plant defense proteins that bind and degrade chitin of fungal cell walls. In this study, we investigated plant responses to such pathogen invasion, and discovered novel inhibitors of fungalysin. We produced several recombinant hevein-like antimicrobial peptides named wheat antimicrobial peptides (WAMPs) containing different amino acids (Ala, Lys, Glu, and Asn) at the nonconserved position 34. An additional Ser at the site of fungalysin proteolysis makes the peptides resistant to the protease. Moreover, an equal molar concentration of WAMP-1b or WAMP-2 to chitinase was sufficient to block the fungalysin activity, keeping the chitinase intact. Thus, WAMPs represent novel protease inhibitors that are active against fungal metalloproteases. According to in vitro antifungal assays WAMPs directly inhibited hyphal elongation, suggesting that fungalysin plays an important role in fungal development. A novel molecular mechanism of dynamic interplay between host defense molecules and fungal virulence factors is suggested., (© 2014 FEBS.)

Yellow sac spiders (Cheiracanthium punctorium, family Miturgidae) are unique in terms of venom composition, because, as we show here, two-domain toxins have replaced the usual one-domain peptides as the major constituents. We report the structure of the two-domain Che. punctorium toxins (CpTx), along with the corresponding cDNA and genomic DNA sequences. At least three groups of insecticidal CpTx were identified, each consisting of several members. Unlike many cone snail and snake toxins, accelerated evolution is not typical of cptx genes, which instead appear to be under the pressure of purifying selection. Both CpTx modules present the inhibitor cystine knot (ICK), or knottin signature; however, the sequence similarity between the domains is low. Conversely, notable similarity was found between separate domains of CpTx and one-domain toxins from spiders of the Lycosidae family. The observed chimerism is a landmark of exon shuffling events, but in contrast to many families of multidomain protein genes no introns were found in the cptx genes. Considering the possible scenarios, we suggest that an early transcription-mediated fusion event between two related one-domain toxin genes led to the emergence of a primordial cptx-like sequence. We conclude that evolution of toxin variability in spiders appears to be quite different from other venomous animals., (© 2014 The Royal Entomological Society.)

In this study, we present the spatial structure of the wheat antimicrobial peptide (AMP) Tk-AMP-X2 studied using NMR spectroscopy. This peptide was found to adopt a disulfide-stabilized α-helical hairpin fold and therefore belongs to the α-hairpinin family of plant defense peptides. Based on Tk-AMP-X2 structural similarity to cone snail and scorpion potassium channel blockers, a mutant molecule, Tk-hefu, was engineered by incorporating the functionally important residues from κ-hefutoxin 1 onto the Tk-AMP-X2 scaffold. The designed peptide contained the so-called essential dyad of amino acid residues significant for channel-blocking activity. Electrophysiological studies showed that although the parent peptide Tk-AMP-X2 did not present any activity against potassium channels, Tk-hefu blocked Kv1.3 channels with similar potency (IC50 ∼ 35 μm) to κ-hefutoxin 1 (IC50 ∼ 40 μm). We conclude that α-hairpinins are attractive in their simplicity as structural templates, which may be used for functional engineering and drug design., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Spiderines are comparatively long polypeptide toxins (∼110 residues) from lynx spiders (genus Oxyopes). They are built of an N-terminal linear cationic domain (∼40 residues) and a C-terminal knottin domain (∼60 residues). The linear domain empowers spiderines with strong cytolytic activity. In the present work we report 16 novel spiderine sequences from Oxyopes takobius and Oxyopes lineatus classified into two subfamilies. Strikingly, negative selection acts on both linear and knottin domains. Genes encoding Oxyopes two-domain toxins were sequenced and found to be intronless. We further discuss a possible scenario of lynx spider modular toxin evolution., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Plant defense against disease is a complex multistage system involving initial recognition of the invading pathogen, signal transduction and activation of specialized genes. An important role in pathogen deterrence belongs to so-called plant defense peptides, small polypeptide molecules that present antimicrobial properties. Using multidimensional liquid chromatography, we isolated a novel antifungal peptide named Sm-AMP-X (33 residues) from the common chickweed (Stellaria media) seeds. The peptide sequence shows no homology to any previously described proteins. The peculiar cysteine arrangement (C(1)X3C(2)XnC(3)X3C(4)), however, allocates Sm-AMP-X to the recently acknowledged α-hairpinin family of plant defense peptides that share the helix-loop-helix fold stabilized by two disulfide bridges C(1)-C(4) and C(2)-C(3). Sm-AMP-X exhibits high broad-spectrum activity against fungal phytopathogens. We further showed that the N- and C-terminal "tail" regions of the peptide are important for both its structure and activity. The truncated variants Sm-AMP-X1 with both disulfide bonds preserved and Sm-AMP-X2 with only the internal S-S-bond left were progressively less active against fungi and presented largely disordered structure as opposed to the predominantly helical conformation of the full-length antifungal peptide. cDNA and gene cloning revealed that Sm-AMP-X is processed from a unique multimodular precursor protein that contains as many as 12 tandem repeats of α-hairpinin-like peptides. Structure of the sm-amp-x gene and two related pseudogenes sm-amp-x-ψ1 and sm-amp-x-ψ2 allows tracing the evolutionary scenario that led to generation of such a sophisticated precursor protein. Sm-AMP-X is a new promising candidate for engineering disease resistance in plants.

In addition to the conventional neurotoxins and cytotoxins, venom of the lynx spider Oxyopes takobius was found to contain two-domain modular toxins named spiderines: OtTx1a, 1b, 2a and 2b. These toxins show both insecticidal activity (a median lethal dose against flesh fly larvae of 75 μg·g(-1)) and potent antimicrobial effects (minimal inhibitory concentrations in the range 0.1-10 μm). Full sequences of the purified spiderines were established by a combination of Edman degradation, mass spectrometry and cDNA cloning. They are relatively large molecules (~ 110 residues, 12.0-12.5 kDa) and consist of two distinct modules separated by a short linker. The N-terminal part (~ 40 residues) contains no cysteine residues, is highly cationic, forms amphipathic α-helical structures in a membrane-mimicking environment, and shows potent cytolytic effects on cells of various origins. The C-terminal part (~ 60 residues) is disulfide-rich (five S-S bonds), and contains the inhibitor cystine knot (ICK/knottin) signature. The N-terminal part of spiderines is very similar to linear cytotoxic peptides found in various organisms, whereas the C-terminal part corresponds to the usual spider neurotoxins. We synthesized the modules of OtTx1a and compared their activity to that of full-length mature toxin produced recombinantly, highlighting the importance of the N-terminal part, which retained full-length toxin activity in both insecticidal and antimicrobial assays. The unique structure of spiderines completes the range of two-domain spider toxins., (© 2013 FEBS.)

A novel family of antifungal peptides was discovered in the wheat Triticum kiharae Dorof. et Migusch. Two members of the family, designated Tk-AMP-X1 and Tk-AMP-X2, were completely sequenced and shown to belong to the α-hairpinin structural family of plant peptides with a characteristic C1XXXC2-X(n)-C3XXXC4 motif. The peptides inhibit the spore germination of several fungal pathogens in vitro. cDNA and gene cloning disclosed unique structure of genes encoding Tk-AMP-X peptides. They code for precursor proteins of unusual multimodular structure, consisting of a signal peptide, several α-hairpinin (4-Cys) peptide domains with a characteristic cysteine pattern separated by linkers and a C-terminal prodomain. Three types of precursor proteins, with five, six or seven 4-Cys peptide modules, were found in wheat. Among the predicted family members, several peptides previously isolated from T. kiharae seeds were identified. Genes encoding Tk-AMP-X precursors have no introns in the protein-coding regions and are upregulated by fungal pathogens and abiotic stress, providing conclusive evidence for their role in stress response. A combined PCR-based and bioinformatics approach was used to search for related genes in the plant kingdom. Homologous genes differing in the number of peptide modules were discovered in phylogenetically-related Triticum and Aegilops species, including polyploid wheat genome donors. Association of the Tk-AMP-X genes with A, B/G or D genomes of hexaploid wheat was demonstrated. Furthermore, Tk-AMP-X-related sequences were shown to be widespread in the Poaceae family among economically important crops, such as barley, rice and maize., (© 2013 FEBS.)

To gain success in the evolutionary "arms race," venomous animals such as scorpions produce diverse neurotoxins selected to hit targets in the nervous system of prey. Scorpion α-toxins affect insect and/or mammalian voltage-gated sodium channels (Na(v)s) and thereby modify the excitability of muscle and nerve cells. Although more than 100 α-toxins are known and a number of them have been studied into detail, the molecular mechanism of their interaction with Na(v)s is still poorly understood. Here, we employ extensive molecular dynamics simulations and spatial mapping of hydrophobic/hydrophilic properties distributed over the molecular surface of α-toxins. It is revealed that despite the small size and relatively rigid structure, these toxins possess modular organization from structural, functional, and evolutionary perspectives. The more conserved and rigid "core module" is supplemented with the "specificity module" (SM) that is comparatively flexible and variable and determines the taxon (mammal versus insect) specificity of α-toxin activity. We further show that SMs in mammal toxins are more flexible and hydrophilic than in insect toxins. Concomitant sequence-based analysis of the extracellular loops of Na(v)s suggests that α-toxins recognize the channels using both modules. We propose that the core module binds to the voltage-sensing domain IV, whereas the more versatile SM interacts with the pore domain in repeat I of Na(v)s. These findings corroborate and expand the hypothesis on different functional epitopes of toxins that has been reported previously. In effect, we propose that the modular structure in toxins evolved to match the domain architecture of Na(v)s.

Human voltage-gated potassium channel Kv1.3 is an important pharmacological target for the treatment of autoimmune and metabolic diseases. Increasing clinical demands stipulate an active search for efficient and selective Kv1.3 blockers. Here we present a new, reliable, and easy-to-use analytical system designed to seek for and study Kv1.3 ligands that bind to the extracellular vestibule of the K(+)-conducting pore. It is based on Escherichia coli spheroplasts with the hybrid protein KcsA-Kv1.3 embedded into the membrane, fluorescently labeled Kv1.3 blocker agitoxin-2, and confocal laser scanning microscopy as a detection method. This system is a powerful alternative to radioligand and patch-clamp techniques. It enables one to search for Kv1.3 ligands both among individual compounds and in complex mixtures, as well as to characterize their affinity to Kv1.3 channel using the "mix and read" mode. To demonstrate the potential of the system, we performed characterization of several known Kv1.3 ligands, tested nine spider venoms for the presence of Kv1.3 ligands, and conducted guided purification of a channel blocker from scorpion venom.

Antichlamydial activity of cyto-insectotoxin 1a (CIT 1a), representative of a unique class of antimicrobial peptides from the venom of the Central Asian spider Lachesana tarabaevi, was studied. A plasmid vector expressing the cit 1a gene controlled by a human cytomegalovirus tetracycline-dependent promoter was constructed. Impressive inhibition of Chlamydia trachomatis infection in HEK 293 cells transfected by the cit 1a-harboring vector was achieved. With the use of various schemes of cell infection and gene expression induction, it was shown for the first time that an antimicrobial peptide exerts its potent antichlamydial action at an early stage of the pathogen life cycle.

Venom of Lachesana tarabaevi (Zodariidae, "ant spiders") exhibits high insect toxicity and serves a rich source of potential insecticides. Five new peptide toxins active against insects were isolated from the venom by means of liquid chromatography and named latartoxins (LtTx). Complete amino acid sequences of LtTx (60-71 residues) were established by a combination of Edman degradation, mass spectrometry and selective proteolysis. Three toxins have eight cysteine residues that form four intramolecular disulfide bridges, and two other molecules contain an additional cystine; three LtTx are C-terminally amidated. Latartoxins can be allocated to two groups with members similar to CSTX and LSTX toxins from Cupiennius salei (Ctenidae) and Lycosa singoriensis (Lycosidae). The interesting feature of the new toxins is their modular organization: they contain an N-terminal cysteine-rich (knottin or ICK) region as in many neurotoxins from spider venoms and a C-terminal linear part alike some cytolytic peptides. The C-terminal fragment of one of the most abundant toxins LtTx-1a was synthesized and shown to possess membrane-binding activity. It was found to assume amphipathic α-helical conformation in membrane-mimicking environment and exert antimicrobial activity at micromolar concentrations. The tails endow latartoxins with the ability to bind and damage membranes; LtTx show cytolytic activity in fly larvae neuromuscular preparations. We suggest a membrane-dependent mode of action for latartoxins with their C-terminal linear modules acting as anchoring devices., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Recently, the novel peptide named purotoxin-1 (PT1) has been identified in the venom of the spider Geolycosa sp. and shown to exert marked modulatory effects on P2X3 receptors in rat sensory neurons. Here we studied another polypeptide from the same spider venom, purotoxin-2 (PT2), and demonstrated that it also affected activity of mammalian P2X3 receptors. The murine and human P2X3 receptors were heterologously expressed in cells of the CHO line, and nucleotide-gated currents were stimulated by CTP and ATP, respectively. Both PT1 and PT2 negligibly affected P2X3-mediated currents elicited by brief pulses of the particular nucleotide. When subthreshold CTP or ATP was added to the bath to exert the high-affinity desensitization of P2X3 receptors, both spider toxins strongly enhanced the desensitizing action of the ambient nucleotides. At the concentration of 50nM, PT1 and PT2 elicited 3-4-fold decrease in the IC(50) dose of ambient CTP or ATP. In contrast, 100nM PT1 and PT2 negligibly affected nucleotide-gated currents mediated by mP2X2 receptors or mP2X2/mP2X3 heteromers. Altogether, our data point out that the PT1 and PT2 toxins specifically target the fast-desensitizing P2X3 receptor, thus representing a unique tool to manipulate its activity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Membrane-active peptides (MAPs) represent a broad variety of molecules, and biological functions of most are directly associated with their ability to interact with membranes. Taking into account the effect of MAPs on living cells they can be nominally divided into three major groups - fusion (FPs), antimicrobial/cytolytic (AMPs/CPs) and cell-penetrating (CPPs) peptides. Although spatial structure of different MAPs varies to a great extent, linear α-helical peptides represent the most studied class. These peptides possess relatively simple structural organization and share a set of similar molecular features, which make them very attractive to both experimental and computational studies. Here, we review different molecular modeling methods in prospective of their applications to study of α-helical MAPs. The most sophisticated of them, such as molecular dynamics simulations, give atomistic information about molecular interactions driving peptide binding to the water-lipid interface, cooperative mechanisms of membrane destabilization and thermodynamics of these processes. Significant progress has been achieved in this field during the last few years, resulting in a possibility to observe computationally MAPs action in realistic peptide-to-lipid ratios and over the microsecond timescale. Other relatively simple but powerful approaches allow assessment of important characteristics of MAPs such as α-helical propensity, amphiphilicity, total hydrophobicity, and spatial distribution of charge and hydrophobic/hydrophilic properties, etc. Altogether, computational methods provide efficient basis for rational design of MAPs with predefined properties and a spectrum of biological activities.