Two recombinant α-like scorpion toxins from Mesobuthus eupeus with differential affinity toward insect and mammalian Na channels

α-Scorpion toxins are modulators of voltage-gated Na⁺ channels (Na_vs), which bind to the receptor site 3 to inhibit the fast inactivation of the channels. MeuNaTxα-12 and MeuNaTxα-13 are two new α-scorpion toxin-like peptides identified by cDNA cloning from the scorpion Mesobuthus eupeus with unknown functions. Here, we report their recombinant production, oxidative refolding, structural and functional features. By in vitro renaturation from bacterial inclusion bodies and further purification through reverse phase high-performance liquid chromatography, we obtained high purity recombinant products with a native-like conformation identified by circular dichroism analysis. Two-electrode voltage clamp recordings on five cloned mammalian Na_v subtypes (rNa_v1.1, rNa_v1.2, rNa_v1.4, rNa_v1.5, and mNa_v1.6) and the insect counterpart DmNa_v1, all expressed in Xenopus laevis oocytes, showed that these two peptides inhibited rapid inactivation of the sensitive Na⁺ channels with significant preference for DmNa_v1. The half maximal effective concentrations (EC₅₀) of MeuNaTxα-12 and MeuNaTxα-13 for this channel are 19.95 ± 2.99 nM and 65.50 ± 7.28 nM, respectively, showing 45 and 38 folds higher affinities than for rNa_v1.1, the most sensitive mammalian channel among the five isoforms. Our functional data confirms that these two peptides belong to the α-like scorpion toxin group. A combined analysis of the site 3 sequences and the pharmacological data illuminates the importance of the loop L_D4:S5–S6 of the channel in interacting with the toxins whereas affinity variations between MeuNaTxα-12 and MeuNaTxα-13 highlight a key functional role of a cationic side chain at position 28 of MeuNaTxα-12. Successful expression together with structural and functional characterization of these two new α-like scorpion toxins lays basis for further studies of their structure–function relationship.     

Inhibition of melanization by a Nasonia defensin-like peptide: Implications for host immune suppression

The parasitic wasp Nasonia vitripennis suppresses host immune mechanisms that include melanization reactions. Melanization is an important immune response of hosts induced by wasp infection and thus its inhibition represents a successful strategy for parasitism. However, the molecular basis associated with such inhibition is largely unknown in N. vitripennis. Here, we report recombinant expression, structural and functional characterization of a Nasonia-derived defensin-like peptide (called nasonin-3) whose recombinant product exerts inhibitory effect on host melanization. The possible role of nasonin-3 in immune suppression is also discussed.     

Structure-activity relationships of the intramolecular disulfide bonds in coprisin,a defensin from the dung beetle

Defensins, which are small cationic molecules produced by organisms as part of their innate immune response, share a common structural scaffold that is stabilized by three disulfide bridges. Coprisin is a 43-amino acid defensin-like peptide from Copris tripartitus. Here, we report the intramolecular disulfide connectivity of cysteine-rich coprisin, and show that it is the same as in other insect defensins. The disulfide bond pairings of coprisin were determined by combining the enzymatic cleavage and mass analysis. We found that the loss of any single disulfide bond in coprisin eliminated all antibacterial, but not antifungal, activity. Circular dichroism (CD) analysis showed that two disulfide bonds, Cys20-Cys39 and Cys24-Cys41, stabilize coprisin’s α-helical region. Moreover, a BLAST search against UniProtKB database revealed that coprisin’s α-helical region is highly homologous to those of other insect defensins. [BMB Reports 2014; 47(11): 625-630]     

AdDLP, a bacterial defensin-like peptide, exhibits anti-Plasmodium activity

Antimicrobial defensins with the cysteine-stabilized α-helical and β-sheet (CSαβ) motif are widely distributed in three eukaryotic kingdoms. However, recent work suggests that bacteria could possess defensin-like peptides (DLPs). Here, we report recombinant expression, in vitro folding, structural and functional characterization of a DLP from the myxobacterium Anaeromyxobacter dehalogenans (AdDLP). Circular dichroism analysis indicates that recombinant AdDLP adopts a typical structural feature of eukaryotic defensins, which is also consistent with an ab initio structure model predicted using I-TASSER algorithm. We found that AdDLP is an antimalarial peptide that led to more than 50% growth inhibition on sexual stages of Plasmodium berghei at micromolar concentrations and killed 100% intraerythrocytic Plasmodium falciparum at 10 μM in a time-dependent manner. These results provide functional evidence for myxobacterial origin of eukaryotic defensins. High-level production of the pure anti-Plasmodium peptide without harming mammalian red blood cells in Escherichia coli makes AdDLP an interesting candidate for antimalarial drug design.     

Antibacterial activity and pore-forming properties of ceratotoxins: a mechanism of action based on the barrel stave model

Ceratotoxins are α-helical cationic peptides isolated from the medfly Ceratitis capitata. These amphipathic peptides were found to display strong antibacterial activity and weak hemolytic activity. When reconstituted into planar lipid bilayers, ceratotoxins developed highly asymmetric I/V curves under voltage ramps and formed, in single-channel experiments, well-defined voltage-dependent ion channels according to the barrel stave model. The antibacterial activity and pore-forming properties of these molecules were well correlated. Similar experiments performed with synthesized truncated fragments showed that the C-terminal domain of ceratotoxins is strongly implicated in the formation of helical bundles in the membrane whereas the largely cationic N-terminal region is likely to anchor ceratotoxins on the lipid surface.     

The folding pathway of a single domain in a multidomain protein is not affected by its neighbouring domain

Domains are the structural, functional, and evolutionary components of proteins. Most folding studies to date have concentrated on the folding of single domains, but more than 70% of human proteins contain more than one domain, and interdomain interactions can affect both the stability and the folding kinetics. Whether the folding pathway is altered by interdomain interactions is not yet known. Here we investigated the effect of a folded neighbouring domain on the folding pathway of spectrin R16 (the 16th α-helical repeat from chicken brain α-spectrin) by using the two-domain construct R1516. The R16 folds faster and unfolds more slowly in the presence of its folded neighbour R15 (the 15th α-helical repeat from chicken brain α-spectrin). An extensive Φ-value analysis of the R16 domain in R1516 was completed to compare the transition state of the R16 domain alone with that of the R16 domain in a multidomain construct. The results indicate that the folding pathways are the same. This result validates the current approach of breaking up larger proteins into domains for the study of protein folding pathways.     

Crystal structure of Bacillus sp. GL1 xanthan lyase complexed with a substrate: insights into the enzyme reaction mechanism

Bacillus sp. GL1 xanthan lyase, a member of polysaccharide lyase family 8 (PL-8), acts exolytically on the side-chains of pentasaccharide-repeating polysaccharide xanthan and cleaves the glycosidic bond between glucuronic acid (GlcUA) and pyruvylated mannose (PyrMan) through a beta-elimination reaction. To clarify the enzyme reaction mechanism, i.e. its substrate recognition and catalytic reaction, we determined crystal structures of a mutant enzyme, N194A, in complexes with the product (PyrMan) and a substrate (pentasacharide) and in a ligand-free form at 1.8, 2.1, and 2.3A resolution. Based on the structures of the mutant in complexes with the product and substrate, we found that xanthan lyase recognized the PyrMan residue at subsite -1 and the GlcUA residue at +1 on the xanthan side-chain and underwent little interaction with the main chain of the polysaccharide. The structure of the mutant-substrate complex also showed that the hydroxyl group of Tyr255 was close to both the C-5 atom of the GlcUA residue and the oxygen atom of the glycosidic bond to be cleaved, suggesting that Tyr255 likely acts as a general base that extracts the proton from C-5 of the GlcUA residue and as a general acid that donates the proton to the glycosidic bond. A structural comparison of catalytic centers of PL-8 lyases indicated that the catalytic reaction mechanism is shared by all members of the family PL-8, while the substrate recognition mechanism differs.     

Haemolytic and cytotoxic action of latarcin Ltc2a

Activity and action mechanisms of latarcin 2a (Ltc2a), an antimicrobial peptide from the venom of the spider Lachesana tarabaevi (Zodariidae), were studied in vitro on human cells. Cytotoxicity of Ltc2a for erythrocytes (EC₅₀ = 3.4 μM), leukocytes (EC₅₀ = 19.5 μM) and erythroleukemia K562 cells (EC₅₀ = 3.3 μM) has been found to be primary related to plasma membrane destabilization. Using fluorescently labeled Ltc2a, three common features are found for erythrocytes and K562 cells: pronounced inhomogeneity of cellular response to Ltc2a; complex multistage character of Ltc2a-cell interactions; a positive feedback between Ltc2a binding to plasma membrane and development of toxic effects. Discocyte - echinocyte - spherocyte - ghost is a sequence of Ltc2a-induced transformations of erythrocytes that are accompanied by multistage enhancement of Ltc2a membrane binding, formation of small (ca. 2.0 nm) membrane pores, osmotic imbalance development and reorganization of the pores into large (ca. 13 nm) membrane openings that are preserved in ghosts. Ltc2a induces membrane blebbing and swelling of K562 cells followed by cell death. Cytotoxic action occurs through formation of membrane pores (ca. 3.7 nm) which show greater permeability for anionic than cationic molecules. The pore formation is accompanied with self-assisted Ltc2a internalization and accumulation in mitochondria, mitochondrion inactivation and apoptosis-independent phosphatidylserine externalization.     

A comparative study of amyloid fibril formation by residues 15-19 of the human calcitonin hormone: a single beta-sheet model with a small hydrophobic core

Experimentally, the human calcitonin hormone (hCT) can form highly stable amyloid protofibrils. Further, a peptide consisting of hCT residues 15-19, DFNKF, was shown to create highly ordered fibrils, similar to those formed by the entire hormone sequence. However, there are limited experimental data regarding the detailed 3D arrangement of either of these fibrils. We have modeled the DFNKF protofibril, using molecular dynamics simulations. We tested the stabilities of single sheet and of various multi sheet models. Remarkably, our most ordered and stable model consists of a parallel-stranded, single beta-sheet with a relatively insignificant hydrophobic core. We investigate the chemical and physical interactions responsible for the high structural organization of this single beta-sheet amyloid fibril. We observe that the most important chemical interactions contributing to the stability of the DFNKF organization are electrostatic, specifically between the Lys and the C terminus, between the Asp and N terminus, and a hydrogen bond network between the Asn side-chains of adjacent strands. Additionally, we observe hydrophobic and aromatic pi stacking interactions. We further simulated truncated filaments, FNKF and DFNK. Our tetra-peptide mutant simulations assume models similar to the penta-peptide. Experimentally, the FNKF does not create fibrils while DFNK does, albeit short and less ordered than DFNKF. In the simulations, the FNKF system was less stable than the DFNK and DFNKF. DFNK also lost many of its original interactions becoming less organized, however, many contacts were maintained. Thus, our results emphasize the role played by specific amino acid interactions. To further study specific interactions, we have mutated the penta-peptide, simulating DANKF, DFNKA and EFNKF. Here we describe the model, its relationship to experiment and its implications to amyloid organization.     

Fast atom bombardment mass spectrometry as a tool for the rapid determination of enantioselective binding of methylated cyclodextrins

The first FABMS study of the enantioselectivity shown during complex formation between per-methylated cyclodextrins and pairs of enantiomeric guest molecules is described. The 1:1 mixtures of the cyclodextrins, both α- and β-, with the guests, the methyl esters of the amino acids tryptophan and phenylalanine, were studied in a 100:50:1 glycerol-thioglycerol-trifluoro-acetic acid matrix. The uncomplexed cyclodextrin peaks were then used as internal standards to determine the preference of the cavity for one or other of the enantiomers. A clear trend for the preferential binding, greater than 5:1 in each case, of the d-enantiomers of the amino acid esters was observed in agreement with literature ¹H NMR experiments. This methodology provides a rapid route to assessing the enantioselectivity shown by the widely used cyclodextrins towards pairs of enantiomeric guests.     

Confirmation of the mode of action of an antibacterial inhibitor using regulated antisense RNA

Summary We previously demonstrated that regulated antisense RNA technology enables us to validate and identify the mode of action for some antibiotics. In this study, we have expanded the application of the regulated antisense approach to track the mode of action for a novel inhibitor of polypeptide deformylase (Pdf), which is an attractive target for the development of novel classes of antibacterial agents. We created a pdf antisense isogenic strain in Staphylococcus aureus using a TetR-regulated expression system. We demonstrated that the partial inhibition of pdf expression significantly increased the susceptibility of S. aureus to Pdf-specific inhibitor. This result provides further evidence that the TetR-regulated antisense technology is a robust tool for tracking the mode of action of novel antibacterial agents.     

Antimicrobial activity of amphiphilic neamine derivatives: Understanding the mechanism of action on Gram-positive bacteria

Amphiphilic aminoglycoside derivatives are potential new antimicrobial agents mostly developed to fight resistant bacteria. The mechanism of action of the 3′,6-dinonyl neamine, one of the most promising derivative, has been investigated on Gram-negative bacteria, including P. aeruginosa. In this study, we have assessed its mechanism of action against Gram-positive bacteria, S. aureus and B. subtilis. By conducting time killing experiments, we assessed the bactericidal effect induced by 3′,6-dinonyl neamine on S. aureus MSSA and MRSA. By measuring the displacement of BODIPY?-TR cadaverine bound to lipoteichoic acids (LTA), we showed that 3′,6-dinonyl neamine interacts with these bacterial surface components. We also highlighted the ability of 3′,6-dinonyl neamine to enhance membrane depolarization and induce membrane permeability, by using fluorescent probes, DiSC₃C(5) and propidium iodide, respectively. These effects are observed for both MSSA and MRSA S. aureus as well as for B. subtilis. By electronic microscopy, we imaged the disruption of membrane integrity of the bacterial cell wall and by fluorescence microscopy, we demonstrated changes in the localization of lipids from the enriched-septum region and the impairment of the formation of septum. At a glance, we demonstrated that 3′,6-dinonyl neamine interferes with multiple targets suggesting a low ability of bacteria to acquire resistance to this agent. In turn, the amphiphilic neamine derivatives are promising candidates for development as novel multitarget therapeutic antibiotics.     

Generation of potential in lipid bilayer membranes as a result of proton-transfer reactions in the unstirred layers

The addition of an uncoupler in the presence of a concentration gradient of weak acids or bases (sodium acetate and ammonium chloride) leads to the generation of a potential on lipid bilayer membranes (LBM) which is positive in sign on the side of the membrane with a high concentration of sodium acetate and negative on the side with a high concentration of ammonium chloride. It is shown that the potential was caused by the pH gradient in the unstirred layers. These effects can be understood in terms of the previously described [Science,182, 1258 (1973)] model for the transfer of weak acids and bases through LBM. This system described may be useful for quantitation of permeabilities for weak acids and bases through bilayer membranes.     

Analysis of the Drosophila betaPS subunit indicates that regulation of integrin activity is a primal function of the C8-C9 loop

Integrin-ligand interactions can be influenced by the sequence in a disulfide-bridged loop between the 8th and 9th beta subunit cysteines. Previous experiments are consistent with C8-C9 loop residues being involved in direct ligand-integrin interactions and/or being important in heterodimer regulation. In betaPS from Drosophila melanogaster and three other dipterans, the C8-C9 loop consists of only two amino acids, and exists in two forms that arise by differential splicing of exon 4. In these species, the betaPS4A isoform has an acidic residue in the first loop position (C8+1), with an alanine or proline in the corresponding position of betaPS4B. Mutations in both isoforms (in combination with alphaPS2) can reduce cell spreading during normal growth, but function is generally restored under conditions that enhance integrin activation. Replacement of the betaPS4A acidic residue with a basic lysine has relatively modest effects on integrin function. Spread cells bearing C8-C9 mutations tend to become less elongated, with reduced frequencies of actin stress fibers. The results indicate that even a minimal, two-residue C8-C9 loop contains structural information that can differentially regulate integrin activity and/or integrin signaling, and that this regulation does not rely on direct molecular interactions involving the variable C8+1 side chains.     

Detergent extraction of membrane proteins related to the action of antidiuretic hormone

Antidiuretic hormone (ADH) induces, in the apical plasma membrane of target cells, the insertion of intramembranous particle aggregates that probably contain water channels. A mild attack of this membrane by a polyoxyethylene nonylphenyl detergent, which reversibly depressed ADH-induced water permeability, has been found to modify aggregate structure while extracting additional proteins. This simple procedure could be a valuable approach to the problem of aggregate isolation and characterization.     

Maintaining the ionic permeability of a cellulose ester membrane

This paper reports the results of a series of experiments designed to test conditions that would permit NaCl to diffuse through 100 Da molecular weight cut-off (MWCO) and 1,000 Da MWCO membranes. For the 100 Da MWCO membrane, the membrane becomes completely impermeable to NaCl when dialyzed against distilled water (DW), but inclusion of one of a variety of different salts in the dialyzing solution maintains membrane permeability to NaCl. A titration experiment revealed that a minimum concentration of 0.1 mM of a salt such as KH2PO4 is required to sustain membrane permeability. In contrast, diffusion through the 1,000 Da MWCO membrane was slightly higher when DW was used as the dialysate. We conclude that the 100 Da MWCO membrane works well for a variety of dialysis applications provided that a maintenance salt is included in all dialyzing solutions.     

The role of the central L- or D-Pro residue on structure and mode of action of a cell-selective alpha-helical IsCT-derived antimicrobial peptide

IsCT-P (ILKKIWKPIKKLF-NH2) is a novel alpha-helical antimicrobial peptide with bacterial cell selectivity designed from a scorpion-derived peptide IsCT. To investigate the role of L- or D-Pro kink on the structure and the mode of action of a short alpha-helical antimicrobial peptide with bacterial cell selectivity, we synthesized IsCT-p, in which D-Pro is substituted for L-Pro8 of IsCT-P. CD spectra revealed that IsCT-P adopted a typical alpha-helical structure in various membrane-mimicking conditions, whereas IsCT-p showed a random structure. This result indicated that D-Pro in the central position of a short alpha-helical peptide provides more remarkable structural flexibility than L-Pro. Despite its higher antibacterial activity, IsCT-p was much less effective at inducing dye leakage in the negatively charged liposome mimicking bacterial membrane and induced no or little membrane potential depolarization of Staphylococcus aureus. Confocal laser scanning microscopy showed that IsCT-p penetrated the bacterial cell membrane and accumulated in the cytoplasm, whereas IsCT-P remained outside or on the cell membrane. These results suggested that the major target of IsCT-P and IsCT-p is the bacterial membranes and intracellular components, respectively. Collectively, our results demonstrated that the central D-Pro kink in alpha-helical antimicrobial peptides plays an important role in penetrating bacterial membrane as well as bacterial cell selectivity.     

Alteration in amino-glycerophospholipids levels in the plasma of children with autism: a potential biochemical diagnostic marker

Currently, there is no biochemical test to assist in the behavioral diagnosis of autism. We observed that levels of phosphatidylethanolamine (PE) were decreased while phosphatidylserine (PS) were increased in the erythrocyte membranes of children with autism as compared to their non-autistic developmentally normal siblings. A new method using Trinitrobenezene sulfonic acid (TNBS) for the quantification of PE and PS (amino-glycerophospholipids, i.e., AGP) in the plasma of children was developed and standardized. Wavelength scans of TNBS-PE and TNBS-PS complexes gave two peaks at 320 nm and 410 nm. When varying concentrations of PS and PE were used, a linear regression line was observed at 410 nm with TNBS. Using this assay, the levels of AGP were found to be significantly increased in the plasma of children with autism as compared to their non-autistic normal siblings. It is proposed that plasma AGP levels may function as a potential diagnostic marker for autism.