Specific Amino Acid Residues in the Three Loops of Snake Cytotoxins Determine Their Membrane Activity and Provide a Rationale for a New Classification of These Toxins.

Cytotoxins (CTs) are three-finger membrane-active toxins present mainly in cobra venom. Our analysis of the available CT amino acid sequences, literature data on their membrane activity, and conformational equilibria in aqueous solution and detergent micelles allowed us to identify specific amino acid residues which interfere with CT incorporation into membranes. They include Pro9, Ser28, and Asn/Asp45 within the N-terminal, central, and C-terminal loops, respectively. There is a hierarchy in the effect of these residues on membrane activity: Pro9 > Ser28 > Asn/Asp45. Taking into account all the possible combinations of special residues, we propose to divide CTs into eight groups. Group 1 includes toxins containing all of the above residues. Their representatives demonstrated the lowest membrane activity. Group 8 combines CTs that lack these residues. For the toxins from this group, the greatest membrane activity was observed. We predict that when solely membrane activity determines the cytotoxic effects, the activity of CTs from a group with a higher number should exceed that of CTs from a group with a lower number. This classification is supported by the available data on the cytotoxicity and membranotropic properties of CTs. We hypothesize that the special amino acid residues within the loops of the CT molecule may indicate their involvement in the interaction with non-lipid targets.

Variability in the Spatial Structure of the Central Loop in Cobra Cytotoxins Revealed by X-ray Analysis and Molecular Modeling.

Cobra cytotoxins (CTs) belong to the three-fingered protein family and possess membrane activity. Here, we studied cytotoxin 13 from Naja naja cobra venom (CT13Nn). For the first time, a spatial model of CT13Nn with both "water" and "membrane" conformations of the central loop (loop-2) were determined by X-ray crystallography. The "water" conformation of the loop was frequently observed. It was similar to the structure of loop-2 of numerous CTs, determined by either NMR spectroscopy in aqueous solution, or the X-ray method. The "membrane" conformation is rare one and, to date has only been observed by NMR for a single cytotoxin 1 from N. oxiana (CT1No) in detergent micelle. Both CT13Nn and CT1No are S-type CTs. Membrane-binding of these CTs probably involves an additional step-the conformational transformation of the loop-2. To confirm this suggestion, we conducted molecular dynamics simulations of both CT1No and CT13Nn in the Highly Mimetic Membrane Model of palmitoiloleoylphosphatidylglycerol, starting with their "water" NMR models. We found that the both toxins transform their "water" conformation of loop-2 into the "membrane" one during the insertion process. This supports the hypothesis that the S-type CTs, unlike their P-type counterparts, require conformational adaptation of loop-2 during interaction with lipid membranes.

Membrane-Disrupting Activity of Cobra Cytotoxins Is Determined by Configuration of the N-Terminal Loop.

In aqueous solutions, cobra cytotoxins (CTX), three-finger folded proteins, exhibit conformational equilibrium between conformers with either cis or trans peptide bonds in the N-terminal loop (loop-I). The equilibrium is shifted to the cis form in toxins with a pair of adjacent Pro residues in this loop. It is known that CTX with a single Pro residue in loop-I and a cis peptide bond do not interact with lipid membranes. Thus, if a cis peptide bond is present in loop-I, as in a Pro-Pro containing CTX, this should weaken its lipid interactions and likely cytotoxic activities. To test this, we have isolated seven CTX from Naja naja and N. haje cobra venoms. Antibacterial and cytotoxic activities of these CTX, as well as their capability to induce calcein leakage from phospholipid liposomes, were evaluated. We have found that CTX with a Pro-Pro peptide bond indeed exhibit attenuated membrane-perturbing activity in model membranes and lower cytotoxic/antibacterial activity compared to their counterparts with a single Pro residue in loop-I.

The omega-loop of cobra cytotoxins tolerates multiple amino acid substitutions.

Cobra cytotoxins (CTs), the three-fingered proteins, feature high amino acid sequence homology in the beta-strands and variations in the loop regions. We selected a pair of cytotoxins from Naja kaouthia crude venom to clarify the sequence-structure relationships. Using chromatography and mass spectroscopy, we separated and identified the mixture of cytotoxins 2 and 3, differentiated by the only Val 41/Ala 41 substitution. Here, using natural abundance 13C, 15N NMR-spectroscopy we performed chemical shift assignments of the signals of the both toxins in aqueous solution in the major and minor forms. Combining NOE and chemical shift data, the toxins' spatial structure was determined. Finally, we proved that the tip of the "finger"-2, or the loop-2 of cytotoxins adopts the shape of an omega-loop with a tightly-bound water molecule in its cavity. Comparison with other NMR and X-ray structures of cytotoxins possessing different amino acid sequences reveals spatial similarity in this family of proteins, including the loop-2 region, previously considered to be flexible.

Stepwise Insertion of Cobra Cardiotoxin CT2 into a Lipid Bilayer Occurs as an Interplay of Protein and Membrane "Dynamic Molecular Portraits".

For many peripheral membrane-binding polypeptides(MBPs), especially ß-structural ones, the precise molecular mechanisms of membrane insertion remain unclear. In most cases, only the terminal water-soluble and membrane-bound states have been elucidated, whereas potential functionally important intermediate stages are still not understood in sufficient detail. In this study, we present one of the first successful attempts to describe step-by-step embedding of the MBP cardiotoxin 2 (CT2) from cobra Naja oxiana venom into a lipid bilayer at the atomistic level. CT2 possesses a highly conservative and rigid ß-structured three-finger fold shared by many other exogenous and endogenous proteins performing a wide variety of functions. The incorporation of CT2 into the lipid bilayer was analyzed via a 2 µs all-atom molecular dynamics (MD) simulation without restraints. This process was shown to occur over a number of distinct steps, while the geometry of initial membrane attachment drastically differs from that of the final equilibrated state. In the latter one, the hydrophobic platform ("bottom") formed by the tips of the three loops is deeply buried into the lipid bilayer. This agrees well with the NMR data obtained earlier for CT2 in detergent micelles. However, the bottom is too bulky to insert itself into the membrane at once. Instead, the gradual immersion of CT2 initiated by the loop-1 was observed. This initial binding stage was also demonstrated in a series of MD runs with varying starting orientations of the toxin with respect to the bilayer surface. Apart from the nonspecific long-range electrostatic attraction and hydrophobic match/mismatch factor, several specific lipid-binding sites were identified in CT2. They were shown to promote membrane insertion by engaging in strong interactions with lipid head groups, fine-tuning the toxin-membrane accommodation. We therefore propose that the toxin insertion relies on the interplay of nonspecific and specific interactions, which are determined by the "dynamic molecular portraits" of the two players, the protein and the membrane. The proposed model does not require protein oligomerization for membrane insertion and can be further employed to design MBPs with predetermined properties with regard to particular membrane targets.

Antibacterial activity of cardiotoxin-like basic polypeptide from cobra venom.

Antibacterial activity of the three-finger toxins from cobra venom, including cytotoxin 3 from N. kaouthia, cardiotoxin-like basic polypeptide A5 from N. naja (CLBP), and alpha-neurotoxin from N. oxiana venom, was investigated. All toxins failed to influence Gram-negative bacteria. The most pronounced activity against Bacillus subtilis was demonstrated by CLBP. The latter is ascribed to the presence of additional Lys-residues within the membrane-binding motif of this toxin.

The role of hydrophobic /hydrophilic balance in the activity of structurally flexible vs. rigid cytolytic polypeptides and analogs developed on their basis.

INTRODUCTION: Being important representatives of various proteomes, membrane-active cationic peptides (CPs) are attractive objects as lead compounds in the design of new antibacterial, anticancer, antifungal, and antiviral molecules. Numerous CPs are found in insect and snake venoms, where many of them reveal cytolytic properties. Due to advances in omics technologies, the number of such peptides is growing dramatically. Areas covered: To understand structure-function relationships for CPs in a living cell, detailed analysis of their hydrophobic/hydrophilic properties is indispensable. We consider two structural classes of membrane-active CPs: latarcins (Ltc) from spider and cardiotoxins (CTXs) from snake venoms. While the former are void off disulfide bonds and conformationally flexible, the latter are structurally rigid and cross-linked with disulfide bonds. In order to elucidate structure-activity relationships behind their antibacterial, anticancer, and hemolytic effects, the properties of these polypeptides are considered on a side-by-side basis. Expert commentary: An ever-increasing number of venom-derived membrane-active polypeptides require new methods for identification of their functional propensities and sequence-based design of novel pharmacological substances. We address these issues considering a number of the designed peptides, based either on Ltc or CTX sequences. Experimental and computer modeling techniques required for these purposes are delineated.

Improving therapeutic potential of antibacterial spider venom peptides: coarse-grain molecular dynamics guided approach.

AIM: Spider venom is a rich source of antibacterial peptides, whose hemolytic activity is often excessive. METHODOLOGY: How to get rid of it? Using latarcins from Lachesana tarabaevi and oxyopinin Oxt 4a from Oxyopes takobius spider venoms we performed coarse-grained molecular dynamics simulations of these peptides in the presence of lipid bilayers, mimicking erythrocyte membranes. This identified hemolytically active fragments within Oxt 4a and latarcins. Then, we synthesized five 20-residue peptides, containing different parts of the Oxt 4a and latarcin-1 sequence, carrying mutations within the identified regions. CONCLUSION: The antibacterial and hemolytic tests suggested that the three of the synthesized peptides demonstrated substantial decrease in hemolytic activity, retaining, or even exceeding antibacterial potential of the parent peptides.

Impact of membrane partitioning on the spatial structure of an S-type cobra cytotoxin.

Cobra cytotoxins (CTs) belong to the three-fingered protein family. They are classified into S- and P-types, the latter exhibiting higher membrane-perturbing capacity. In this work, we investigated the interaction of CTs with phospholipid bilayers, using coarse-grained (CG) and full-atom (FA) molecular dynamics (MD). The object of this work is a CT of an S-type, cytotoxin I (CT1) from N.oxiana venom. Its spatial structure in aqueous solution and in the micelles of dodecylphosphocholine (DPC) were determined by 1H-NMR spectroscopy. Then, via CG- and FA MD-computations, we evaluated partitioning of CT1 molecule into palmitoyloleoylphosphatidylcholine (POPC) membrane, using the toxin spatial models, obtained either in aqueous solution, or detergent micelle. The latter model exhibits minimal structural changes upon partitioning into the membrane, while the former deviates from the starting conformation, loosing the tightly bound water molecule in the loop-2. These data show that the structural changes elicited by CT1 molecule upon incorporation into DPC micelle take place likely in the lipid membrane, although the mode of the interaction of this toxin with DPC micelle (with the tips of the all three loops) is different from its mode in POPC membrane (primarily with the tip of the loop-1 and both the tips of the loop-1 and loop-2).

Structural and Dynamic "Portraits" of Recombinant and Native Cytotoxin I from Naja oxiana: How Close Are They?

Today, recombinant proteins are quite widely used in biomedical and biotechnological applications. At the same time, the question about their full equivalence to the native analogues remains unanswered. To gain additional insight into this problem, intimate atomistic details of a relatively simple protein, small and structurally rigid recombinant cardiotoxin I (CTI) from cobra Naja oxiana venom, were characterized using nuclear magnetic resonance (NMR) spectroscopy and atomistic molecular dynamics (MD) simulations in water. Compared to the natural protein, it contains an additional Met residue at the N-terminus. In this work, the NMR-derived spatial structure of uniformly 13C- and 15N-labeled CTI and its dynamic behavior were investigated and subjected to comparative analysis with the corresponding data for the native toxin. The differences were found in dihedral angles of only a single residue, adjacent to the N-terminal methionine. Microsecond-long MD traces of the toxins reveal an increased flexibility in the residues spatially close to the N-Met. As the detected structural and dynamic changes of the two CTI models do not result in substantial differences in their cytotoxicities, we assume that the recombinant protein can be used for many purposes as a reasonable surrogate of the native one. In addition, we discuss general features of the spatial organization of cytotoxins, implied by the results of the current combined NMR and MD study.

Latarcins: versatile spider venom peptides.

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.

Antiproliferative activity of cobra venom cytotoxins.

Cytotoxins (or cardiotoxins, CTs) are small rigid membrane-active proteins of the three-finger toxin (TFT) family. They comprise about 60 amino acid residues, stabilized by four disulphide bridges. CTs, the most abundant proteins in cobra venom are able to kill cancer cells in a dose and time-dependent manner. The present review summarizes the current data on the molecular pathways of cancer cell death, induced by CTs. A relationship between structural characteristics of CTs and mechanism of their antiproliferative activity is reviewed as well. The CT molecules are rigid and their structure does not change significantly, when they interact with their molecular targets. This rigidity facilitates identification of molecular entities, responsible for antiproliferative activity of the toxins. We demonstrate that consideration of a net electrostatic charge and recently introduced HTL (Hydrophobicity of the Tips of the Loops) score allows distinguishing between the two mechanisms of cell death. The first is related to membrane destabilization by the toxins. The second involves their capture inside the cells, followed by interrogation into signal cascades mediated by the proteins, essential for cell life. Via addressing to antibacterial activity of CTs, which is supposed to arise from the plasma membrane damage, we demonstrate that, if membrane deterioration is involved in malignant cell death, the toxic activity of CTs correlates with their HTL scores and net electrostatic charge. We assume the relationship found may be used for rational design of antiproliferative compounds.

Unusual titration of the membrane-bound artificial hemagglutinin fusion peptide.

E5 is a 20-residue-long analog of the fusion peptide from influenza hemagglutinin (GLFEAIAEFIEGGWEGLIEG). It has been suggested that two of its five glutamates, Glu11and Glu15, are critical in its pH-dependent membrane perturbation. To reveal their specific involvement, a pair of analogs with substitution of either Glu11 or Glu15 for Ala were synthesized. By analysis of the pH-dependence of the chemical shifts of protons of these peptides bound to dodecylphosphocholine micelles we found: (1) the peptides adopt an amphiphilic alpha-helical structure within residues 2-18, similar to the parent peptide; (2) the helix is significantly more disordered at neutral pH than at acidic pH for E5 peptide only; and (3) in E5 and mutant peptides the Glu11 and 15 residues have similar pK (a) values, higher than those of the other glutamates. This excludes their mutual interaction in E5, being a source of the elevated pK (a) values. We attribute this phenomenon to the presence of minor states caused by deepening of the Glu11 and 15 side-chains in the hydrophobic environment of the membrane. As the mid-pH of membrane-perturbation activity of E5 matches the pK (a) value of these glutamates, we conclude their presence contributes to the plasticity of the peptide and determines the pH-dependence of membrane perturbation caused by E5.

Structure and dynamics of cardiotoxins.

Cytotoxins (or cardiotoxins; CTs) are toxins from cobra venom characterized by the three-finger (TF) fold. CTs are on average 60-residue-long peptides, possessing as many as 4 disulfide bonds. The elements of antiparallel ß-structure take origin from the hydrophobic core formed by the disulfides. The ß-strands adopt the shape of the three loops, giving the name of the fold. While neurotoxins (NTs) - also TF proteins from snake venom - exert their effect through specific interactions with protein receptors, no specific protein target has been found for CTs. Unlike NTs, CTs are amphiphilic and cytotoxic against a variety of cells, including cancer ones. Thus, the hypothesis that the activity of CTs is caused by their interactions with lipid membranes is currently central. To understand molecular basis behind variations in toxicities of CTs highly homologous in their sequences, detailed knowledge of their structure and dynamics is required. The present review summarizes experimental and computational data on the spatial organization of CTs and their dynamics in various environments (aqueous solution, membranous milieus).

Novel lynx spider toxin shares common molecular architecture with defense peptides from frog skin.

A unique 30-residue cationic peptide oxyopinin 4a (Oxt 4a) was identified in the venom of the lynx spider Oxyopes takobius (Oxyopidae). Oxt 4a contains a single N-terminally located disulfide bond, Cys4-Cys10, and is structurally different from any spider toxin studied so far. According to NMR findings, the peptide is disordered in water, but assumes a peculiar torpedo-like structure in detergent micelles. It features a C-terminal amphipathic α-helical segment (body; residues 12-25) and an N-terminal disulfide-stabilized loop (head; residues 1-11), and has an unusually high density of positive charge in the head region. Synthetic Oxt 4a was produced and shown to possess strong and broad-spectrum cytolytic and antimicrobial activity. cDNA cloning showed that the peptide is synthesized in the form of a conventional prepropeptide with an acidic prosequence. Unlike other arachnid toxins, Oxt 4a exhibits striking similarity with defense peptides from the skin of ranid frogs that contain the so-called Rana-box motif (a C-terminal disulfide-enclosed loop). Parallelism or convergence is apparent on several levels: the structure, function and biosynthesis of a lynx spider toxin are mirrored by those of Rana-box peptides from frogs.

Solution structure of a defense peptide from wheat with a 10-cysteine motif.

Hevein, a well-studied lectin from the rubber tree Hevea brasiliensis, is the title representative of a broad family of chitin-binding polypeptides. WAMP-1a, a peptide isolated from the wheat Triticum kiharae, shares considerable similarity with hevein. The peptide possesses antifungal, antibacterial activity and is thought to play an important role in the defense system of wheat. Importantly, it features a substitution of the conserved serine residue to glycine reducing its carbohydrate-binding capacity. We used NMR spectroscopy to derive the spatial structure of WAMP-1a in aqueous solution. Notably, the mutation was found to strengthen amphiphilicity of the molecule, associated with its mode of action, an indication of the hevein domain multi-functionality. Both primary and tertiary structure of WAMP-1a suggest its evolutionary origin from the hevein domain of plant chitinases.

Three-dimensional structure/hydrophobicity of latarcins specifies their mode of membrane activity.

Latarcins, linear peptides from the Lachesana tarabaevi spider venom, exhibit a broad-spectrum antimicrobial activity, likely acting on the bacterial cytoplasmic membrane. We study their spatial structures and interaction with model membranes by a combination of experimental and theoretical methods to reveal the structure-activity relationship. In this work, a 26 amino acid peptide, Ltc1, was investigated. Its spatial structure in detergent micelles was determined by (1)H nuclear magnetic resonance (NMR) and refined by Monte Carlo simulations in an implicit water-octanol slab. The Ltc1 molecule was found to form a straight uninterrupted amphiphilic helix comprising 8-23 residues. A dye-leakage fluorescent assay and (31)P NMR spectroscopy established that the peptide does not induce the release of fluorescent marker nor deteriorate the bilayer structure of the membranes. The voltage-clamp technique showed that Ltc1 induces the current fluctuations through planar membranes when the sign of the applied potential coincides with the one across the bacterial inner membrane. This implies that Ltc1 acts on the membranes via a specific mechanism, which is different from the carpet mode demonstrated by another latarcin, Ltc2a, featuring a helix-hinge-helix structure with a hydrophobicity gradient along the peptide chain. In contrast, the hydrophobic surface of the Ltc1 helix is narrow-shaped and extends with no gradient along the axis. We have also disclosed a number of peptides, structurally homologous to Ltc1 and exhibiting similar membrane activity. This indicates that the hydrophobic pattern of the Ltc1 helix and related antimicrobial peptides specifies their activity mechanism. The latter assumes the formation of variable-sized lesions, which depend upon the potential across the membrane.

Spatial structure and activity mechanism of a novel spider antimicrobial peptide.

Latarcins (Ltc), linear peptides (ca. 25 amino acid long) isolated from the venom of the Lachesana tarabaevi spider, exhibit a broad-spectrum antibacterial activity, most likely acting on the bacterial plasmatic membrane. We study the structure-activity relationships in the series of these compounds. At the first stage, we investigated the spatial structure of one of the peptides, Ltc2a, and its mode of membrane perturbation. This was done by a combination of experimental and theoretical methods. The approach includes (i) structural study of the peptide by CD spectroscopy in phospholipid liposomes and by (1)H NMR in detergent micelles, (ii) determination of the effect on the liposomes by a dye leakage fluorescent assay and (31)P NMR spectroscopy, (iii) refinement of the NMR-derived spatial structure via Monte Carlo simulations in an implicit water-octanol slab, and (iv) calculation of the molecular hydrophobicity potential. The molecule of Ltc2a was found to consist of two helical regions (residues 3-9 and 13-21) connected via a poorly ordered fragment. The effect of the peptide on the liposomes suggests the carpet mechanism of the membrane deterioration. This is also supported by the analysis of hydrophobic/hydrophilic characteristics of Ltc2a and homologous antimicrobial peptides. These peptides exhibiting a helix-hinge-helix structural motif are characterized by a distinct and feebly marked amphiphilicity of their N- and C-terminal helices, respectively, and by a hydrophobicity gradient along the peptide chain. The approach we suggested may be useful in studying not only other latarcins but also a wider class of membrane-active peptides.

Modeling of 31P-NMR spectra of magnetically oriented phospholipid liposomes: A new analytical solution.

31P-NMR spectroscopy is widely used for studies of phospholipid liposomes, a commonly used model of a biological membrane. For the correct analysis of 31P-NMR spectra of the liposomes it is necessary to take into account that they are deformed by the magnetic field of the spectrometer. The liposomes become ellipsoidal and this affects the lineshape of the spectrum. In the present communication we suggest a new analytical formula for modeling of 31P-NMR spectra of the prolate phospholipid liposomes. The formula assumes a Lorentzian broadening function and exactly ellipsoidal shape of the liposomes. Based on the formula a program called P-FIT is designed for the practical analysis of the experimental multicomponent spectra of the prolate liposomes. The versatility of the program developed in a Mathematica environment is demonstrated by simulations of a number of 31P-NMR spectra with different complexity.