Amino acid sequence of toxin VII,A β-toxin from the venom of the scorpton Tityus serrulatus

The sequence of the 61 amino acids of toxin VII, a β-toxin from the venom of the South American scorpion Tityus serrulatus, has been determined by automatic sequencing of the reduced and S-[¹⁴C] car?ymethylated protein and of tryptic peptides obtained before or after citraconylation of this protein. This toxin, the most active β-toxin from this venom, is the first Tityus toxin to be fully sequenced. The results clearly show that toxin VII belongs to the structural group of scorpion toxins originating from Central and North America.     

Do cardiotoxins possess a functional site? Structural and chemical modification studies reveal the functional site of the cardiotoxin from Naja nigricollis

Examination of the literature has revealed that regarding the amino acid sequences, cardiotoxins constitute a family of homogeneous compounds. In contrast, cardiotoxins appear heterogeneous as far as their biological and spectroscopic properties are concerned. As a result, comparison between these molecules with a view to establishing structure-activity correlations is complicated. We have therefore reviewed recent works aiming at identifying the functional site of a defined cardiotoxin, ie toxin gamma from the venom of the spitting cobra Naja nigricollis. The biological and structural properties of toxin gamma are first described. In particular, a model depicting the 3-dimensional structure of the toxin studied by NMR spectroscopy is proposed. The toxin polypeptide chain is folded into 3 adjacent loops rich in beta-sheet structure connected to a small globular core containing the 4 disulfide bonds. A number of derivatives chemically modified at a single aromatic or amino group have been prepared. The structure of each derivative was probed by emission fluorescence, circular dichroism and NMR spectroscopy. Also tested was the ability of the derivatives to kill mice, depolarize excitable cell membranes and lyse epithelial cells. Modification of some residues in the first loop, in particular Lys-12 and at the base of the second loop substantially affected biological properties, with no sign of concomitant structural modifications other than local changes. Modifications in other regions much less affected the biological properties of the toxin. A plausible functional site for toxin gamma involving loop I and the base of loop II is presented. It is stressed that the functional site of other cardiotoxins may be different.     

Residue 544 in Domain III of the <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry1Ac Toxin is Involved in Protein Structure Stability

A unique residue W544 in the β18–β19 loop of the Bacillus thuringiensis Cry1Ac toxin has been implicated in its toxicity. In this study, the effects of mutations at this residue on protein stability during protease treatment, UV irradiation, and preservation were examined. Residue 544 of Cry1Ac was involved in maintaining structural stability, and substitution of a polar group at this position was unfavorable to protein stability. One mutant, W544F, produced larger crystals and was more stable. This mutant showed greater resistance to UV radiation than the wild type Cry1Ac but retained equal toxicity. This is the first report showing that residue 544 in the Cry1Ac domain III plays a significant role in toxin structural stability. Our W544F mutant is a significant development in terms of field applications of Cry1Ac toxin.     

Do structural deviations between toxins adopting the same fold reflect functional differences?

Three-finger proteins form a structurally related family of compounds that exhibit a great variety of biological properties. To address the question of the prediction of functional areas on their surfaces, we tentatively conferred the acetylcholinesterase inhibitory activity of fasciculins on a short-chain curaremimetic toxin. For this purpose, we assimilated the three-dimensional structure of fasciculin 2 with the one of toxin alpha. This comparison revealed that the tips of the first and second loops, together with the C terminus residue, deviated most. A first recombinant fasciculin/toxin alpha chimera was designed by transferring loop 1 in its entirety together with the tip of loop 2 of fasciculin 2 into the toxin alpha scaffold. A second chimera (rChII) was obtained by adding the point Asn-61 --> Tyr substitution. Comparison of functional and structural properties of both chimeras show that rChII can accommodate the imposed modifications and displays nearly all the acetylcholinesterase-blocking activities of fasciculins. The three-dimensional structure of rChII demonstrates that rChII adopts a typical three-fingered fold with structural features of both parent toxins. Taken together, these results emphasize the great structural flexibility and functional adaptability of that fold and confirm that structural deviations between fasciculins and short-chain neurotoxins do indeed reflect functional diversity.     

Understanding structural/functional properties of immunoconjugates for cancer therapy by computational approaches

Monoclonal antibodies coupled to highly toxic molecules (immunoconjugates) are currently being developed for cancer therapy. We have used an in silico procedure for evaluating some physicochemical properties of two tumor-targeting anti-HER2 immunoconjugates: (a) the single-chain antibody scFv(FRP5) linked to a bacterial toxin, that has been recently progressed to phase I clinical trial in human cancer; (b) the putative molecule formed by the intrinsically stable scFv(800E6), which has been proposed as toxin carrier to cancer cells in human therapy, joined to the same toxin of (a). Structural models of the immunoconjugates have been built by homology modeling and assessed by molecular dynamics simulations. The trajectories have been analyzed to extract some biochemical properties and to assess the potential effects of the toxin on the structure and dynamics of the anti-HER2 antibodies. The results of the computational approach indicate that the antibodies maintain their correct folding even in presence of the toxin, whereas a certain stiffness in correspondence of some structural regions is observed. Furthermore, the toxin does not seem to affect the antibody solubility, whereas it enhances the structural stability. The proposed computational approach represent a promising tool for analyzing some physicochemical properties of immunoconjugates and for predicting the effects of the linked toxin on structure, dynamics, and functionality of the antibodies.     

Isolation and characterization of the polypeptide fragments obtained by limited proteolysis of botulinic toxin type A

A limited proteolysis of the botulinic toxin of A type by subtylopeptidase A resulted in two high molecular weight non-toxic fragments. The peptide with mol. weight of 100,000 is made up of two subunits with mol. weights of 52,000 and 48,000. The second peptide whose mol. weight is 40,000 is a single-chained one. The high molecular weight peptide has one S--S bond and two SH-groups, whereas the one with a lower molecular weight--no S--S bond and 1.3--1.5 SH-groups. Dansylation of the first fragment revealed two N-terminal amino acids (histidine, arginine) in toxin, which suggests the localization of the first fragment at the N-end of the toxin molecule. Using immunochemical analysis with monospecific antiserum against original toxin and antifragment sera, the antigenic determinants from the fragments were shown to be serologically different. A structural model of botulinic toxin of A type is proposed.     

Construction of recombinant plasmids encoding the biosynthesis of the beta-subunit of cholera toxin

The structure of the cholera toxin operon and the location of A and B toxin subunits have been studied by the Southern blot hybridization on filters. The gene coding for the synthesis of the cholera toxin B-subunit has been cloned in the vector plasmid pBR322. The structural gene of A-subunit has been partially deleted by the restriction endonuclease Bal31 digestion. The size of the 250 b. p. deletion has been defined by electron microscopy. The production of the cholera toxin B-subunit in Escherichia coli K12 cells has been studied.     

Solution structure of BmKK4, the first member of subfamily alpha-KTx 17 of scorpion toxins

BmKK4 is a 30 amino acid peptide purified from the venom of the Chinese scorpion Buthus martensi Karsch. It has been classified as the first member of scorpion toxin subfamily alpha-KTx 17. The 3D structure of BmKK4 in solution has been determined by 2D NMR spectroscopy. This toxin adopts a common alpha/beta-motif, but shows a distinctive local conformation. The most novel feature is that the regular arrangements of the side chains of the residues involved in the beta-sheet of BmKK4 are distorted by a classic beta-bulge structure, which involves two residues (Asp18 and Arg19) in the first strand opposite a single residue (Tyr26) in the second strand. The bulge produces two main changes in the structure of the antiparallel beta-sheet: (1) It disrupts the normal alteration of the side chain direction; the side chain of Asp18 turns over to form a salt bridge with that of Arg19. (2) It accentuates the twist of the sheet, and alters the direction of the antiparallel beta-sheet. The unusual structural feature of the toxin is attributed to the shorter peptide segment (Leu15-Arg19) between the third and fourth Cys residues and two unique residues (Asp18 and Arg19) at the position preceding the fourth Cys. In addition, the lower affinity of the peptide for the Kv channel is correlated to the structural features: residue Arg19 instead of a Lys residue at the critical position for binding and the salt bridge formed between residues Arg19 and Asp18.     

The alpha-helix 4 residue, Asn135, is involved in the oligomerization of Cry1Ac1 and Cry1Ab5 Bacillus thuringiensis toxins.

The insecticidal Cry toxins produced by the bacterium Bacillus thuringiensis are comprised of three structural domains. Domain I, a seven-helix bundle, is thought to penetrate the insect epithelial cell plasma membrane through a hairpin composed of alpha-helices 4 and 5, followed by the oligomerization of four hairpin monomers. The alpha-helix 4 has been proposed to line the lumen of the pore, whereas some residues in alpha-helix 5 have been shown to be responsible for oligomerization. Mutation of the Cry1Ac1 alpha-helix 4 amino acid Asn135 to Gln resulted in the loss of toxicity to Manduca sexta, yet binding was still observed. In this study, the equivalent mutation was made in the Cry1Ab5 toxin, and the properties of both wild-type and mutant toxin counterparts were analyzed. Both mutants appeared to bind to M. sexta membrane vesicles, but they were not able to form pores. The ability of both N135Q mutants to oligomerize was also disrupted, providing the first evidence that a residue in alpha-helix 4 can contribute to toxin oligomerization.     

The effect of pH on the conformation and stability of the structure of plant toxin-ricin

The effect of pH on the conformation of ricin and its A- and B-chains has been studied by measuring their intrinsic fluorescence. At pH 5.0 and 7.5, the structural stability of toxin and subunits was estimated according to the denaturing action of guanidine hydrochloride. It was demonstrated that the fluorescence of native toxin and catalytic A-subunit does not depend significantly on pH in the range pH 3-8, whereas ricin B-chain undergoes a structural transition at pH less than 5.0. The structural stability of ricin and isolated chains differs significantly at pH 7.5 and 5.0; the structural stability of ricin and the A-chain increases, whereas that of the B-chain decreases.     

Reduced pH causes structural changes in the potent mitogenic toxin of Pasteurella multocida

Pasteurella multocida toxin is a potent mitogen that is believed to act intracellularly. On transverse urea gradient gels at pH 8.0 the toxin displayed one major unfolding transition at 4 M urea. However, at pH 6.1 the unfolding transition took place at 3.5 M urea. Circular dichroism spectra also indicated that a structural change took place at acidic pH. In addition it was found that the toxin that had been denatured in 8 M urea refolded in solution with a high recovery of biological activity. These findings are discussed in terms of the likely domain structure of the P. multocida toxin.     

Four Distinct Structural Domains in Clostridium difficile Toxin B Visualized Using SAXS

Clostridium difficile is a nosocomial bacterial pathogen causing antibiotic-associated diarrhea and fatal pseudomembranous colitis. Key virulence factors are toxin A and toxin B (TcdB), two highly related toxins that are members of the large clostridial toxin family. These large multifunctional proteins disrupt cell function using a glucosyltransferase domain that is translocated into the cytosol after vesicular internalization of intact holotoxin. Although substantial information about the biochemical mechanisms of intoxication exists, research has been hampered by limited structural information, particularly of intact holotoxin. Here, we used small-angle X-ray scattering (SAXS) methods to obtain an ab initio low-resolution structure of native TcdB, which demonstrated that this molecule is monomeric in solution and possesses a highly asymmetric shape with a maximum dimension of ∼ 275 Å. Combining this SAXS information with crystallographic or modeled structures of individual functional domains of TcdB reveals for the first time that the three-dimensional structure of TcdB is organized into four distinct structural domains. Structures of the N-terminal glucosyltransferase, the cysteine protease, and the C-terminal repeat region can be aligned within three domains of the SAXS envelope. A fourth domain, predicted to be involved in the translocation of the glucosyltransferase, appears as a large solvent-exposed protrusion. Knowledge of the shapes and relative orientations of toxin domains provides new insight into defining functional domain boundaries and provides a framework for understanding how potential intra-domain interactions enable conformational changes to propagate between domains to facilitate intoxication processes.     

Accumulation of the insecticidal crystal protein of Bacillus thuringiensis subsp. kurstaki in post-exponential-phase Bacillus subtilis

A gene from Bacillus thuringiensis subsp. kurstaki that codes for a Lepidoptera-specific insecticidal toxin (delta-endotoxin) was engineered for expression in Bacillus subtilis. A low-copy-number plasmid vector that replicates in Escherichia coli and B. subtilis was constructed to transform B. subtilis with gene fusions first isolated and characterized in E. coli. Naturally occurring promoter sequences from B. subtilis (43, veg, ctc, and spoVG) were inserted upstream from the plasmid-borne structural gene. In the most prolific case, when the sporulation-specific spoVG promoter was fused to the heterologous toxin gene, the toxin product accumulated during postexponential growth to greater than 25% of the total cell protein. However, the resulting specific activity of the insecticidal toxin product was not commensurate with the abundance of the protein.     

Accumulation of the insecticidal crystal protein of Bacillus thuringiensis subsp. kurstaki in post-exponential-phase Bacillus subtilis.

A gene from Bacillus thuringiensis subsp. kurstaki that codes for a Lepidoptera-specific insecticidal toxin (delta-endotoxin) was engineered for expression in Bacillus subtilis. A low-copy-number plasmid vector that replicates in Escherichia coli and B. subtilis was constructed to transform B. subtilis with gene fusions first isolated and characterized in E. coli. Naturally occurring promoter sequences from B. subtilis (43, veg, ctc, and spoVG) were inserted upstream from the plasmid-borne structural gene. In the most prolific case, when the sporulation-specific spoVG promoter was fused to the heterologous toxin gene, the toxin product accumulated during postexponential growth to greater than 25% of the total cell protein. However, the resulting specific activity of the insecticidal toxin product was not commensurate with the abundance of the protein.     

Structural mapping of the voltage-dependent sodium channel. Distance between the tetrodotoxin and Centruroides suffusus suffusus II beta-scorpion toxin receptors

A 7- dimethylaminocoumarin -4-acetate fluorescent derivative of toxin II from the venom of the scorpion Centruroides suffusus suffusus (Css II) has been prepared to study the structural, conformational, and cellular properties of the beta-neurotoxin receptor site on the voltage-dependent sodium channel. The derivative retains high affinity for its receptor site on the synaptosomal sodium channel with a KD of 7 nM and site capacity of 1.5 pmol/mg of synaptosomal protein. The fluorescent toxin is very environmentally sensitive and the fluorescence emission upon binding indicates that the Css II receptor is largely hydrophobic. Binding of tetrodotoxin or batrachotoxin does not alter the spectroscopic properties of bound Css II, whereas toxin V from Leiurus quinquestriatus effects a 10-nm blue shift to a more hydrophobic environment. This is the first direct indication of conformational coupling between these separate neurotoxin receptor sites. The distance between the tetrodotoxin and Css II scorpion toxin receptors on the sodium channel was measured by fluorescence resonance energy transfer. Efficiencies were measured by both donor quenching and acceptor-sensitized emission. The distance between these two neurotoxin sites is about 34 A. The implications of these receptor locations together with other known molecular distances are discussed in terms of a molecular structure of the voltage-dependent sodium channel.     

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

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

Structural requirements of the unique disulphide bond and the proline-rich motif within the alpha4-alpha5 loop for larvicidal activity of the Bacillus thuringiensis Cry4Aa delta-endotoxin

Both the disulphide bond (Cys192-Cys199) and the proline-rich motif (Pro193ProAsnPro196) in the long loop connecting the alpha4-alpha5 transmembrane hairpin of the Cry4Aa mosquito-larvicidal protein have been found to be unique among the Bacillus thuringiensis Cry delta-endotoxins. In this study, their structural requirements for larvicidal activity of the Cry4Aa toxin were investigated. C192A and C199A mutant toxins were initially generated and over-expressed in Escherichia coli cells as 130-kDa protoxins at levels comparable to that of the wild-type toxin. When their activities against Aedes aegypti larvae were determined, Escherichia coli cells expressing each mutant toxin retained the high-level toxicity. Further mutagenic analysis of the PPNP motif revealed that an almost complete loss in larvicidal activity was observed for the C199A/P193A double mutant, whereas a small reduction in toxicity was shown for the C199A/P194A and C199A/P196A mutants. Increasing the flexibility of the alpha4-alpha5 loop through C199A/P193G, C199A/P194G/P196A, C199A/P194A/P196G, and C199A/P194G/P196G mutations significantly decreased the larvicidal activity. Similar to the wild-type protoxin, all mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. These findings are the first biological evidence for a structural function in larvicidal activity of the unique disulphide bridge as well as the proline-rich motif within the alpha4-alpha5 loop of the Cry4Aa toxin.     

E. coli hemolysin E (HlyE, ClyA, SheA): X-ray crystal structure of the toxin and observation of membrane pores by electron microscopy

Hemolysin E (HlyE) is a novel pore-forming toxin of Escherichia coli, Salmonella typhi, and Shigella flexneri. Here we report the X-ray crystal structure of the water-soluble form of E. coli HlyE at 2.0 A resolution and the visualization of the lipid-associated form of the toxin in projection at low resolution by electron microscopy. The crystal structure reveals HlyE to be the first member of a new family of toxin structures, consisting of an elaborated helical bundle some 100 A long. The electron micrographs show how HlyE oligomerizes in the presence of lipid to form transmembrane pores. Taken together, the data from these two structural techniques allow us to propose a simple model for the structure of the pore and for membrane interaction.