The unique pharmacology of the scorpion alpha-like toxin Lqh3 is associated with its flexible C-tail

The affinity of scorpion alpha-toxins for various voltage-gated sodium channels (Na(v)s) differs considerably despite similar structures and activities. It has been proposed that key bioactive residues of the five-residue-turn (residues 8-12) and the C-tail form the NC domain, whose topology is dictated by a cis or trans peptide-bond conformation between residues 9 and 10, which correlates with the potency on insect or mammalian Na(v)s. We examined this hypothesis using Lqh3, an alpha-like toxin from Leiurus quinquestriatus hebraeus that is highly active in insects and mammalian brain. Lqh3 exhibits slower association kinetics to Na(v)s compared with other alpha-toxins and its binding to insect Na(v)s is pH-dependent. Mutagenesis of Lqh3 revealed a bi-partite bioactive surface, composed of the Core and NC domains, as found in other alpha-toxins. Yet, substitutions at the five-residue turn and stabilization of the 9-10 bond in the cis conformation did not affect the activity. However, substitution of hydrogen-bond donors/acceptors at the NC domain reduced the pH-dependency of toxin binding, while retaining its high potency at Drosophila Na(v)s expressed in Xenopus oocytes. Based on these results and the conformational flexibility and rearrangement of intramolecular hydrogen-bonds at the NC domain, evident from the known solution structure, we suggest that acidic pH or specific mutations at the NC domain favor toxin conformations with high affinity for the receptor by stabilizing the bound toxin-receptor complex. Moreover, the C-tail flexibility may account for the slower association rates and suggests a novel mechanism of dynamic conformer selection during toxin binding, enabling alpha-like toxins to affect a broad range of Na(v)s.     

Miniaturization of scorpion beta-toxins uncovers a putative ancestral surface of interaction with voltage-gated sodium channels

The bioactive surface of scorpion beta-toxins that interact with receptor site-4 at voltage-gated sodium channels is constituted of residues of the conserved betaalphabetabeta core and the C-tail. In an attempt to evaluate the extent by which residues of the toxin core contribute to bioactivity, the anti-insect and anti-mammalian beta-toxins Bj-xtrIT and Css4 were truncated at their N and C termini, resulting in miniature peptides composed essentially of the core secondary structure motives. The truncated beta-toxins (DeltaDeltaBj-xtrIT and DeltaDeltaCss4) were non-toxic and did not compete with the parental toxins on binding at receptor site-4. Surprisingly, DeltaDeltaBj-xtrIT and DeltaDeltaCss4 were capable of modulating in an allosteric manner the binding and effects of site-3 scorpion alpha-toxins in a way reminiscent of that of brevetoxins, which bind at receptor site-5. While reducing the binding and effect of the scorpion alpha-toxin Lqh2 at mammalian sodium channels, they enhanced the binding and effect of LqhalphaIT at insect sodium channels. Co-application of DeltaDeltaBj-xtrIT or DeltaDeltaCss4 with brevetoxin abolished the brevetoxin effect, although they did not compete in binding. These results denote a novel surface at DeltaDeltaBj-xtrIT and DeltaDeltaCss4 capable of interaction with sodium channels at a site other than sites 3, 4, or 5, which prior to the truncation was masked by the bioactive surface that interacts with receptor site-4. The disclosure of this hidden surface at both beta-toxins may be viewed as an exercise in "reverse evolution," providing a clue as to their evolution from a smaller ancestor of similar scaffold.     

The putative bioactive surface of insect-selective scorpion excitatory neurotoxins

Scorpion neurotoxins of the excitatory group show total specificity for insects and serve as invaluable probes for insect sodium channels. However, despite their significance and potential for application in insect-pest control, the structural basis for their bioactivity is still unknown. We isolated, characterized, and expressed an atypically long excitatory toxin, Bj-xtrIT, whose bioactive features resembled those of classical excitatory toxins, despite only 49% sequence identity. With the objective of clarifying the toxic site of this unique pharmacological group, Bj-xtrIT was employed in a genetic approach using point mutagenesis and biological and structural assays of the mutant products. A primary target for modification was the structurally unique C-terminal region. Sequential deletions of C-terminal residues suggested an inevitable significance of Ile73 and Ile74 for toxicity. Based on the bioactive role of the C-terminal region and a comparison of Bj-xtrIT with a Bj-xtrIT-based model of a classical excitatory toxin, AaHIT, a conserved surface comprising the C terminus is suggested to form the site of recognition with the sodium channel receptor.     

The impact of the fourth disulfide bridge in scorpion toxins of the alpha-KTx6 subfamily

Animal toxins are highly reticulated and structured polypeptides that adopt a limited number of folds. In scorpion species, the most represented fold is the alpha/beta scaffold in which an helical structure is connected to an antiparallel beta-sheet by two disulfide bridges. The intimate relationship existing between peptide reticulation and folding remains poorly understood. Here, we investigated the role of disulfide bridging on the 3D structure of HsTx1, a scorpion toxin potently active on Kv1.1 and Kv1.3 channels. This toxin folds along the classical alpha/beta scaffold but belongs to a unique family of short-chain, four disulfide-bridged toxins. Removal of the fourth disulfide bridge of HsTx1 does not affect its helical structure, whereas its two-stranded beta-sheet is altered from a twisted to a nontwisted configuration. This structural change in HsTx1 is accompanied by a marked decrease in Kv1.1 and Kv1.3 current blockage, and by alterations in the toxin to channel molecular contacts. In contrast, a similar removal of the fourth disulfide bridge of Pi1, another scorpion toxin from the same structural family, has no impact on its 3D structure, pharmacology, or channel interaction. These data highlight the importance of disulfide bridging in reaching the correct bioactive conformation of some toxins.     

作用于钾离子通道蝎毒素的结构特征及活性表面研究进展

对作用于钾离子通道的蝎毒素的空间结构特点进行了简要归纳,发现高含量碱性残基在不同结构单元广泛分布而少量酸性残基特征性分布等新特点。蝎毒素活性表面研究进展表明,利用空间结构的分子模建结合残基突变是确定活性表面的有效方法。基于少量酸性残基特征分布与活性表面取向的相关性,提出酸性残基为活性调节残基的新观点和简单的“拇指”规则预测钾毒素活性表面的方法,从而可望加速蝎毒素的结构与功能关系研究。     

Dissection of the functional surface of an anti-insect excitatory toxin illuminates a putative "hot spot" common to all scorpion beta-toxins affecting Na+ channels

Scorpion beta-toxins affect the activation of voltage-sensitive sodium channels (NaChs). Although these toxins have been instrumental in the study of channel gating and architecture, little is known about their active sites. By using an efficient system for the production of recombinant toxins, we analyzed by point mutagenesis the entire surface of the beta-toxin, Bj-xtrIT, an anti-insect selective excitatory toxin from the scorpion Buthotus judaicus. Each toxin mutant was purified and analyzed using toxicity and binding assays, as well as by circular dichroism spectroscopy to discern the differences among mutations that caused structural changes and those that specifically affected bioactivity. This analysis highlighted a functional discontinuous surface of 1405 A(2), which was composed of a number of non-polar and three charged amino acids clustered around the main alpha-helical motif and the C-tail. Among the charged residues, Glu(30) is a center of a putative "hot spot" in the toxin-receptor binding-interface and is shielded from bulk solvent by a hydrophobic "gasket" (Tyr(26) and Val(34)). Comparison of the Bj-xtrIT structure with that of other beta-toxins that are active on mammals suggests that the hot spot and an adjacent non-polar region are spatially conserved. These results highlight for the first time structural elements that constitute a putative "pharmacophore" involved in the interaction of beta-toxins with receptor site-4 on NaChs. Furthermore, the unique structure of the C-terminal region most likely determines the specificity of excitatory toxins for insect NaChs.     

Trisoxazole macrolide toxins mimic the binding of actin-capping proteins to actin

Marine macrolide toxins of trisoxazole family target actin with high affinity and specificity and have promising pharmacological properties. We present X-ray structures of actin in complex with two members of this family, kabiramide C and jaspisamide A, at a resolution of 1.45 and 1.6 A, respectively. The structures reveal the absolute stereochemistry of these toxins and demonstrate that their trisoxazole ring interacts with actin subdomain 1 while the aliphatic side chain is inserted into the hydrophobic cavity between actin subdomains 1 and 3. The binding site is essentially the same as the one occupied by the actin-capping domain of the gelsolin superfamily of proteins. The structural evidence suggests that actin filament severing and capping by these toxins is also analogous to that of gelsolin. Consequently, these macrolides may be viewed as small molecule biomimetics of an entire class of actin-binding proteins.     

Common features in the functional surface of scorpion beta-toxins and elements that confer specificity for insect and mammalian voltage-gated sodium channels

Scorpion beta-toxins that affect the activation of mammalian voltage-gated sodium channels (Navs) have been studied extensively, but little is known about their functional surface and mode of interaction with the channel receptor. To enable a molecular approach to this question, we have established a successful expression system for the anti-mammalian scorpion beta-toxin, Css4, whose effects on rat brain Navs have been well characterized. A recombinant toxin, His-Css4, was obtained when fused to a His tag and a thrombin cleavage site and had similar binding affinity for and effect on Na currents of rat brain sodium channels as those of the native toxin isolated from the scorpion venom. Molecular dissection of His-Css4 elucidated a functional surface of 1245 A2 composed of the following: 1) a cluster of residues associated with the alpha-helix, which includes a putative "hot spot" (this cluster is conserved among scorpion beta-toxins and contains their "pharmacophore"); 2) a hydrophobic cluster associated mainly with the beta2 and beta3 strands, which is likely to confer the specificity for mammalian Navs; 3) a single bioactive residue (Trp-58) in the C-tail; and 4) a negatively charged residue (Glu-15) involved in voltage sensor trapping as inferred from our ability to uncouple toxin binding from activity upon its substitution. This study expands our understanding about the mode of action of scorpion beta-toxins and illuminates differences in the functional surfaces that may dictate their specificities for mammalian versus insect sodium channels.     

Phospholipase-D activity and inflammatory response induced by brown spider dermonecrotic toxin: endothelial cell membrane phospholipids as targets for toxicity

Brown spider dermonecrotic toxins (phospholipases-D) are the most well-characterized biochemical constituents of Loxosceles spp. venom. Recombinant forms are capable of reproducing most cutaneous and systemic manifestations such as dermonecrotic lesions, hematological disorders, and renal failure. There is currently no direct confirmation for a relationship between dermonecrosis and inflammation induced by dermonecrotic toxins and their enzymatic activity. We modified a toxin isoform by site-directed mutagenesis to determine if phospholipase-D activity is directly related to these biological effects. The mutated toxin contains an alanine substitution for a histidine residue at position 12 (in the conserved catalytic domain of Loxosceles intermedia Recombinant Dermonecrotic Toxin - LiRecDT1). LiRecDT1H12A sphingomyelinase activity was drastically reduced, despite the fact that circular dichroism analysis demonstrated similar spectra for both toxin isoforms, confirming that the mutation did not change general secondary structures of the molecule or its stability. Antisera against whole venom and LiRecDT1 showed cross-reactivity to both recombinant toxins by ELISA and immunoblotting. Dermonecrosis was abolished by the mutation, and rabbit skin revealed a decreased inflammatory response to LiRecDT1H12A compared to LiRecDT1. Residual phospholipase activity was observed with increasing concentrations of LiRecDT1H12A by dermonecrosis and fluorometric measurement in vitro. Lipid arrays showed that the mutated toxin has an affinity for the same lipids LiRecDT1, and both toxins were detected on RAEC cell surfaces. Data from in vitro choline release and HPTLC analyses of LiRecDT1-treated purified phospholipids and RAEC membrane detergent-extracts corroborate with the morphological changes. These data suggest a phospholipase-D dependent mechanism of toxicity, which has no substrate specificity and thus utilizes a broad range of bioactive lipids.     

Lachesis muta (Viperidae) cDNAs reveal diverging pit viper molecules and scaffolds typical of cobra (Elapidae) venoms: implications for snake toxin repertoire evolution

Efforts to describe toxins from the two major families of venomous snakes (Viperidae and Elapidae) usually reveal proteins belonging to few structural types, particular of each family. Here we carried on an effort to determine uncommon cDNAs that represent possible new toxins from Lachesis muta (Viperidae). In addition to nine classes of typical toxins, atypical molecules never observed in the hundreds of Viperidae snakes studied so far are highly expressed: a diverging C-type lectin that is related to Viperidae toxins but appears to be independently originated; an ohanin-like toxin, which would be the third member of the most recently described class of Elapidae toxins, related to human butyrophilin and B30.2 proteins; and a 3FTx-like toxin, a new member of the widely studied three-finger family of proteins, which includes major Elapidae neurotoxins and CD59 antigen. The presence of these common and uncommon molecules suggests that the repertoire of toxins could be more conserved between families than has been considered, and their features indicate a dynamic process of venom evolution through molecular mechanisms, such as multiple recruitments of important scaffolds and domain exchange between paralogs, always keeping a minimalist nature in most toxin structures in opposition to their nontoxin counterparts.     

Crystal structures of protein phosphatase-1 bound to motuporin and dihydromicrocystin-LA: elucidation of the mechanism of enzyme inhibition by cyanobacterial toxins

The microcystins and nodularins are tumour promoting hepatotoxins that are responsible for global adverse human health effects and wildlife fatalities in countries where drinking water supplies contain cyanobacteria. The toxins function by inhibiting broad specificity Ser/Thr protein phosphatases in the host cells, thereby disrupting signal transduction pathways. A previous crystal structure of a microcystin bound to the catalytic subunit of protein phosphatase-1 (PP-1c) showed distinct changes in the active site region when compared with protein phosphatase-1 structures bound to other toxins. We have elucidated the crystal structures of the cyanotoxins, motuporin (nodularin-V) and dihydromicrocystin-LA bound to human protein phosphatase-1c (gamma isoform). The atomic structures of these complexes reveal the structural basis for inhibition of protein phosphatases by these toxins. Comparisons of the structures of the cyanobacterial toxin:phosphatase complexes explain the biochemical mechanism by which microcystins but not nodularins permanently modify their protein phosphatase targets by covalent addition to an active site cysteine residue.     

Expression and mutagenesis of the sea anemone toxin Av2 reveals key amino acid residues important for activity on voltage-gated sodium channels

Type I sea anemone toxins are highly potent modulators of voltage-gated Na-channels (Na(v)s) and compete with the structurally dissimilar scorpion alpha-toxins on binding to receptor site-3. Although these features provide two structurally different probes for studying receptor site-3 and channel fast inactivation, the bioactive surface of sea anemone toxins has not been fully resolved. We established an efficient expression system for Av2 (known as ATX II), a highly insecticidal sea anemone toxin from Anemonia viridis (previously named A. sulcata), and mutagenized it throughout. Each toxin mutant was analyzed in toxicity and binding assays as well as by circular dichroism spectroscopy to discern the effects derived from structural perturbation from those related to bioactivity. Six residues were found to constitute the anti-insect bioactive surface of Av2 (Val-2, Leu-5, Asn-16, Leu-18, and Ile-41). Further analysis of nine Av2 mutants on the human heart channel Na(v)1.5 expressed in Xenopus oocytes indicated that the bioactive surfaces toward insects and mammals practically coincide but differ from the bioactive surface of a structurally similar sea anemone toxin, Anthopleurin B, from Anthopleura xanthogrammica. Hence, our results not only demonstrate clear differences in the bioactive surfaces of Av2 and scorpion alpha-toxins but also indicate that despite the general conservation in structure and importance of the Arg-14 loop and its flanking residues Gly-10 and Gly-20 for function, the surface of interaction between different sea anemone toxins and Na(v)s varies.     

Structural analysis of the Laetiporus sulphureus hemolytic pore-forming lectin in complex with sugars

LSL is a lectin produced by the parasitic mushroom Laetiporus sulphureus, which exhibits hemolytic and hemagglutinating activities. Here, we report the crystal structure of LSL refined to 2.6-A resolution determined by the single isomorphous replacement method with the anomalous scatter (SIRAS) signal of a platinum derivative. The structure reveals that LSL is hexameric, which was also shown by analytical ultracentrifugation. The monomeric protein (35 kDa) consists of two distinct modules: an N-terminal lectin module and a pore-forming module. The lectin module has a beta-trefoil scaffold that bears structural similarities to those present in toxins known to interact with galactose-related carbohydrates such as the hemagglutinin component (HA1) of the progenitor toxin from Clostridium botulinum, abrin, and ricin. On the other hand, the C-terminal pore-forming module (composed of domains 2 and 3) exhibits three-dimensional structural resemblances with domains 3 and 4 of the beta-pore-forming toxin aerolysin from the Gram-negative bacterium Aeromonas hydrophila, and domains 2 and 3 from the epsilon-toxin from Clostridium perfringens. This finding reveals the existence of common structural elements within the aerolysin-like family of toxins that could be directly involved in membrane-pore formation. The crystal structures of the complexes of LSL with lactose and N-acetyllactosamine reveal two dissacharide-binding sites per subunit and permits the identification of critical residues involved in sugar binding.     

Looking over toxin-K(+) channel interactions. Clues from the structural and functional characterization of α-KTx toxin Tc32, a Kv1.3 channel blocker

α-KTx toxin Tc32, from the Amazonian scorpion Tityus cambridgei, lacks the dyad motif, including Lys27, characteristic of the family and generally associated with channel blockage. The toxin has been cloned and expressed for the first time. Electrophysiological experiments, by showing that the recombinant form blocks Kv1.3 channels of olfactory bulb periglomerular cells like the natural Tc32 toxin, when tested on the Kv1.3 channel of human T lymphocytes, confirmed it is in an active fold. The nuclear magnetic resonance-derived structure revealed it exhibits an α/β scaffold typical of the members of the α-KTx family. TdK2 and TdK3, all belonging to the same α-KTx 18 subfamily, share significant sequence identity with Tc32 but diverse selectivity and affinity for Kv1.3 and Kv1.1 channels. To gain insight into the structural features that may justify those differences, we used the recombinant Tc32 nuclear magnetic resonance-derived structure to model the other two toxins, for which no experimental structure is available. Their interaction with Kv1.3 and Kv1.1 has been investigated by means of docking simulations. The results suggest that differences in the electrostatic features of the toxins and channels, in their contact surfaces, and in their total dipole moment orientations govern the affinity and selectivity of toxins. In addition, we found that, regardless of whether the dyad motif is present, it is always a Lys side chain that physically blocks the channels, irrespective of its position in the toxin sequence.     

Chironex fleckeri (Box Jellyfish) Venom Proteins: EXPANSION OF A CNIDARIAN TOXIN FAMILY THAT ELICITS VARIABLE CYTOLYTIC AND CARDIOVASCULAR EFFECTS*

The box jellyfish Chironex fleckeri produces extremely potent and rapid-acting venom that is harmful to humans and lethal to prey. Here, we describe the characterization of two C. fleckeri venom proteins, CfTX-A (∼40 kDa) and CfTX-B (∼42 kDa), which were isolated from C. fleckeri venom using size exclusion chromatography and cation exchange chromatography. Full-length cDNA sequences encoding CfTX-A and -B and a third putative toxin, CfTX-Bt, were subsequently retrieved from a C. fleckeri tentacle cDNA library. Bioinformatic analyses revealed that the new toxins belong to a small family of potent cnidarian pore-forming toxins that includes two other C. fleckeri toxins, CfTX-1 and CfTX-2. Phylogenetic inferences from amino acid sequences of the toxin family grouped CfTX-A, -B, and -Bt in a separate clade from CfTX-1 and -2, suggesting that the C. fleckeri toxins have diversified structurally and functionally during evolution. Comparative bioactivity assays revealed that CfTX-1/2 (25 μg kg⁻¹) caused profound effects on the cardiovascular system of anesthetized rats, whereas CfTX-A/B elicited only minor effects at the same dose. Conversely, the hemolytic activity of CfTX-A/B (HU₅₀ = 5 ng ml⁻¹) was at least 30 times greater than that of CfTX-1/2. Structural homology between the cubozoan toxins and insecticidal three-domain Cry toxins (δ-endotoxins) suggests that the toxins have a similar pore-forming mechanism of action involving α-helices of the N-terminal domain, whereas structural diversification among toxin members may modulate target specificity. Expansion of the cnidarian toxin family therefore provides new insights into the evolutionary diversification of box jellyfish toxins from a structural and functional perspective.     

Structural basis for the voltage-gated Na+ channel selectivity of the scorpion alpha-like toxin BmK M1

Scorpion alpha-like toxins are proteins that act on mammalian and insect voltage-gated Na+ channels. Therefore, these toxins constitute an excellent target for examining the foundations that underlie their target specificity. With this motive we dissected the role of six critical amino acids located in the five-residue reverse turn (RT) and C-tail (CT) of the scorpion alpha-like toxin BmK M1. These residues were individually substituted resulting in 11 mutants and were subjected to a bioassay on mice, an electrophysiological characterization on three cloned voltage-gated Na+ channels (Nav1.2, Nav1.5 and para), a CD analysis and X-ray crystallography. The results reveal two molecular sites, a couplet of residues (8-9) in the RT and a hydrophobic surface consisting of residues 57 and 59-61 in the CT, where the substitution with specific residues can redirect the alpha-like characteristics of BmK M1 to either total insect or much higher mammal specificity. Crystal structures reveal that the pharmacological ramification of these mutants is accompanied by the reshaping of the 3D structure surrounding position 8. Furthermore, our results also reveal that residues 57 and 59-61, located at the CT, enclose the critical residue 58 in order to form a hydrophobic "gasket". Mutants of BmK M1 that interrupt this hydrophobic surface significantly gain insect selectivity.     

The crystal structure of exfoliative toxin B: a superantigen with enzymatic activity.

The exfoliative toxins (ETs) cause staphylococcal scalded skin syndrome, a disease characterized by specific separation of layers of the skin. Evidence suggests that the toxins act as serine proteases, though the specific substrate and mode of action are not known for certain. The crystal structure of exfoliative toxin A (ETA) was reported earlier and shown to be similar to that of the chymotrypsin-like serine proteases. Here, we report the 2.4 A resolution crystal structure of the other exfoliative toxin, ETB, which is 40% identical to ETA. The overall structures of ETA and ETB are similar including the positions of key residues within the active site. The structure of ETB supports the previous findings that the ETs are serine proteases that cleave substrates after glutamic acid residues. In this study we also discuss a number of structural differences including a large 14 residue loop insertion which may be a key feature involved in the differing biological properties of the ETs, particularly the pyrogenic and lethal activities of ETB not shared by ETA.     

Dynamic Diversification from a Putative Common Ancestor of Scorpion Toxins Affecting Sodium, Potassium, and Chloride Channels

Scorpions have survived successfully over millions of years without detectable changes in their morphology. Instead, they have developed an efficient alomonal machinery and a stinging device supporting their needs for prey and defense. They produce a large variety of polypeptidic toxins that bind and modulate ion channel conductance in excitable tissues. The binding site, mode of action, and chemical properties of many toxins have been studied extensively, but little is known about their genomic organization and diversity. Genes representing each of the major classes of Buthidae scorpion toxins, namely, ``long' toxins, affecting sodium channels (alpha, depressant, and excitatory), and ``short' toxins, affecting potassium and chloride channels, were isolated from a single scorpion segment and analyzed. Each toxin type was found to be encoded by a gene family. Regardless of toxin length, 3-D structure, and site of action, all genes contain A+T-rich introns that split, at a conserved location, an amino acid codon of the signal sequence. The introns vary in length and sequence but display identical boundaries, agree with the GT/AG splice junctions, and contain T-runs downstream of a putative branch point, 5′-TAAT-3′. Despite little sequence similarity among all toxin classes, the conserved gene organization, intron features, and common cysteine-stabilized α-helical (CSH) core connecting an α-helix to a three-stranded β-sheet suggest, that they all evolved from an ancestral common progenitor. Furthermore, the vast diversity found among genomic copies, cDNAs, and their protein products for each toxin suggests an extensive evolutionary process of the scorpion ``pharmaceutical factory,' whose success is due, most likely, to the inherent permissiveness of the toxin exterior to structural alterations. Received: 16 March 1998 / Accepted: 30 July 1998     

Computer modelling of the NAD binding site of ADP-ribosylating toxins: active-site structure and mechanism of NAD binding

Five ADP-ribosylating bacterial toxins, pertussis toxin, cholera toxin, diphtheria toxin, Escherichia LT toxin and Pseudomonas exotoxin A, show significant homology in selected segments of their sequence. Site-directed mutagenesis and chemical modification of residues within these regions cause loss of catalytic activity and of NAD binding. On the basis of these results and of molecular modelling based on the three-dimensional structure of exotoxin A, the geometry of an NAD binding site common to all the toxins is deduced and described in the paper. For diphtheria toxin, sequence similarity with exotoxin A is such that its preliminary structure can be computed by molecular modelling, whereas for the other toxins similarity appears to be restricted to the NAD binding site. Moreover, an analysis of molecular fitting of the NAD molecule into its binding cavity suggests a new model for the conformation of the bound NAD that better accounts for all available experimental information.     

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.