Solution structure of native and recombinant expressed toxin CssII from the venom of the scorpion Centruroides suffusus suffusus, and their effects on Nav1.5 Sodium channels (original) (raw)
Related papers
2012
The three-dimensional structures of the long-chain mammalian scorpion β-toxin CssII from Centruroides suffusus suffusus and of its recombinant form, HisrCssII, were determined by NMR. The neurotoxin CssII (nCssII) is a 66 amino acid long peptide with four disulfide bridges; it is the most abundant and deadly toxin from the venom of this scorpion. Both native and recombinant CssII structures were determined by nuclear magnetic resonance using a total of 828 sequential distance constraints derived from the volume integration of the cross peaks observed in 2D NOESY spectra. Both nCssII and HisrCssII structures display a mixed α/β fold stabilized by four disulfide bridges formed between pairs of cysteines: C1-C8, C2-C5, C3-C6, and C4-C7 (the numbers indicate the relative positions of the cysteine residues in the primary structure), with a distortion induced by two cis-prolines in its C-terminal part. The native CssII electrostatic surface was compared to both the recombinant one and to the Cn2 toxin, from the scorpion Centruroides noxius, which is also toxic to mammals. Structural features such N-and C-terminal differences could influence toxin specificity and affinity towards isoforms of different sub-types of Na v channels.
J Mol Biol, 1999
We have determined the solution structure of Cn2, a β-toxin extracted from the venom of the New World scorpion Centruroides noxius Hoffmann. Cn2 belongs to the family of scorpion toxins that affect the sodium channel activity, and is very toxic to mammals (LD50 = 0.4 μg/20 g mouse mass). The three-dimensional structure was determined using 1H-1H two-dimensional NMR spectroscopy, torsion angle dynamics, and restrained energy minimization. The final set of 15 structures was calculated from 876 experimental distance constraints and 58 angle constraints. The structures have a global r.m.s.d. of 1.38 Å for backbone atoms and 2.21 Å for all heavy atoms. The overall fold is similar to that found in the other scorpion toxins acting on sodium channels. It is made of a triple-stranded antiparallel β-sheet and an α-helix, and is stabilized by four disulfide bridges. A cis-proline residue at position 59 induces a kink of the polypeptide chain in the C-terminal region. The hydrophobic core of the protein is made up of residues L5, V6, L51, A55, and by the eight cysteine residues. A hydrophobic patch is defined by the aromatic residues Y4, Y40, Y42, W47 and by V57 on the side of the β-sheet facing the solvent. A positively charged patch is formed by K8 and K63 on one edge of the molecule in the C-terminal region. Another positively charged spot is represented by the highly exposed K35. The structure of Cn2 is compared with those of other scorpion toxins acting on sodium channels, in particular Aah II and CsE-v3. This is the first structural report of an anti-mammal β-scorpion toxin and it provides the necessary information for the design of recombinant mutants that can be used to probe structure-function relationships in scorpion toxins affecting sodium channel activity.
Journal of Molecular Biology, 1999
We have determined the solution structure of Cn2, a b-toxin extracted from the venom of the New World scorpion Centruroides noxius Hoffmann. Cn2 belongs to the family of scorpion toxins that affect the sodium channel activity, and is very toxic to mammals (LD 50 0.4 mg/20 g mouse mass). The three-dimensional structure was determined using 1 H-1 H two-dimensional NMR spectroscopy, torsion angle dynamics, and restrained energy minimization. The ®nal set of 15 structures was calculated from 876 experimental distance constraints and 58 angle constraints. The structures have a global r.m.s.d. of 1.38 A Ê for backbone atoms and 2.21 A Ê for all heavy atoms. The overall fold is similar to that found in the other scorpion toxins acting on sodium channels. It is made of a triple-stranded antiparallel b-sheet and an a-helix, and is stabilized by four disul®de bridges. A cis-proline residue at position 59 induces a kink of the polypeptide chain in the C-terminal region. The hydrophobic core of the protein is made up of residues L5, V6, L51, A55, and by the eight cysteine residues. A hydrophobic patch is de®ned by the aromatic residues Y4, Y40, Y42, W47 and by V57 on the side of the b-sheet facing the solvent. A positively charged patch is formed by K8 and K63 on one edge of the molecule in the C-terminal region. Another positively charged spot is represented by the highly exposed K35. The structure of Cn2 is compared with those of other scorpion toxins acting on sodium channels, in particular Aah II and CsE-v3. This is the ®rst structural report of an anti-mammal b-scorpion toxin and it provides the necessary information for the design of recombinant mutants that can be used to probe structure-function relationships in scorpion toxins affecting sodium channel activity.
Journal of Biological Chemistry, 2004
Scorpion -toxins that affect the activation of mammalian voltage-gated sodium channels (Na v s) 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 -toxin, Css4, whose effects on rat brain Na v s 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 Å 2 composed of the following: 1) a cluster of residues associated with the ␣-helix, which includes a putative "hot spot" (this cluster is conserved among scorpion -toxins and contains their "pharmacophore"); 2) a hydrophobic cluster associated mainly with the 2 and 3 strands, which is likely to confer the specificity for mammalian Na v s; 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 -toxins and illuminates differences in the functional surfaces that may dictate their specificities for mammalian versus insect sodium channels.
Journal of Biological Chemistry, 2013
Background: Scorpion ␣-toxins affect voltage-gated sodium channels in both mammals and insects. Results: We perform thorough computational analyses of ␣-toxin molecular architecture and structure-function relationship. Conclusion: Taxon specificity of "orphan" toxins can be predicted from a structural perspective. Significance: The proposed surface mapping technique is a new tool to analyze protein-protein complexes. The abbreviations used are: Na v , voltage-gated sodium channel; MD, molecular dynamics; MHP, molecular hydrophobicity potential; PD, pore domain; RMSF, root mean square fluctuation; RMSF-NM, RMSF along the first eigenvector in the normal mode analysis; SM, specificity module; Trx, thioredoxin; VSD, voltage-sensing domain; RT, reverse turn.
Biochemistry, 2008
Voltage-gated sodium channels (Na v s) are large transmembrane proteins that initiate action potential in electrically excitable cells. This central role in the nervous system has made them a primary target for a large number of neurotoxins. Scorpion R-neurotoxins bind to Na v s with high affinity and slow their inactivation, causing a prolonged action potential. Despite the similarity in their mode of action and three-dimensional structure, R-toxins exhibit great variations in selectivity toward insect and mammalian Na v s, suggesting differences in the binding surfaces of the toxins and the channels. The scorpion R-toxin binding site, termed neurotoxin receptor site 3, has been shown to involve the extracellular S3-S4 loop in domain 4 of the R-subunit of voltage-gated sodium channels (D4/S3-S4). In this study, the binding site for peptides corresponding to the D4/S3-S4 loop of the para insect Na v was mapped on the highly insecticidal R-neurotoxin, LqhRIT, from the scorpion Leiurus quinquestriatus hebraeus, by following changes in the toxin amide 1 H and 15 N chemical shifts upon binding. This analysis suggests that the five-residue turn (residues Lq K8-Lq C12) of LqhRIT and those residues in its vicinity interact with the D4/S3-S4 loop of Na v . Residues Lq R18, Lq W38, and Lq A39 could also form a patch contributing to the interaction with D4/S3-S4. Moreover, a new bioactive residue, Lq V13, was identified as being important for Na v binding and specifically for the interaction with the D4/S3-S4 loop. The contribution of Lq V13 to Na V binding was further verified by mutagenesis. Future studies involving other extracellular regions of Na v s are required for further characterization of the structure of the LqhRIT-Na v s binding site.
Scorpion toxins specific for Na+-channels
European Journal of Biochemistry, 1999
Na + -channel specific scorpion toxins are peptides of 60±76 amino acid residues in length, tightly bound by four disulfide bridges. The complete amino acid sequence of 85 distinct peptides are presently known. For some toxins, the three-dimensional structure has been solved by X-ray diffraction and NMR spectroscopy. A constant structural motif has been found in all of them, consisting of one or two short segments of a-helix plus a triplestranded b-sheet, connected by variable regions forming loops (turns). Physiological experiments have shown that these toxins are modifiers of the gating mechanism of the Na + -channel function, affecting either the inactivation (a-toxins) or the activation (b-toxins) kinetics of the channels. Many functional variations of these peptides have been demonstrated, which include not only the classical a-and b-types, but also the species specificity of their action. There are peptides that bind or affect the function of Na + -channels from different species (mammals, insects or crustaceans) or are toxic to more than one group of animals. Based on functional and structural features of the known toxins, a classification containing 10 different groups of toxins is proposed in this review. Attempts have been made to correlate the presence of certain amino acid residues or`active sites' of these peptides with Na + -channel functions. Segments containing positively charged residues in special locations, such as the five-residue turn, the turn between the second and the third b-strands, the C-terminal residues and a segment of the N-terminal region from residues 2±11, seems to be implicated in the activity of these toxins. However, the uncertainty, and the limited success obtained in the search for the site through which these peptides bind to the channels, are mainly due to the lack of an easy method for expression of cloned genes to produce a well-folded, active peptide. Many scorpion toxin coding genes have been obtained from cDNA libraries and from polymerase chain reactions using fragments of scorpion DNAs, as templates. The presence of an intron at the DNA level, situated in the middle of the signal peptide, has been demonstrated.
Toxicon, 2005
Scorpion venoms contain a large number of bioactive components. Several of the long-chain peptides were shown to be responsible for neurotoxic effects, due to their ability to recognize Na C channels and to cause impairment of channel functions. Here, we revisited the basic paradigms in the study of these peptides in the light of recent data concerning their structure-function relationships, their functional divergence and extant biodiversity. The reviewed topics include: the criteria for classification of long-chain peptides according to their function, and a revision of the state-of-the-art knowledge concerning the surface areas of contact of these peptides with known Na C channels. Additionally, we compiled a comprehensive list encompassing 191 different amino acid sequences from long-chain peptides purified from scorpion venoms. With this dataset, a phylogenetic tree was constructed and discussed taking into consideration their documented functional divergence. A critical view on problems associated with the study of these scorpion peptides is presented, drawing special attention to the points that need revision and to the subjects under intensive research at this moment, regarding scorpion toxins specific for Na C channels and the other related long-chain peptides recently described.
Molecular Requirements for Recognition of Brain Voltage-gated Sodium Channels by Scorpion α-Toxins
Journal of Biological Chemistry, 2009
The scorpion ␣-toxin Lqh2 (from Leiurus quinquestriatus hebraeus) is active at various mammalian voltage-gated sodium channels (Na v s) and is inactive at insect Na v s. To resolve the molecular basis of this preference we used the following strategy: 1) Lqh2 was expressed in recombinant form and key residues important for activity at the rat brain channel rNa v 1.2a were identified by mutagenesis. These residues form a bipartite functional surface made of a conserved "core domain" (residues of the loops connecting the secondary structure elements of the molecule core), and a variable "NC domain" (five-residue turn and the C-tail) as was reported for other scorpion ␣-toxins. 2) The functional role of the two domains was validated by their stepwise construction on the similar scaffold of the anti-insect toxin Lqh␣IT. Analysis of the activity of the intermediate constructs highlighted the critical role of Phe 15 of the core domain in toxin potency at rNa v 1.2a, and has suggested that the shape of the NC-domain is important for toxin efficacy. 3) Based on these findings and by comparison with other scorpion ␣-toxins we were able to eliminate the activity of Lqh2 at rNa v 1.4 (skeletal muscle), hNa v 1.5 (cardiac), and rNa v 1.6 channels, with no hindrance of its activity at Na v 1.1-1.3. These results suggest that by employing a similar approach the design of further target-selective sodium channel modifiers is imminent.