Solution structure of toxin 2 from Centruroides noxius Hoffmann, a β-scorpion neurotoxin acting on sodium channels (original) (raw)
Related papers
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.
A new type of scorpion Na+-channel-toxin-like polypeptide active on K+ channels
Biochemical Journal, 2005
We have purified and characterized two peptides, named KAaH1 and KAaH2 (AaH polypeptides 1 and 2 active on K + channels, where AaH stands for Androctonus australis Hector), from the venom of A. australis Hector scorpions. Their sequences contain 58 amino acids including six half-cysteines and differ only at positions 26 (Phe/Ser) and 29 (Lys/Gln). Although KAaH1 and KAaH2 show important sequence similarity with anti-mammal β toxins specific for voltage-gated Na + channels, only weak β-like effects were observed when KAaH1 or KAaH2 (1 µM) were tested on brain Nav1.2 channels. In contrast, KAaH1 blocks Kv1.1 and Kv1.3 channels expressed in Xenopus oocytes with IC 50 values of 5 and 50 nM respectively, whereas KAaH2 blocks only 20 % of the current on Kv1.1 and is not active on Kv1.3 channels at a 100 nM concentration. KAaH1 is thus the first member of a new subfamily of long-chain toxins mainly active on voltage-gated K + channels. NMR spectra of KAaH1 and KAaH2 show good dispersion of signals but broad lines and poor quality. Self-diffusion NMR experiments indicate that lines are broadened due to a conformational exchange on the millisecond time scale. NMR and CD indicate that both polypeptides adopt a similar fold with α-helical and β-sheet structures. Homology-based molecular models generated for KAaH1 and KAaH2 are in accordance with CD and NMR data. In the model of KAaH1, the functionally important residues Phe 26 and Lys 29 are close to each other and are located in the α-helix. These residues may constitute the so-called functional dyad observed for short α-KTx scorpion toxins in the β-sheet.
European journal of biochemistry, 1989
Chemical modifications of tyrosine and tryptophan residues of scorpion a-neurotoxins I1 and I11 from Androctonus australis Hector were performed as well as modification of the two arginines and the a-amino group of toxin I. The pharmacological potencies of each derivative were assessed in vivo by LDS0 measurement and in vitro by competition experiments with '251-toxin for synaptosomal receptors. Arginine residues in positions 2 and 60 and the a-amino group of Androctonus toxin 1 were derivatized by p-hydroxyphenylglyoxal; the corresponding modified toxins exhibit low pharmacological potencies. Tryptophan 38 of toxin I1 and tryptophan 45 of toxin 111 were modified by nitrophenylsulfenyl chloride, leading respectively to a poorly and a fully active derivative. The tetranitromethane modification of tyrosine residues in positions 60, 5 and 14 of toxin 111 induced respectively 60%, 40% and 30% of loss of biological activity. Circular dichroic analysis indicated that for every derivative, except the nitrophenylsulfenyl derivative of Trp-45 of AaH 111, the conformation of the toxin was not altered by derivatization. Conformational integrity was also confirmed by full activity of the derivatives in radioimmunoassays. Taken together, the results suggest that aromatic residues belonging to the conserved hydrophobic surface, to the C-terminal and to the loop region 37-44 are involved in the molecular mechanisms by which scorpion atoxins act. Charged residues in the N-terminal and C-terminal also contribute to the high efficacy of the binding process. It appears that all important residues are clustered on one face of the toxin, suggesting a multipoint interaction with the proteins of the sodium channel. Scorpion toxins are known to interact specifically with the voltage-dependent sodium channel 11, 21. They form a family of structurally related proteins made of a single polypeptide chain and having a molecular mass of about 7 kDa (60-70 amino acid residues) [3] (for a review on isolation and structure, see [4]). They can be divided into mammal and insect toxins according to their specificity toward mammalian and insect nervous systems [5]. Two types (a and p) of mammal scorpion toxins have been described according to their pharmacological properties and their binding to two different sites [6]. These toxins act at the level of the voltage-dependent sodium channel in different ways. Thus a-toxins bind to the receptor in a potential-dependent manner whereas p-toxins do not; a-toxins induce a prolongation of the repolarization phase of the action potential while p-toxins promote a repetitive firing after a unique stimulation [7]. The three-dimensional structures of a protein from the venom of Centruroides sculpturatus Ewing (CsE V,) [8] and of toxin I1 from the venom of Androctonus australis Hector (AaH 11) [9] were elucidated at high resolution by X-ray crystallography and may be taken as structural models for respectively Pand a-toxins. In both cases an a-helix structure and three short strands of P-sheet are found in similar regions.
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.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 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.
Journal of Molecular Biology, 1999
The crystal structures of two group III a-like toxins from the scorpion Buthus martensii Karsch, BmK M1 and BmK M4, were determined at 1.7 A Ê and 1.3 A Ê resolution and re®ned to R factors of 0.169 and 0.166, respectively. The ®rst high-resolution structures of the a-like scorpion toxin show some striking features compared with structures of thè`c lassical'' a-toxin. Firstly, a non-proline cis peptide bond between residues 9 and 10 unusually occurs in the ®ve-member reverse turn 8-12. Secondly, the cis peptide 9-10 mediates the spatial relationship between the turn 8-12 and the C-terminal stretch 58-64 through a pair of mainchain hydrogen bonds between residues 10 and 64 to form a unique tertiary arrangement which features the special orientation of the terminal residues 62-64. Finally, in consequence of the peculiar orientation of the C-terminal residues, the functional groups of Arg58, which are crucial for the toxin-receptor interaction, are exposed and accessible in BmK M1 and M4 rather than buried as in the classical a-toxins. Sequence alignment and characteristics analysis suggested that the above structural features observed in BmK M1 and M4 occur in all group III a-like toxins. Recently, some group III a-like toxins were demonstrated to occupy a receptor site different from the classical a-toxin. Therefore, the distinct structural features of BmK M1 and M4 presented here may provide the structural basis for the newly recognized toxin-receptor binding site selectivity. Besides, the non-proline cis peptide bonds found in these two structures play a role in the formation of the structural characteristics and in keeping accurate positions of the functionally crucial residues. This manifested a way to achieve high levels of molecular speci®city and atomic precision through the strained backbone geometry.
Journal of Molecular Biology, 1999
The crystal structures of two group III a-like toxins from the scorpion Buthus martensii Karsch, BmK M1 and BmK M4, were determined at 1.7 A Ê and 1.3 A Ê resolution and re®ned to R factors of 0.169 and 0.166, respectively. The ®rst high-resolution structures of the a-like scorpion toxin show some striking features compared with structures of thè`c lassical'' a-toxin. Firstly, a non-proline cis peptide bond between residues 9 and 10 unusually occurs in the ®ve-member reverse turn 8-12. Secondly, the cis peptide 9-10 mediates the spatial relationship between the turn 8-12 and the C-terminal stretch 58-64 through a pair of mainchain hydrogen bonds between residues 10 and 64 to form a unique tertiary arrangement which features the special orientation of the terminal residues 62-64. Finally, in consequence of the peculiar orientation of the C-terminal residues, the functional groups of Arg58, which are crucial for the toxin-receptor interaction, are exposed and accessible in BmK M1 and M4 rather than buried as in the classical a-toxins. Sequence alignment and characteristics analysis suggested that the above structural features observed in BmK M1 and M4 occur in all group III a-like toxins. Recently, some group III a-like toxins were demonstrated to occupy a receptor site different from the classical a-toxin. Therefore, the distinct structural features of BmK M1 and M4 presented here may provide the structural basis for the newly recognized toxin-receptor binding site selectivity. Besides, the non-proline cis peptide bonds found in these two structures play a role in the formation of the structural characteristics and in keeping accurate positions of the functionally crucial residues. This manifested a way to achieve high levels of molecular speci®city and atomic precision through the strained backbone geometry.
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.
Solution Structure of a β-Neurotoxin from the New World ScorpionCentruroides sculpturatusEwing
Biochemical and Biophysical Research Communications, 1999
We report the detailed solution structure of the 7.2 kDa protein CsE-I, a -neurotoxin from the New World scorpion Centruroides sculpturatus Ewing. This toxin binds to sodium channels, but unlike the ␣-neurotoxins, shifts the voltage of activation toward more negative potentials causing the membrane to fire spontaneously. Sequence-specific proton NMR assignments were made using 600 MHz 2D-NMR data. Distance geometry and dynamical simulated annealing refinements were performed using experimental distance and torsion angle constraints from NOESY and pH-COSY data. A family of 40 structures without constraint violations was generated, and an energy-minimized average structure was computed. The backbone conformation of the CsE-I toxin shows similar secondary structural features as the prototypical ␣-neurotoxin, CsE-v3, and is characterized by a short 2½-turn ␣-helix and a 3-strand antiparallel -sheet, both held together by disulfide bridges. The RMSD for the backbone atoms between CsE-I and CsE-v3 is 1.48 Å. Despite this similarity in the overall backbone folding, the these two proteins show some important differences in the primary structure (sequence) and electrostatic potential surfaces. Our studies provide a basis for unravelling the role of these differences in relation to the known differences in the receptor sites on the voltage sensitive sodium channel for the ␣and -neurotoxins.