The Putative Bioactive Surface of Insect-selective Scorpion Excitatory Neurotoxins

Abstract

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 Ile 73 and Ile 74 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.

Common Features in the Functional Surface of Scorpion β-Toxins and Elements That Confer Specificity for Insect and Mammalian Voltage-gated Sodium Channels

“…However, large quantities of recombinant toxin, His-Css4, were obtained using the expression vector pET-14b (see "Materials and Methods"). The recombinant protein was recovered from inclusion bodies, folded in vitro, and purified using a procedure similar to that reported for the excitatory, anti-insect ␤-toxin Bj-xtrIT (9). Analysis of the amino acid composition of His-Css4 was compatible with the deduced sequence of the construct made of Css4 with an N-terminal addition containing a His tag and a thrombin cleavage site (9905 Da).…”

“…DNA Manipulations-A synthetic Css4 gene was constructed using eight overlapping oligonucleotide primers designed with a codon usage based on the nucleotide sequences of long-chain scorpion toxin genes that have been successfully expressed in E. coli (Table I) (9,17,18). A mixture of 1 M of each of primers 1-8 (Table I) heated to 95°C was cooled stepwise to 4°C for annealing.…”

“…Bacterial Strains-Escherichia coli DH5␣ was used for plasmid constructions, and the BL21 strain was used for toxin expression using the pET-14b vector in a protocol similar to that described elsewhere (9).…”

“…Css4 -Protocols that were previously shown to be useful for the production and extensive mutagenesis of a number of long-chain scorpion toxins using the expression vector pET-11c (9,10,17,18,26) were unsuccessful in attempts to overexpress Css4. However, large quantities of recombinant toxin, His-Css4, were obtained using the expression vector pET-14b (see "Materials and Methods").…”

“…However, Bj-xtrIT differs prominently from anti-mammalian ␤-toxins (e.g. Cn2 from Centruroides noxius, Css2, and Css4) in specificity (5,9,(12)(13)(14) and structure (8,15,16). It shares only 27% sequence identity with Css4, and the spatial arrangement of the fourth disulfide bridge and C-tail configuration vary greatly ( Fig.…”