Structure–function studies of ω‐atracotoxin, a potent antagonist of insect voltage‐gated calcium channels

Abstract

The v-atracotoxins are a family of 36 to 37-residue peptide neurotoxins that block insect but not mammalian voltage-gated calcium channels. The high phylogenetic specificity of these toxins recommends them as lead compounds for targeting insects that have developed resistance to chemical pesticides. We have begun to examine structure±function relationships in the v-atracotoxins in order to explore the molecular basis of their activity and phylogenetic specificity. By probing the venom of the Blue Mountains funnel-web spider, Hadronyche versuta, for insecticidal toxins with masses close to that of v-atracotoxin-Hv1a (v-ACTX-Hv1a), we have isolated and sequenced five additional v-atracotoxins. Five of the six v-atracotoxins isolated from the venom of H. versuta (v-ACTX-Hv1a to -Hv1e) differ from one another by only 1±3 residues and have similar insecticidal potencies. In contrast, v-ACTX-Hv1f differs from the other toxins by up to 10 residues and it has markedly reduced insecticidal potency, thus providing information on key functional residues. The new atracotoxin sequences have revealed that the three N-terminal residues are highly conserved. Despite the fact that these residues are structurally disordered in solution we show here, by a series of N-terminal truncations, that they contribute significantly to insecticidal potency. However, loss of activity does not correlate with deletion of highly conserved residues, which leads us to propose that the disposition of the N-terminal charge, rather than the chemical properties of the N-terminal residues themselves, may be critical for the activity of v-atracotoxin on insect calcium channels.

Scanning Mutagenesis of ω-Atracotoxin-Hv1a Reveals a Spatially Restricted Epitope That Confers Selective Activity against Insect Calcium Channels

“…Sequencing of venom peptides (1,3) and analysis of venomgland cDNA libraries (4,21) has revealed that each species of Australian funnel-web spider typically expresses a complement of 3-6 -ACTX-Hv1a homologs in its venom. Despite significant intra-and interspecific variations in the sequences of these homologs, the five pharmacophore residues (Gln 9 , Pro 10 , Tyr 13 , Asn 27 , and Arg 35 ) are identical in all -ACTX-1 orthologs that have been sequenced to date (see Fig.…”

Functional Significance of the β-Hairpin in the Insecticidal Neurotoxin ω-Atracotoxin-Hv1a

“…The Gly-Gly linker was predicted to induce formation of a ␤-turn at the tip of the small residual ␤-hairpin. We previously showed that excision of the three N-terminal residues caused a small (15-fold) but not critical reduction in biological activity (10), and hence these residues were also excluded from the final construct.…”

“…Insect Toxicity Assays-Insecticidal activity was tested by injecting peptides dissolved in insect saline (14) into house crickets (Acheta domesticus Linnaeus, sex undetermined, mass 50 -100 mg) as described previously (10). Control insects received injections of insect saline.…”

Discovery and Structure of a Potent and Highly Specific Blocker of Insect Calcium Channels

“…Once purified to Ͼ98% homogeneity, peptides were lyophilized and stored at Ϫ20°C until further use. Cysteine residues were alkylated before sequencing (7).…”

“…Insect and Vertebrate Toxicity Assays-Insecticidal activity was tested by injecting peptides into house crickets (Acheta domesticus Linnaeus) as described previously (7). Vertebrate activity was assayed as described previously (8) using vertebrate smooth (vas deferens) and skeletal (biventer cervicis) nerve-muscle preparations; tissue contractions were recorded in the absence of additives or after injection of peptides directly into the bath buffer.…”