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

Abstract

We constructed a complete panel of alanine mutants of the insect-specific calcium channel blocker -atracotoxin-Hv1a. Lethality assays using these mutant toxins identified three spatially contiguous residues, Pro 10 , Asn 27 , and Arg 35 , that are critical for insecticidal activity against flies (Musca domestica) and crickets (Acheta domestica). Competitive binding assays using radiolabeled -atracotoxin-Hv1a and neuronal membranes prepared from the heads of American cockroaches (Periplaneta americana) confirmed the importance of these three residues for binding of the toxin to target calcium channels presumably expressed in the insect membranes. At concentrations up to 10 M, -atracotoxinHv1a had no effect on heterologously expressed rat Ca v 2.1, Ca v 2.2, and Ca v 1.2 calcium channels, consistent with the previously reported insect selectivity of the toxin. 30 M -atracotoxin-Hv1a inhibited rat Ca v currents by 10 -34%, depending on the channel subtype, and this low level of inhibition was essentially unchanged when Asn 27 and Arg 35 , which appears to be critical for interaction of the toxin with insect Ca v channels, were both mutated to alanine. We propose that the spatially contiguous epitope formed by Pro 10 , Asn 27 , and Arg 35 confers specific binding to insect Ca v channels and is largely responsible for the remarkable phyletic selectivity of -atracotoxin-Hv1a. This epitope provides a structural template for rational design of chemical insecticides that selectively target insect Ca v channels.The first peptide neurotoxins isolated from the venom of Australian funnel-web spiders (genera Atrax and Hadronyche) and shown to have selective activity against insects were members of the -atracotoxin-1 (ACTX) 1 family (1-3). These toxins comprise 36 -37 residues with six strictly conserved cysteine residues that are paired to form three disulfide bridges (4). The best studied family member, -ACTX-Hv1a, is one of the most potent insecticidal peptide toxins discovered so far (4, 5); it has proved lethal to all insect orders that have been tested, including coleopterans, dictyopterans, hemipterans, orthopterans, and refractory lepidopteran pests such as the tobacco budworm Heliothis virescens and the cotton bollworm Helicoverpa armigera (1, 2, 5).The phyletic specificity of these toxins appears to reside in their ability to block insect, but not vertebrate, voltage-gated calcium channels (2). Although nanomolar concentrations of -ACTX-Hv1a are sufficient to block high voltage-activated (HVA) calcium currents in the central nervous system of the fruit fly Drosophila melanogaster and the American cockroach Periplaneta americana (2, 5), 1 M toxin does not block HVA calcium currents in vertebrate neurons (2) and the toxin is harmless when injected subcutaneously into newborn mice (1).With the exception of the organophosphate and carbamate insecticides (which target acetylcholinesterase), the vast majority of synthetic insecticides are directed against voltagegated sodium channels, nicotinic acetylcholine receptors, or ...

Optimising expression of the recombinant fusion protein biopesticide ω-hexatoxin-Hv1a/GNA inPichia pastoris:sequence modifications and a simple method for the generation of multi-copy strains

“…Hv1a is a member of the -ACTX-1 family of 36-37 insecticidal residue peptides isolated from the Australian funnel web spider that block insect but not vertebrate, voltage-gated calcium channels ( [20] Fletcher et al, 1997). Previous alanine scanning mutagenesis studies by Tedford et al, [21] 2004 have identified 3 key functional residues (Pro10, Asn27, and Arg35) that determine specific binding to insect calcium channels. Here replacement of the 34th lysine residue in Hv1a with a glutamine residue by site directed mutagenesis was selected as glutamine is known to be present at this position of other members of the -ACTX-1 family ( [22] Tedford et al 2004) and was thus unlikely to disrupt biological function of the recombinant toxin.…”

Engineering insect-resistant plants by transgenic expression of an insecticidal spider-venom peptide

“…The three disulfide bonds form an ICK motif in which the loop formed by the Cys11-Cys22 and Cys4-Cys18 SS bonds and the intervening sections of the peptide backbone is pierced by the Cys17-Cys37 SS bond ( Fig. 1.2B) (Tedford, 2001;Tedford et al, 2004a). The binding site of Hv1a on insect Ca v channels is not known but three residues on the toxin (Pro10, Asn27 and Arg35; see Fig.…”

“…The binding site of Hv1a on insect Ca v channels is not known but three residues on the toxin (Pro10, Asn27 and Arg35; see Fig. 1.2C) mediate its interaction with Ca V channels (Tedford, 2001;Tedford et al, 2004a;King, 2007b;King et al, 2008).…”

“…There are three main regions within the three-dimensional structure of Hv1a ( Fig. 1.2A): a structurally disordered N-terminus (residues 1-3), an SS-rich globular core (residues 4-21) and a β-hairpin at the C-terminus (residues 22-37) that protrudes from the SS-rich core (Tedford, 2001;Tedford et al, 2004a). The three disulfide bonds form an ICK motif in which the loop formed by the Cys11-Cys22 and Cys4-Cys18 SS bonds and the intervening sections of the peptide backbone is pierced by the Cys17-Cys37 SS bond ( Fig.…”

“…Hv1a specifically targets Ca v channels in the insect CNS but is harmless to vertebrates (Fletcher et al, 1997;Tedford et al, 2004a;Tedford et al, 2004b;Chong et al, 2007). This 37-residue peptide has potent insecticidal activity in a wide range of insect orders, including Lepidoptera, Homoptera, and Diptera (Khan et al, 2006;Mukherjee et al, 2006), but it is harmless to honeybees (Nakasu et al, 2014).…”