Scanning Mutagenesis of a Janus-faced Atracotoxin Reveals a Bipartite Surface Patch That Is Essential for Neurotoxic Function

Abstract

The Janus-faced atracotoxins (J-ACTXs) are a family of insect-specific excitatory neurotoxins isolated from the venom of Australian funnel web spiders. In addition to a strikingly asymmetric distribution of charged residues, from which their name is derived, these toxins contain an extremely rare vicinal disulfide bond. To shed light on the mechanism of action of these toxins and to enhance their utility as lead compounds for insecticide development, we developed a recombinant expression system for the prototypic family member, J-ACTX-Hv1c, and mapped the key functional residues using site-directed mutagenesis. An alanine scan using a panel of 24 mutants provided the first complete map of the bioactive surface of a spider toxin and revealed that the entire J-ACTX-Hv1c pharmacophore is restricted to seven residues that form a bipartite surface patch on one face of the toxin. However, the primary pharmacophore, or hot spot, is formed by just five residues (Arg 8 , Pro 9 , Tyr 31 , and the Cys 13 -Cys 14 vicinal disulfide). The Arg 8 -Tyr 31 diad in J-ACTX-Hv1c superimposes closely on the Lys-(Tyr/Phe) diad that is spatially conserved across a range of structurally dissimilar K ؉ channel blockers, which leads us to speculate that the JACTXs might target an invertebrate K ؉ channel.As well as destroying an estimated 20 -30% of the world's food supply (1), arthropod pests are responsible for the transmission of numerous human diseases. Mosquitoes, for example, transmit dengue fever, yellow fever, West Nile virus, filariasis, and malaria, with malaria causing more than two million deaths annually (2). These phytophagous and hematophagous insect pests have traditionally been controlled by spraying broad spectrum chemical pesticides. However, the emergence of insecticide-resistant insect populations (3, 4), as well as increasing concerns about the environmental and human health risks associated with certain chemical pesticides (5, 6), has stimulated the search for new pest control strategies.The pioneering work of Olivera and colleagues (7, 8) has revealed that the venoms of aquatic cone snails (Conus spp.) are essentially highly evolved combinatorial peptide libraries. We have argued that spider venoms can be analogously viewed as preoptimized combinatorial libraries of insecticidal compounds (9), and therefore we decided to exploit these venoms in the search for insect-specific peptide toxins. By screening the venom of the lethal funnel web spider Hadronyche versuta (10), we discovered three families of insect-specific toxins (9,11,12), including an unusual family of excitatory neurotoxins that we named the Janus-faced atracotoxins (J-ACTXs; Fig. 1).1 In addition to being excellent biopesticide candidates, these toxins have allowed validation of new insecticide targets, and consequently they could be invaluable leads for the development of novel chemical insecticides (13).In addition to a strikingly asymmetric distribution of charged residues, from which the toxin name is derived, the J-ACTXs contain an extremely rare...

A Novel Family of Insect-Selective Peptide Neurotoxins Targeting Insect Large-Conductance Calcium-Activated K+ Channels Isolated from the Venom of the Theraphosid Spider Eucratoscelus constrictus

“…-HXTX-Hv1c exhibits lethal activities in a number of insect orders, including Coleoptera, Diptera, Lepidoptera, and Orthoptera, whereas lacking activity in mouse, rabbit, rat, and chick preparations (Maggio and King, 2002). Thus -HXTX-Hv1c validates K ϩ channels as potential insecticidal targets.…”

Evolutionary Diversification of Mesobuthus α-Scorpion Toxins Affecting Sodium Channels

“…In addition, MeuNaTx␣-3 contains an unusual C-terminal sequence His-Leu-Gln, as compared with other ␣-toxins. Based on the size of the J-loop preceding the conserved ␣-helix, MeuNaTx␣s can be divided into three distinct groups: (1) MeuNaTx␣-1, -2, -7, -9 and -12, which all have a five-residue J-loop, as oberved in most known ␣-toxins; (2) MeuNaTx␣-4, -5, -6, -8, -10, -11, and -13, which contains an insertion of two hydrophobic residues (Ala-Phe) in the J-loop relative to the first group; (3) MeuNaTx␣-3 whose J-loop extends a hydrophilic eight-residue segment ( 19 GluTyr-Thr-Tyr-Ile-Gly-Thr-Ser 26 ). Given the J-loop carries crucial functional residues in several ␣-toxins (e.g.…”

“…Insect Toxicity Assays-Insect toxicity assays were performed according to the method of Maggio and King (26). Briefly, MeuNaTx␣-4 with twofold dilution by insect saline was injected into house flies (Musca domestica TJS, deltamethrin-sensitive strain; and M. domestica BJD, deltamethrin-resistant strain) (27) for toxicity assays.…”

Lethal effects of an insecticidal spider venom peptide involve positive allosteric modulation of insect nicotinic acetylcholine receptors

“…Furthermore, a significant enhancement of I Glu-Cl amplitude (p < 0.05) was only evident at a concentration of 1 µM, which seems unlikely to be effective enough to correlate with the potent toxicity seen in insect bioassays (Gunning et al, 2008;Maggio and King, 2002b;Tedford et al, 2007;Wang et al, 2000).…”