Aphicidal efficacy of scorpion- and spider-derived neurotoxins.

Insect-specific neurotoxins that act within the insect hemocoel (body cavity) represent an untapped resource for insect pest management. On the basis of recent advances made in development of appropriate delivery systems for transport of these toxins from the insect gut, across the gut epithelium to their target site, we screened neurotoxins derived from scorpion or spider venom for efficacy against the pea aphid, Acyrthosiphon pisum, and the green peach aphid, Myzus persicae. Toxins were selected to represent different modes of electrophysiological action, including activity on voltage-gated calcium channels (ω-TRTX-Gr1a, ω-agatoxin Aa4a, ω-hexatoxin-Hv1a), calcium- and voltage-activated potassium channels (charybdotoxin, maurotoxin), chloride channels (chlorotoxin) and voltage-gated sodium channels (LqhαIT). The Bacillus thuringiensis-derived toxin Cyt1Aa was also tested as a positive control for toxicity. In per os bioassays with both aphid species, toxicity was only seen for ω-TRTX-Gr1a and Cyt1Aa. On injection into the hemocoel of A. pisum, LD50 values ranged from 1 to 8 ng/mg body weight, with ω-hexatoxin-Hv1a being the most toxic (1.02 ng/mg body weight). All neurotoxins caused rapid paralysis, with charybdotoxin, maurotoxin and chlorotoxin also causing melanization of injected aphids. These data represent the first comprehensive screen of neurotoxins against aphids, and highlight the potential for practical use of the insect-specific toxin ω-hexatoxin-Hv1a in aphid management.

Isolation, solution structure, and insecticidal activity of kalata B2, a circular protein with a twist: do Möbius strips exist in nature?

A large number of macrocyclic miniproteins with diverse biological activities have been isolated from the Rubiaceae, Violaceae, and Cucurbitaceae plant families in recent years. Here we report the three-dimensional structure determined using (1)H NMR spectroscopy and demonstrate potent insecticidal activity for one of these peptides, kalata B2. This peptide is one of the major components of an extract from the leaves of the plant Oldenlandia affinis. The structure consists of a distorted triple-stranded beta-sheet and a cystine knot arrangement of the disulfide bonds and is similar to those described for other members of the cyclotide family. The unique cyclic and knotted nature of these molecules makes them a fascinating example of topologically complex proteins. Examination of the sequences reveals that they can be separated into two subfamilies, one of which contains a larger number of positively charged residues and has a bracelet-like circularization of the backbone. The second subfamily contains a backbone twist due to a cis-peptidyl-proline bond and may conceptually be regarded as a molecular Mobius strip. Kalata B2 is the second putative member of the Mobius cyclotide family to be structurally characterized and has a cis-peptidyl-proline bond, thus validating the suggested name for this subfamily of cyclotides. The observation that kalata B2 inhibits the growth and development of Helicoverpa armigera larvae suggests a role for the cyclotides in plant defense. A comparison of the sequences and structures of kalata B1 and B2 provides insight into the biological activity of these peptides.

Conserved structural and sequence elements implicated in the processing of gene-encoded circular proteins.

The cyclotides are the largest family of naturally occurring circular proteins. The mechanism by which the termini of these gene-encoded proteins are linked seamlessly with a peptide bond to form a circular backbone is unknown. Here we report cyclotide-encoding cDNA sequences from the plant Viola odorata and compare them with those from an evolutionarily distinct species, Oldenlandia affinis. Individual members of this multigene family encode one to three mature cyclotide domains. These domains are preceded by N-terminal repeat regions (NTRs) that are conserved within a plant species but not between species. We have structurally characterized peptides corresponding to these NTRs and show that, despite them having no sequence homology, they form a structurally conserved alpha-helical motif. This structural conservation suggests a vital role for the NTR in the in vivo folding, processing, or detoxification of cyclotide domains from the precursor protein.

Twists, knots, and rings in proteins. Structural definition of the cyclotide framework.

In recent years an increasing number of miniproteins containing an amide-cyclized backbone have been discovered. The cyclotide family is the largest group of such proteins and is characterized by a circular protein backbone and six conserved cysteine residues linked by disulfide bonds in a tight core of the molecule. These form a cystine knot in which an embedded ring formed by two of the disulfide bonds and the connecting backbone segment is threaded by a third disulfide bond. In the current study we have undertaken high resolution structural analysis of two prototypic cyclotides, kalata B1 and cycloviolacin O1, to define the role of the conserved residues in the sequence. We provide the first comprehensive analysis of the topological features in this unique family of proteins, namely rings (a circular backbone), twists (a cis-peptide bond in the Möbius cyclotides) and knots (a knotted arrangement of the disulfide bonds).