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

Hugo W Tedford,Jamie I Fletcher,Glenn F King

doi:10.1074/jbc.m102199200

Hugo W Tedford, Jamie I Fletcher + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m102199200

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Abstract

omega-Atracotoxin-Hv1a is an insect-specific neurotoxin whose phylogenetic specificity derives from its ability to antagonize insect, but not vertebrate, voltage-gated calcium channels. In order to help understand its mechanism of action and to enhance its utility as a lead compound for insecticide development, we used a combination of protein engineering and site-directed mutagenesis to probe the toxin for key functional regions. First, we constructed a Hairpinless mutant in which the C-terminal beta-hairpin, which is highly conserved in this family of neurotoxins, was excised without affecting the fold of the residual disulfide-rich core of the toxin. The Hairpinless mutant was devoid of insecticidal activity, indicating the functional importance of the hairpin. We subsequently developed a highly efficient system for production of recombinant toxin and then probed the hairpin for key functional residues using alanine-scanning mutagenesis followed by a second round of mutagenesis based on initial "hits" from the alanine scan. This revealed that two spatially proximal residues, Asn(27) and Arg(35), form a contiguous molecular surface that is essential for toxin activity. We propose that this surface of the beta-hairpin is a key site for interaction of the toxin with insect calcium channels.

Highlights

Public disquiet about the environmental and human health risks (1, 2) associated with chemical pesticides has stimulated the search for “environmentally friendly” pest control strategies
In order to help understand its mechanism of action and to enhance its utility as a lead compound for insecticide development, we used a combination of protein engineering and site-directed mutagenesis to probe the toxin for key functional regions
The cystine knot toxins essentially comprise a cystine framework onto which four loops are tethered, each bounded by half-cystines. ␻-ACTX-Hv1a differs from the well known conotoxin members of this family (28) in that one of the loops forms an unusually long ␤-hairpin that is highly conserved among the ␻-atracotoxin-1 family (Fig. 1A)

Summary

EXPERIMENTAL PROCEDURES

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). Purification of Synthetic and Native Toxin—Synthetic ␻-ACTX-Hv1a was purified and oxidized/folded as described previously (9). Dihedral angle restraints were derived from 3JHNH␣ coupling constants measured from either high resolution one-dimensional NMR spectra or from inverse Fourier transforms of in-phase NOESY multiplets (19). The PCR-amplified synthetic gene was digested with BamHI and EcoRI and subcloned using standard methods into BamHI/EcoRI-digested pGEX-2T (Amersham Pharmacia Biotech) to give the plasmid pHWT1, in which ␻-ACTX-Hv1a is encoded as an in-frame fusion to the C terminus of Schistosoma japonicum glutathione S-transferase (with an intervening thrombin cleavage site). The recombinant toxin was purified from the soluble cell fraction using affinity chromatography on GSH-Sepharose (Amersham Pharmacia Biotech) followed by on-column thrombin cleavage as described previously (25) except that phenylmethylsulfonyl fluoride and dithiothreitol were not present in the lysis buffer, and bovine thrombin (Sigma) was used instead of human thrombin. Data Base Files—Coordinates and experimental restraints for the ensemble of 20 Hairpinless structures have been deposited in the Protein Data Bank (PDB accession code 1HVW), and 1H chemical shifts have been deposited in BioMagResBank (accession number 4937)

RESULTS

Experimental restraints

All heavy atoms

Toxin or mutant

DISCUSSION