Functional significance of the beta hairpin in the insecticidal neurotoxin omega-atracotoxin-Hv1a.
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.
Tedford
H W
HW
Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032, USA.
Fletcher
J I
JI
King
G F
GF
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004926:Escherichia coli; D007306:Insecticides / Q000378:metabolism / Q000633:toxicity; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D013111:Spider Venoms / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D013329:Structure-Activity Relationship
D007306:Insecticides; D013111:Spider Venoms; C107148:omega-atracotoxin-HV1