Scanning mutagenesis of a Janus-faced atracotoxin reveals a bipartite surface patch that is essential for neurotoxic function.
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 J-ACTXs might target an invertebrate K(+) channel.
Maggio
Francesco
F
Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032, USA.
King
Glenn F
GF
D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D002460:Cell Line; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D007313:Insecta; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017354:Point Mutation; D011188:Potassium / Q000378:metabolism; D011392:Proline / Q000737:chemistry; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013111:Spider Venoms / Q000235:genetics* / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry; D014466:Ultraviolet Rays
D004220:Disulfides; D011994:Recombinant Proteins; D013111:Spider Venoms; C406870:delta-atracotoxin-HV1; D014443:Tyrosine; D001120:Arginine; D011392:Proline; D005982:Glutathione Transferase; D003545:Cysteine; D000409:Alanine; D011188:Potassium