title: Dissection of the functional surface of an anti-insect excitatory toxin illuminates a putative "hot spot" common to all scorpion beta-toxins affecting Na+ channels.
abstract: Scorpion beta-toxins affect the activation of voltage-sensitive sodium channels (NaChs). Although these toxins have been instrumental in the study of channel gating and architecture, little is known about their active sites. By using an efficient system for the production of recombinant toxins, we analyzed by point mutagenesis the entire surface of the beta-toxin, Bj-xtrIT, an anti-insect selective excitatory toxin from the scorpion Buthotus judaicus. Each toxin mutant was purified and analyzed using toxicity and binding assays, as well as by circular dichroism spectroscopy to discern the differences among mutations that caused structural changes and those that specifically affected bioactivity. This analysis highlighted a functional discontinuous surface of 1405 A(2), which was composed of a number of non-polar and three charged amino acids clustered around the main alpha-helical motif and the C-tail. Among the charged residues, Glu(30) is a center of a putative "hot spot" in the toxin-receptor binding-interface and is shielded from bulk solvent by a hydrophobic "gasket" (Tyr(26) and Val(34)). Comparison of the Bj-xtrIT structure with that of other beta-toxins that are active on mammals suggests that the hot spot and an adjacent non-polar region are spatially conserved. These results highlight for the first time structural elements that constitute a putative "pharmacophore" involved in the interaction of beta-toxins with receptor site-4 on NaChs. Furthermore, the unique structure of the C-terminal region most likely determines the specificity of excitatory toxins for insect NaChs.
authors.affiliation: Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel.
authors.forename: Lior
authors.initials: L
authors.lastname: Cohen
authors.forename: Izhar
authors.initials: I
authors.lastname: Karbat
authors.forename: Nicolas
authors.initials: N
authors.lastname: Gilles
authors.forename: Oren
authors.initials: O
authors.lastname: Froy
authors.forename: Gerardo
authors.initials: G
authors.lastname: Corzo
authors.forename: Ruthie
authors.initials: R
authors.lastname: Angelovici
authors.forename: Dalia
authors.initials: D
authors.lastname: Gordon
authors.forename: Michael
authors.initials: M
authors.lastname: Gurevitz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D019476:Insect Proteins; D007313:Insecta / Q000378:metabolism; D007306:Insecticides / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D012605:Scorpions / Q000737:chemistry*; D016415:Sequence Alignment; D015222:Sodium Channels / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: C115530:Bj-xtrIT toxin; D019476:Insect Proteins; D007306:Insecticides; D009498:Neurotoxins; D011994:Recombinant Proteins; D015222:Sodium Channels