TY - JOUR
T1 - Dissection of the Functional Surface of an Anti-insect Excitatory Toxin Illuminates a Putative "Hot Spot" Common to All Scorpion β-Toxins Affecting Na+ Channels
AU - Cohen, Lior
AU - Karbat, Izhar
AU - Gilles, Nicolas
AU - Froy, Oren
AU - Corzo, Gerardo
AU - Angelovici, Ruthie
AU - Gordon, Dalia
AU - Gurevitz, Michael
PY - 2004/2/27
Y1 - 2004/2/27
N2 - Scorpion β-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 β-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 Å2, which was composed of a number of non-polar and three charged amino acids clustered around the main α-helical motif and the C-tail. Among the charged residues, Glu30 is a center of a putative "hot spot" in the toxin-receptor binding-interface and is shielded from bulk solvent by a hydrophobic "gasket" (Tyr26 and Val34). Comparison of the Bj-xtrIT structure with that of other β-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 β-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.
AB - Scorpion β-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 β-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 Å2, which was composed of a number of non-polar and three charged amino acids clustered around the main α-helical motif and the C-tail. Among the charged residues, Glu30 is a center of a putative "hot spot" in the toxin-receptor binding-interface and is shielded from bulk solvent by a hydrophobic "gasket" (Tyr26 and Val34). Comparison of the Bj-xtrIT structure with that of other β-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 β-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.
UR - http://www.scopus.com/inward/record.url?scp=1542289623&partnerID=8YFLogxK
U2 - 10.1074/jbc.M307531200
DO - 10.1074/jbc.M307531200
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C2 - 14672947
AN - SCOPUS:1542289623
SN - 0021-9258
VL - 279
SP - 8206
EP - 8211
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 9
ER -