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

Abstract

Scorpion ␤-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 ␤-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 Å 2 , which was composed of a number of non-polar and three charged amino acids clustered around the main ␣-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 ␤-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 ␤-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.

Solution Structure and Alanine Scan of a Spider Toxin That Affects the Activation of Mammalian Voltage-gated Sodium Channels

“…S5. Values of S Ͼ 0.8 generally indicate that an angle is well ordered within the family of structures; the backbone and angles were well defined, as measured by this parameter, over the majority of residues, the exceptions being Cys 16 and Gly 17 . The side chain 1 angles for residues Lys 3 -Thr 7 in the N-terminal region were well defined, as were those of Cys 15 The structure of Magi 5 is well defined except for the N-and C-terminal residues, with a pairwise root mean square deviation of 0.40 Å over the backbone heavy atoms (N, C ␣ , C) of Cys 2 -Pro 28 .…”

“…There was also a hydrogen bond between the amide of Ala 25 and the oxygen of Ala 22 in 14 of 20 structures; taken together with the and angles for Leu 23 and Gly 24 (Ϫ76°/53°and 72°/5°, respectively), this hydrogen bond suggests that the chain reversal around residues 22-25 has some characteristics of a distorted type II ␤-turn. Hydrogen bonds between the amide of Glu 14 and the oxygen of Asn 11 , the amide of Cys 16 and the oxygen of Lys 3 , the amide of Met 27 and the oxygen of Ala 20 were present in 14, 19, and 20 structures, respectively. The Glu 14 to Asn 11 hydrogen bond, together with the and angles for Lys 12 and Lys 13 (Ϫ67°/Ϫ32°and Ϫ82°/5°, respectively), indicate that the chain reversal around residues 11-14 is a type I ␤-turn.…”

“…A family of 20 structures determined with no disulfide bonds formed was used to determine the disulfide bonding pattern, because spectral overlap was problematic in the identification of Cys-Cys NOEs. It was clear from these initial structure calculations that Cys 2 and Cys 16 were much closer to one another than to other Cys side chains, so this disulfide was formed in subsequent rounds of structure calculation. To confirm the other two disulfide connectivities, XPLOR-NIH calculations were repeated with two possible combinations of cysteine pairings: Cys 2 -Cys 16 26 , with the lowest energies obtained and fewest NMR restraint violations.…”

“…It was clear from these initial structure calculations that Cys 2 and Cys 16 were much closer to one another than to other Cys side chains, so this disulfide was formed in subsequent rounds of structure calculation. To confirm the other two disulfide connectivities, XPLOR-NIH calculations were repeated with two possible combinations of cysteine pairings: Cys 2 -Cys 16 26 , with the lowest energies obtained and fewest NMR restraint violations. This 1-4/2-5/3-6 pattern of disulfide pairings is consistent with those found in polypeptides that show the inhibitory cystine knot (ICK) fold (40,41).…”

“…The Glu 14 to Asn 11 hydrogen bond, together with the and angles for Lys 12 and Lys 13 (Ϫ67°/Ϫ32°and Ϫ82°/5°, respectively), indicate that the chain reversal around residues 11-14 is a type I ␤-turn. The chain reversal around residues 15-18 does not fit the description of any of the classical turn types, possibly because of distortions associated with the disulfide links to Cys 15 and Cys 16 . However, it should be noted that there is a dearth of NOEs around this region, and the backbone angu- lar order parameters are less well defined than for the rest of the structure (supplemental Fig.…”