Biochemistry kurtoxin. Several charged residues, including Asp3, Lys21, and Lys56, surround the major surface hydrophobic patch, while Lys13, Arg14, and Asp25 are situated near the minor hydrophobic patch (Figure 4D). Structural Comparison of Kurtoxin with Scorpion α- Toxins. The amino acid sequence of kurtoxin is homologous with those of the scorpion α-toxins targeting Na+ channels (Figure 1), which is consistent with the finding that kurtoxin can also interact with voltage-gated Na+ channels.55 The three- dimensional structures of the scorpion α-toxins have been determined by 1H 2D NMR spectroscopy and X-ray crystallography.91−101 Their overall folds are remarkably similar to that of kurtoxin and consist of an α-helix and three β-strands stabilized by four disulfide bridges, which has been termed the CSαβ (cysteine-stabilized α-helix and β-sheet) motif.102 In addition, like kurtoxin, the α-helices of the scorpion α-toxins contain 2.5−3 helical turns and are connected to the central β- stand by two disulfide bridges. Figure 5 shows the geometric average backbone structure from the 20 NMR kurtoxin models superimposed on the backbones of AaH II (antimammal α-toxin), LqhαIT (anti- insect α-toxin), and BmK M1 (α-like toxin). Comparison of the kurtoxin backbone with those of the scorpion α-toxins clearly highlights three regions of structural difference: the first long loop region (Asp8−Ile15), the β-hairpin loop (Gly39−Leu42), and the C-terminal segment (Arg57−Ala63). The structural differences among these regions are strongly correlated with a marked difference in sequence (Figure 1). Among the eight residues extending from Asp8 to Ile15, only two (Ans11 and Cys12) are conserved in the amino acid sequences of these toxins. The 9th and 10th residues are variably polar or hydrophobic in the scorpion α-toxins, with a non-proline cis peptide bond in BMK M1.99 On the other hand, they are bulky hydrophobic residues (Tyr9 and Trp10) with a common trans peptide bond in kurtoxin. In addition, the structurally well- defined hydrophobic residues Val13 and Tyr14 found in LqhαIT and BmK M1 are replaced with disordered positively charged residues Lys13 and Arg14, respectively, in kurtoxin. The sequence of the hairpin loop (Gly39−Leu42) also differs between kurtoxin and the scorpion α-toxins. The length of the loop in kurtoxin (four residues) is shorter than in other toxins (approximately seven residues). In addition, whereas the loop is disordered and involved in the formation of the major hydrophobic patch in kurtoxin, it protrudes from the CSαβ core and turns toward the C-terminal segments in the scorpion α-toxins (Figure 5). Because the C-terminal segments are disordered in both kurtoxin and scorpion α-toxins, it is difficult to assess structural differences in that region. However, site- directed mutagenesis studies and functional assays of scorpion α-toxins have shown that there is a functional site composed of the five-residue reverse turn (Asp8−Cys12) and the C-terminal segment, and that the conserved hydrophobic surface may be involved in maintaining the stability of the protein and its biological activity.103−107 Taken together, these findings indicate that the core region of kurtoxin (i.e., the CSαβ motif) is well-defined and superimposes well on those of the scorpion α-toxins, but the Asp8−Ile15, Gly39−Leu42, and C- terminal segments of kurtoxin are structurally different from those of the scorpion α-toxins, suggesting it is these regions that are responsible for the functional differences between kurtoxin and scorpion α-toxins. Comparison of the Surface Profiles of Kurtoxin and Scorpion α-Toxins. Kurtoxin contains five negatively charged Article Figure 5. Stereopairs showing the superposition of the kurtoxin structure on scorpion α-toxin structures. The backbone (C, Cα, and N) atoms of kurtoxin are superimposed on those of the scorpion α- toxins. The top panel shows the superposition of the backbone of kurtoxin on that of the anti-mammal α-toxin AaH II (PDB entry 1PTX). The middle panel shows the superposition on the backbone of the anti-insect α-toxin LqhαIT (PDB entry 1LQH). The bottom panel shows the superposition on the backbone of the α-like toxin Bmk M1 (PDB entry 1DJT). The backbone structures of kurtoxin and scorpion α-toxins are shown as thick and thin lines, respectively. N and C indicate the N- and C-terminal positions, respectively. Labeling shows the kurtoxin residues in regions of structural difference between the two backbones. The backbone rmsd values are 3.13, 2.98, and 2.62 Å for AaH II, LqhαIT, and Bmk M1, respectively. and 11 positively charged residues in its amino acid sequence (Figure 1), and all of these charged residues except Asp8 are highly exposed on the water-accessible surface of the molecule. The side chain oxygen of Asp8 in AaH II forms a hydrogen bond with the amide proton of Val10, and the side chain of Gln8 in Lqh III forms a hydrogen bond with the oxygen of Val13.100 Asp8 of kurtoxin is directed toward Lys13 and Arg14 (Figure 6A). Although there are no experimental data for the hydrogen bond interactions between Asp8 and any other residues in kurtoxin, some side chain oxygens of Asp8 in 20 ensemble structures are close enough to form hydrogen bonds with Lys13 and/or Arg14 in the determined kurtoxin structures. Along the α-helix, the positive and negative charges align toward the solvent-accessible region of the molecule in both scorpion α- toxins and kurtoxin (Figure 6), suggesting that this feature may be involved in ion channel binding and determining 1867 dx.doi.org/10.1021/bi201633j | Biochemistry 2012, 51, 1862−1873