Biochemistry Article Figure 6. Ribbon diagrams and heavy atom side chains of kurtoxin (A) and Lqq III (B). The surface hydrophobic patches and charged residues are indicated: red for the negatively charged amino acids are colored, blue for the positively charged amino acids, purple for the major surface hydrophobic amino acids, and orange for the minor surface hydrophobic amino acids. The left and right figures are rotated 180° relative to one another about a vertical axis. selectivity.100 Kurtoxin is highly basic, as compared to the α- scorpion toxins; the net charge of kurtoxin is +6, while the others have net charges ranging from −2 to +3. As shown in Figure 1, the two hydrophobic residues (Val13 and Tyr14) conserved in all scorpion α-toxins except AaH II are replaced with two positively charged residues (Lys13 and Arg14, respectively) in kurtoxin. Figure 6B shows that the side chains of Val13 and Tyr14 in Lqq III (an anti-insect α-toxin) are largely buried in the molecular core. By contrast, Lys13 and Arg14 in kurtoxin are exposed to solvent (Figure 6A) and form a local electropositive surface (Figure 4D). In addition, a large 2 electropositive patch (surface area of 660 Å ) is formed by the five positively charged C-terminal residues (Arg54, Lys56, Arg57, Arg60, and Arg62). This is in contrast to the C-terminal structure of Lqq III, which contains only two positively charged residues (Arg58 and Lys62). The water-exposed, positively charged residues of kurtoxin form a distinctive large electro- positive surface, which is located around the five-residue reverse turn and C-terminal segment and is the proposed Na+ channel binding site in scorpion α-toxins.104 A surface hydrophobic patch is a conserved feature of all scorpion α-toxins and is involved in mediating their interaction with Na+ channels.108,109 The orthogonal arrangement of the aromatic side chains in the surface hydrophobic patch, termed a “herringbone” structure, is found in all scorpion α-toxins and has been identified as the lowest-energy configuration of solvent-exposed aromatic rings.110 There are two hydrophobic patches in scorpion α-toxins, a major patch commonly composed of five residues (Tyr5, Tyr35, Tyr42, Trp47, and Tyr49) (Figure 6B) with a surface area of ∼280 Å2 and a minor one with a surface area of ∼210 Å2. Kurtoxin exhibits a larger hydrophobic surface than the scorpion α-toxins. The major hydrophobic patch of kurtoxin consists of the six solvent- exposed side chains of Tyr5, Tyr36, Trp38, Trp40, Leu42, and Tyr45 and has a surface area of 600 Å2 (Figure 6A). The side chains are well-packed on each other, creating a compact hydrophobic patch on the protein surface. The minor 2 hydrophobic patch (surface area of 500 Å ) is centered on Trp17, Tyr18, and Tyr22 and also includes the δ-methyls of Leu26 (Figure 6A). Overall, it appears that kurtoxin shows a distinct surface profile, composed of both positive and hydrophobic residues, compared to other scorpion α-toxins. Kurtoxin Binding Site on Cav3 (T-type) Ca2+ Channels. Voltage-gated ion channels consist of a central ion conduction pore (segments S5 and S6) surrounded by voltage sensors (segments S1−S4), which form “voltage sensor paddles” that move in response to changes in membrane voltage.4,43 The overall structure of the voltage sensor paddles includes hydrophobic, cationic, and helix−turn−helix structures formed by the S3b−S4 segment, and it has been suggested that it is the voltage sensor paddles that are recognized by gating modifier toxins.17−19,38−40,42,45 Kurtoxin has been identified as the first high-affinity (Kd = 15 nM) gating modifier of Cav3.1 (α1G T- type) Ca2+ channels, and also the first to show cross-reactivity with voltage-gated Na+ channels.55 This is similar to HaTx and GrTx, which exhibit cross-reactivity with the Kv2.1 K+ channel and P/Q-type Ca2+ channel, modifying the energetics of their gating.38 It has been suggested that hydrophobic and negatively charged residues (Ile273, Phe274, and Glu277) in the Kv2.1 channel form the binding site for HaTx and GrTx.38,111 Glu1613 in the rat brain IIA Na+ channel is equivalent to Glu277 in the Kv2.1 channel, and mutation of Glu1613 has a large effect on the affinity of scorpion α-toxins for Na+ channels.17 In addition, Glu1658, situated at the end of S3, within repeat IV of the P/Q- type Ca2+ channel, contributes to the binding of ω-Aga IVA.19 These findings prompt us to speculate that kurtoxin may bind to the S3b−S4 motif in domain IV of both Cav3 (T-type) Ca2+ channels and Na+ channels, a region that has some conservation 1868 dx.doi.org/10.1021/bi201633j | Biochemistry 2012, 51, 1862−1873