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A new K+-channel blocking peptide identified from the scorpion venom of Tityus cambridgei (Tc1) is composed of 23 amino acid residues linked with three disulfide bridges. Tc1 is the shortest known toxin from scorpion venom that recognizes the Shaker B K+ channels and the voltage-dependent K+ channels in the brain. Synthetic Tc1 was produced using solid-phase synthesis, and its activity was found to be the same as that of native Tc1. The pairings of three disulfide bridges in the synthetic Tc1 were identified by NMR experiments. The NMR solution structures of Tc1 were determined by simulated annealing and energy-minimization calculations using the X-PLOR program. The results showed that Tc1 contains an α-helix and a 310-helix at N-terminal Gly4–Lys10 and a double-stranded β-sheet at Gly13–Ile16 and Arg19–Tyr23, with a type I′ β-turn at Asn17–Gly18. Superposition of each structure with the best structure yielded an average root mean square deviation of 0.26 ± 0.05 Å for the backbone atoms and of 1.40 ± 0.23 Å for heavy atoms in residues 2 to 23. The three-dimensional structure of Tc1 was compared with two structurally and functionally related scorpion toxins, charybdotoxin (ChTx) and noxiustoxin (NTx). We concluded that the C-terminal structure is the most important region for the blocking activity of voltage-gated (Kv-type) channels for scorpion K+-channel blockers. We also found that some of the residues in the larger scorpion K+-channel blockers (31 to 40 amino acids) are not involved in K+-channel blocking activity.
Ion channels are involved in diverse biological processes and play essential roles in the physiology of all cells. An increasing number of human and animal diseases have been identified as relating to the defective function of ion channels. Scorpion venoms contain various polypeptides with distinct biological functions that particularly affect the permeability of ion channels in cell membranes (Catterall 1980; Valdivia et al. 1992; Garcia et al. 1997). These polypeptides possess the potency to recognize ion channels and receptors in excitable membranes and are classified into four groups on the basis of ion-channel types: (1) group I modulates Na+-channel activity (Possani et al. 1999) and contains peptides of 60 to 70 amino acids linked by four disulfide bridges; (2) group II blocks K+ channels (Miller 1995; Romi-Lebrun et al. 1997) and are short peptides with 31 to 41 amino acid residues with three or four disulfide bonds; (3) group III supposedly inhibits Cl−channels (DeBin et al. 1993) and contains short-chain insect toxin peptides of ∼36 amino acids with four disulfide bonds; and (4) group IV includes peptides that modulate ryanodine-sensitive Ca2+ channels (Valdivia and Possani 1998). It is believed that the toxin has a unique tertiary structure that may provide valuable information for understanding channels. Thus, understanding the structural basis of the specificity of scorpion toxins for these receptors could lead to the design of new ligands with controlled activity and potency with potential for clinical applications.
Scorpion K+-channel blockers of group II, named α-KTx, have been classified into 12 subfamilies (Miller 1995; Tytgat et al. 1999). These K+-channel blockers block two major classes of K+ channels: voltage-gated (Kv-type) and high-conductance Ca2+-activated (BK-type) K+ channels. The three-dimensional structures of several scorpion K+-channel blockers have been determined by NMR spectroscopy; these include charybdotoxin (ChTx; Bontems et al. 1991), iberiotoxin (IbTx; Johnson et al. 1992), noxiustoxin (NTx; Dauplais et al. 1995), PO5-NH2 (Meunier et al. 1993), kaliotoxin (KTx; Fernandez et al. 1994), margatoxin (MgTx; Johnson et al. 1994), and tityustoxin K-α (TsTx-Kα; Ellis et al. 2001). Although the overall fold of these α-KTx toxins is very similar, there are subtle variations among them in amino acid sequence, the size of the β-sheet, the type of β-turn, or the type of α-helix (i.e., α-helix versus 310-helix). These differences in toxin structure affect the placement of side-chain moieties. Thus, the selectivity that various scorpion toxins have for the outer vestibule of different K+ channels is typically quite distinct. Previously, Doyle et al. (1998) applied X-ray crystallographic methods to determine the three-dimensional structure of the KcsA bacterial K+ channel, which may serve as a good model for understanding the binding site of scorpion toxin on Kv-type channels.
Recently, a new K+-channel blocker was identified from the scorpion venom of Tityus cambridgei (Tc1; Batista et al. 2000). Tc1 contains 23 amino acids linked with three disulfide bridges and is the smallest K+-channel blocker toxin from scorpion venoms. All previously known K+-channel blockers from scorpion venoms are longer than 30 amino acid residues and are classified into 12 subfamilies as described above. Tc1 is classified as the first member of the new subfamily 13. In K+-channel blocking activity, Tc1 recognizes the Shaker B K+ channels with a dissociation constant (Kd) of 65 nM and competes with NTx for binding to the synaptosomal membranes, with an inhibitory concentration 50% (IC50) value in the order of 200 nM (Batista et al. 2000). Tc1 is a highly basic peptide because it contains seven positively charged residues with a pI value of 9.50. The sequence alignment of Tc1 with eight other K+-channel blockers from scorpion toxins is shown in Figure 1. We found that six cysteine residues (Cys2, Cys5, Cys9, Cys15, Cys20, and Cys22), Gly13, and Lys14 (Tc1 numbering) are conserved, and the C-terminal regions are highly similar among these toxins. In addition, the sequence of Tc1 shows some unique properties. For example, Tc1 possesses Arg at position 19, whereas the corresponding residue in the other toxins is Lys. At position 16, Tc1 has Ile, whereas the other toxins, with the exception of the PO5 peptide, have Met at the corresponding position. Furthermore, Tc1 contains dense positively charged residues at residues 5–10. Unlike other scorpion toxins, Tc1 does not contain either negatively charged residues or proline. These properties make Tc1 an excellent candidate for three-dimensional structure determination and site-directed mutagenesis and for gaining clearer understanding of K+ channels.
In this study, synthetic Tc1 was made by conventional solid-phase peptide synthesis and folded into its active conformation. We checked the channel-blocking activity of the synthesized Tc1 and found that both synthetic and native Tc1 possess similar blocking activity against the Shaker K+ channel. Next, we applied circular dichrosim (CD) and NMR techniques to solve the solution structure of Tc1. To further understand the various structure-function relationships among the K+-channel blockers from scorpion venoms, we compared the three-dimensional structure of Tc1 and those of other structurally and functionally related scorpion toxins, ChTx and NTx. We concluded that the C-terminal structure is the most important region for the blocking activity of Kv-type channels for scorpion K+-channel blockers. In addition, we also asserted that some of the residues in the larger scorpion K+-channel blockers, which contain 31 to 40 amino acids, are clearly not involved in K+-channel blocking activity.
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- Materials and methods
Our studies on the effects of synthetic Tc1 on Shaker GH4 K+ channels indicated that its functional property is the same as the natural scorpion toxin Tc1. We then used CD and NMR techniques to perform our structural study of the synthetic Tc1. CD spectra at different temperatures and pH values showed that Tc1 is a thermostable peptide with a conformation that is independent of pH values in the range of 3.0 to 7.0. The three-dimensional NMR solution structure of Tc1 showed that it is comprised of an α-helix and a 310-helix at N-terminal Gly4–Lys10, a double-stranded antiparallel β-sheet at Gly13–Ile16 and Arg19–Tyr23, with a type I′ β-turn at residues Asn17–Gly18. Because the NMR data obtained at pH 3.0 and 275 K were well resolved and showed many medium- and long-range NOEs, high resolutions of Tc1 structures were generated. We found that the overall structures of Tc1 and other α-KTx toxins are similar, although Tc1 only possesses 23 amino acids compared with >30 for the other scorpion toxins.
To gain further insight into the structural and functional relationships among the K+-channel blockers from scorpion venoms, we proceeded to a detailed comparison of the three-dimensional structure of Tc1 with two structurally and functionally related scorpion toxins, ChTx (37 amino acids) and NTx (39 amino acids). ChTx is the first member of subfamily 1 of α-KTx and shows a much higher affinity for the Ca2+-activated K+ channels (BK) than for Kv1.3. In contrast, NTx is the first K+-channel blocker isolated from scorpion venoms and displays a strong binding affinity for Kv1.3, whereas it exerts a weaker affinity for BK. Tc1 also has a higher binding affinity for the Shaker B channel than for BK, similar to NTx. In the secondary structure motifs, we found that the N-terminal α-helix, the two C-terminal β-strands, and the β-turn are all located in similar regions based on the sequence alignment of these toxins. However, some variations were observed in the type or length of the secondary elements. For example, Tc1 has a shorter helical conformation, and this helix begins with a regular α-helix and ends with a 310-helix, whereas the helix in NTx begins with a 310-helix and ends with a regular α-helix. Also, because of the presence of a Pro residue in the α-helical region, the α-helix in NTx displays a high degree of curvature. The bending of the α-helix in both Tc1 and ChTx, however, is weak because of the lack of a proline residue in the sequence. Tc1 and NTx have the same type I′ β-turn at Asn17–Gly18 and Asn31–Gly32, respectively, whereas ChTx possesses a type I β-turn at Asn30–Lys31. NTx not only contains an extra N-terminal β-strand but also possess longer C-terminal β-strands. Thus, there is a remarkable plasticity within the α/β-scaffold for the α-KTx toxins.
The specificity of scorpion toxins for the various potassium channels has been investigated through the generation of mutants of both receptors and toxins. Mutational analysis of ChTx showed that eight residues (Ser10, Trp14, Arg25, Lys27, Met29, Asn30, Arg34, and Tyr36) are important for the binding of ChTx to BK (Stampe et al. 1994). Five of these eight residues (Lys27, Met29, Asn30, Arg34, and Tyr36) were shown to be critical for the recognition of a voltage-dependent K+ channel (Shaker B) in ChTx (Goldstein et al. 1994). Among these residues, Lys27 is the most important because a mutation of this lysine to arginine destabilizes the toxin by >1000-fold (Miller 1995). The inhibition of the channel permeation is the result of a physical occlusion of the pore-forming region of the channel. Thus, the Lys27 of ChTx is suspected to directly plug into the pore.
The characteristics of five corresponding residues for the recognition of the Shaker B channel in Tc1 (Lys14, Ile16, Asn17, Lys21, and Tyr23) are similar to those in ChTx. However, the properties of other three residues (Ser10, Trp14, and Arg25), which are also important for the binding of ChTx to BK-type channels, were found to be very different in Tc1. The corresponding residues in Tc1 for the first two are Ser4 and Arg6, respectively, and there is no residue occupied for the third. Thus, the different property in these residues offers a possible explanation for the weak affinity of Tc1 to BK-type channels. For the recognition of the Shaker B channel in Tc1, we found that the side-chains of these residues were all exposed to the solvent on the same side (see Fig. 6A). Interestingly, Lys14, which corresponds to Lys27 in ChTx, showed a rigid side-chain conformation and highly protruded into the solvent. Thus, we suggest that Lys14 in Tc1 is the key residue to have electronic interaction with the negative charge in the pore region of the K+ channel.
In addition, IbTx was found to be inactive against the Kv1.3 channel. A sequence comparison between ChTx and IbTx indicates that Asn30 (ChTx numbering) is replaced with Gly in IbTx. Therefore, Asn30 appears to be important for the two types of voltage-dependent channels; in fact, this residue can be found in all the short-chain scorpion toxins that bind Kv1.3 (ChTx, NTx, MgTx, KTx, and Tc1 in Fig. 1). Two scorpion toxins in subfamily 7, Pi2 and Pi3, have only one amino acid difference at position 7 (a proline for a glutamic acid) in their sequence. However, Pi2 binds the Shaker B K+ channels with a Kd of 8.2 nM, but Pi3 has a much lower affinity of 140 nM (Gomez-Lagunas et al. 1996). The difference in binding affinity supports that the N-terminal residues are part of the domain that recognize Shaker B K+ channels. Interestingly, there is no residue occupied at the corresponding position in Tc1, which has a Kd of 65 nM. Therefore, it is certain that the negative charge at this position disrupts the inhibition of the Shaker B K+ channel.
The surface structures of Tc1, NTx, and ChTx (plot not shown) all indicated that there is a positively charged region at the C terminus and that this region plays an important role for blocking activity. At the N terminus, Tc1 contains a denser positively charged region composed by Arg6, Lys7, Lys8, and Lys10 (Fig. 6B) compared with NTx and ChTx. At present, we do not know whether this region interacts with the K+-channel or whether it plays an important role for activity. We are currently performing mutational studies on Tc1 to further understand its structural-functional relationships.
In addition, Figure 7 shows the comparison of the ribbon structures of Tc1, NTx, and ChTx. Superposition of backbone atoms (N, Cα, and C′) between Gly13and Tyr23 of Tc1 and between Ala27 and Tyr37 of NTx gave a RMSD of 0.76 Å. However, the RMSD became 0.96 Å between Gly13–Tyr23 of Tc1 and Gly26–Tyr36 of ChTx. Thus, Tc1 is much like NTx, especially at the C-terminal β-sheet and β-turn. Because both Tc1 and NTx contain higher activity for the Shaker B channel, we concluded that the C-terminal structure is the most important region for controling the blocking activity of the Kv-type for scorpion K+-channel blockers. Furthermore, based on the structural data and sequence alignment between Tc1 and NTx, we suggest that for NTx the N-terminal region—including the first β-strand (Thr1-Val5), some residues in the α-helix (Lys11, Gln12, Glu19, and Leu20), some residues in the loop region (Tyr21, Ser23, and Ala25), and some residues in the C-terminal region (Ala27, Asn38, and Asn39)—might not be required for channel-blocking activity. Therefore, we are also in the process of studying the structural and functional relationships of a 24-residue peptide that involves the deletion of the above 15 residues from NTx.