Computational simulations of interactions of scorpion toxins with the voltage-gated potassium ion channel

Abstract

Based on a homology model of the Kv1.3 potassium channel, the recognitions of the six scorpion toxins, viz. agitoxin2, charybdotoxin, kaliotoxin, margatoxin, noxiustoxin, and Pandinus toxin, to the human Kv1.3 potassium channel have been investigated by using an approach of the Brownian dynamics (BD) simulation integrating molecular dynamics (MD) simulation. Reasonable three-dimensional structures of the toxin-channel complexes have been obtained employing BD simulations and triplet contact analyses. All of the available structures of the six scorpion toxins in the Research Collaboratory for Structural Bioinformatics Protein Data Bank determined by NMR were considered during the simulation, which indicated that the conformations of the toxin significantly affect both the molecular recognition and binding energy between the two proteins. BD simulations predicted that all the six scorpion toxins in this study use their beta-sheets to bind to the extracellular entryway of the Kv1.3 channel, which is in line with the primary clues from the electrostatic interaction calculations and mutagenesis results. Additionally, the electrostatic interaction energies between the toxins and Kv1.3 channel correlate well with the binding affinities (-logK(d)s), R(2) = 0.603, suggesting that the electrostatic interaction is a dominant component for toxin-channel binding specificity. Most importantly, recognition residues and interaction contacts for the binding were identified. Lys-27 or Lys-28, residues Arg-24 or Arg-25 in the separate six toxins, and residues Tyr-400, Asp-402, His-404, Asp-386, and Gly-380 in each subunit of the Kv1.3 potassium channel, are the key residues for the toxin-channel recognitions. This is in agreement with the mutation results. MD simulations lasting 5 ns for the individual proteins and the toxin-channel complexes in a solvated lipid bilayer environment confirmed that the toxins are flexible and the channel is not flexible in the binding. The consistency between the results of the simulations and the experimental data indicated that our three-dimensional models of the toxin-channel complex are reasonable and can be used as a guide for future biological studies, such as the rational design of the blocking agents of the Kv1.3 channel and mutagenesis in both toxins and the Kv1.3 channel. Moreover, the simulation result demonstrates that the electrostatic interaction energies combined with the distribution frequencies from BD simulations might be used as criteria in ranking the binding configuration of a scorpion toxin to the Kv1.3 channel.

FIGURE 1

Sequence alignments of of KcsA (1BL8) channel with the Kv1.3 channel generated by CLUSTAL W (Thompson et al., 1994). In the sequences, an asterisk indicates an identical or conserved residue, a colon indicates a conserved substitution, and a dot indicates a semiconserved substitution.

FIGURE 2

Molecular graphics side views of the scorpion toxin AgTX and Kv1.3 potassium channel complex embedded in a POPC membrane solvated by water. These snapshots were taken near the beginning of the trajectory. (a) Side view of the system. (b) Top view of the system with water molecules hidden. The toxin was colored by cyan.

FIGURE 3

Electrostatic potential contour maps for the Kv1.3 channel and the six scorpion toxins at an ionic strength of 0.1 M. The red contours represent isopotential surfaces where charge 1 e possesses electrostatic potential energy equal to −2.5 kT; the blue isopotential surfaces are for energy +2.5 kT. Arrows indicate the directions of the dipoles in the proteins. The picture was generated with the program GRASP (Nicholls et al., 1991).

FIGURE 4

Distribution of distances between the two monitors for all complexes of the most favorably docked structures of the scorpion toxins associating with the Kv1.3 channel. The monitor distances <30 Å were recorded.

FIGURE 5

Distribution of the structures of AgTX formed encounter complex around the extracelluar mouth of the Kv1.3 channel. The Kv1.3 channel is represented as C^α trace. Each ball represents the center of mass of the toxin in an encounter snapshot with the Kv1.3 channel.

FIGURE 6

Correlation between the electrostatic interaction energies and the values of −logK_d of the six scorpion toxins to Kv1.3 channel.

FIGURE 7

(a) Sequence alignments of the six scorpion toxins generated by CLUSTAL W (Thompson et al., 1994). In the sequences, an asterisk indicates an identical or conserved residue, a colon indicates a conserved substitution, and a dot indicates a semiconserved substitution. (b) Structure alignment of the six scorpion toxins with some of the AgTX2 residues shown.

FIGURE 8

Flexibilities of the AgTX2 and Kv1.3 channel in their binding deduced from the 5-ns MD simulations. (a) RMSDs of Kv1.3 and AgTX2 in their separated state. (b) RMS fluctuations of the atoms of AgTX2 in the complex. (c) RMS fluctuations of the atoms of Kv1.3 (only shows one monomer) in the complex.

FIGURE 9

Optimized structure of the AgTX2-kv1.3 channel complex. (a) Side view of the structure. The two proteins were represented as ribbon structure. R24, K27, and R31 in AgTX2 formed three hydrogen bonds with D402, Y400, and D386 in Kv1.3, respectively. (b) Top view of the structure. The molecular surface of Kv1.3 channel is colored by electrostatic potential (red, negative; blue, positive, white, uncharged). The toxin is represented as C^α trace, and the important residues in binding are represented as ball-and-stick model.