Protein surface topography as a tool to enhance the selective activity of a potassium channel blocker

Abstract

Tk-hefu is an artificial peptide designed based on the α-hairpinin scaffold, which selectively blocks voltage-gated potassium channels K_v1.3. Here we present its spatial structure resolved by NMR spectroscopy and analyze its interaction with channels using computer modeling. We apply protein surface topography to suggest mutations and increase Tk-hefu affinity to the K_v1.3 channel isoform. We redesign the functional surface of Tk-hefu to better match the respective surface of the channel pore vestibule. The resulting peptide Tk-hefu-2 retains K_v1.3 selectivity and displays ∼15 times greater activity compared with Tk-hefu. We verify the mode of Tk-hefu-2 binding to the channel outer vestibule experimentally by site-directed mutagenesis. We argue that scaffold engineering aided by protein surface topography represents a reliable tool for design and optimization of specific ion channel ligands.

Keywords: alpha-hairpinin; hefutoxin; ion channel; molecular dynamics; neurotoxin; nuclear magnetic resonance (NMR); peptides; pore blocker; potassium channel; protein motif; protein structure.

Figure 1.

Tk-hefu structure determined by NMR and its comparison with the parent peptide Tk-AMP-X2. A and B, ensembles of 10 independently derived NMR structures of Tk-hefu (A; PDB ID 5LM0) and Tk-AMP-X2 (B; PDB ID 2M6A) with the fewest restrain violations. N termini are labeled. Cystine side chains are in yellow. C, ribbon representation of Tk-hefu. The orientation of the molecule is as in A. The N-terminal α-helix is shown in green; C-terminal helix, sky blue; loops are presented as a gray string. S-S bonds are presented as yellow sticks, and other side chains are shown as lines; hydrophobic aliphatic and aromatic residues are orange; hydrophilic uncharged residues are magenta; positively charged residues, blue; and negatively charged, red. N and C termini and side chains are labeled. D, the same representation of Tk-hefu as in C but showing all atoms. Gray dashed lines denote the long-range NOESY connectivities between the protons of Tk-hefu. E and F, comparison of Tk-hefu (E) and Tk-AMP-X2 (F) structures. The color code is as in C, and the 3₁₀ helix in Tk-AMP-X2 is in smudge green. Side chains of residues discussed in the text are shown as lines and labeled. In C–F, the first structures from the set of 10 are presented (having the fewest restrain violations).

Figure 2.

Molecular model of the K_v1.3–Tk-hefu complex suggests rational design of the Tk-hefu-2 high-affinity peptide. A, overall structure of the K_v1.3–Tk-hefu complex after 200-ns MD simulation inside a hydrated lipid bilayer membrane. K_v1.3 is in gray; the pore domain helices of the second protomer and voltage-sensing domain (VSD) of the fourth protomer, as well as extended extracellular loops of the VSDs are omitted for clarity. Lipids are shown in a space-filling representation; atoms are colored: oxygen, red; phosphorus, orange; nitrogen, blue; carbon of phospholipids (POPC and POPE), yellow; carbon of cholesterol, orange. Some lipids are omitted for clarity. Tk-hefu is presented in pink; the functional dyad residues Tyr-6 and Lys-22, as well as Glu-23 are shown as sticks. B, close-up view on Tk-hefu from A overlaid with Tk-hefu-2 for comparison. Asp-383 and Asp-399 of the channel are shown. Tk-hefu-2 is presented in semi-transparent blue, and lipids are omitted for clarity. C–E, protein surface topography maps showing ELP distribution in K_v1.3–Tk-hefu/Tk-hefu-2 complexes: C, Tk-hefu-2; D, Tk-hefu; E, K_v1.3. Semi-transparent green areas in C and D show contact areas with the K_v1.3 channel in complexes. Black boxes highlight the area, where ELP complementarity may be improved by the E23K substitution in Tk-hefu yielding Tk-hefu-2.

Figure 3.

Interaction energy profiles of Tk-hefu, Tk-hefu-2, -3, and -4 in complex with K_v1.3. Bar chart showing the contribution of amino acid residues to the interaction energy averaged over MD simulation. Error bars indicate standard deviations.

Figure 4.

Tk-hefu-2, -3, and -4 block of human K_v1.3 channels studied by electrophysiology. A, activity of Tk-hefu mutants on K_v1.3 channels expressed in Xenopus laevis oocytes. Traces shown are representatives of at least six independent experiments (n ≥ 6). The dotted line indicates the zero current level. The asterisk (*) distinguishes the steady-state current after application of 10 μm peptide. B, concentration-response curves for Tk-AMP-X2, Tk-hefu, Tk-hefu-2, -3 and -4 on K_v1.3 channels obtained by plotting the percentage of remaining current as a function of increasing ligand concentrations. Error bars indicate the mean ± S.E. C, current-voltage relationship of K_v1.3 channels. Closed symbols, control condition; open symbols, after application of 2 μm Tk-hefu-2. D, percentage of inhibition upon application of 2 μm Tk-hefu-2 at a broad range of potentials is shown.

Figure 5.

Binding surface of Tk-hefu-2 is similar to ChTx. Protein surface topography permitted comparison of ChTx (left column; A, C, and E) and Tk-hefu-2 (right column; B, D, and F) surfaces with respect to ELP (A and B), molecular hydrophobicity potential (C and D), and relief (E and F). All map pairs are colored according to the scales below; panel B is the same as panel C in Fig. 2. Contact area with K_v1.3 is shaded green.