Conformational exchange in the potassium channel blocker ShK

Abstract

ShK is a 35-residue disulfide-linked polypeptide produced by the sea anemone Stichodactyla helianthus, which blocks the potassium channels Kv1.1 and Kv1.3 with pM affinity. An analogue of ShK has been developed that blocks Kv1.3 > 100 times more potently than Kv1.1, and has completed Phase 1b clinical trials for the treatment of autoimmune diseases such as psoriasis and rheumatoid arthritis. Previous studies have indicated that ShK undergoes a conformational exchange that is critical to its function, but this has proved difficult to characterise. Here, we have used high hydrostatic pressure as a tool to increase the population of the alternative state, which is likely to resemble the active form that binds to the Kv1.3 channel. By following changes in chemical shift with pressure, we have derived the chemical shift values of the low- and high-pressure states, and thus characterised the locations of structural changes. The main difference is in the conformation of the Cys17-Cys32 disulfide, which is likely to affect the positions of the critical Lys22-Tyr23 pair by twisting the 21-24 helix and increasing the solvent exposure of the Lys22 sidechain, as indicated by molecular dynamics simulations.

Figure 1

¹⁵N HSQC spectra of ShK acquired at pressures from 1 to 2500 bar. Unlabeled peaks are from sidechains.

Figure 2

Differences in the fitted values of ¹H, ¹⁵N, ¹³C′, ¹³Cα and ¹³Cβ shifts between ground state and pressure-induced excited state. Regions of regular secondary structure are shaded. For carbons that have two protons attached, there are often two different shift values.

Figure 3

TALOS-N analysis of backbone dihedral angles in ground and pressure-induced excited states of ShK. (a) φ, (b) ψ backbone dihedral angles. Ground state in black, and excited state in red. Only those residues are shown where the TALOS-N prediction is confident.

Figure 4

The distance between Cα of Cys17 and Cα of Arg24, in an enhanced sampling molecular dynamics trajectory in which each iteration represents 0.2 ns. The blue line is for a negative Cys17-Cys32 χ₃, as in the crystal structure, while the orange line is for a positive χ₃. The downward excursions of the distance (eg at 22.6 ns for the negative angle) are due to changes in the position of the disulfide, rather than a change in the chirality of the angle.

Figure 5

Views of ShK. (a) Overall structure of ShK, colored from blue at the N-terminus to red at the C-terminus, using the PDB file 4LFQ. The six cystines are indicated, plus three residues thought to interact with the Kv1.3 potassium channel (Lys22, Tyr23 and Arg24). (b) Closeup view of the C17-C32 disulfide. The structures shown are iterations 200 from the molecular dynamics simulations, with the ground-state (negative χ₃) in green, and the alternative conformation (positive χ₃) in cyan. (c) End-on view of helix 21–24, showing the positions of Lys22, Tyr23 and Arg24 in iterations 200 from the molecular dynamics simulations. The Cys17-Cys32 disulfide is just visible at the back of the helix at the bottom.