Stichodactyla helianthus Neurotoxin (ShK)

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.

ShK toxin is a cysteine-rich 35-residue protein ion-channel ligand isolated from the sea anemone Stichodactyla helianthus. In this work, we studied the effect of inverting the side chain stereochemistry of individual Thr or Ile residues on the properties of the ShK protein. Molecular dynamics simulations were used to calculate the free energy cost of inverting the side-chain stereochemistry of individual Thr or Ile residues. Guided by the computational results, we used chemical protein synthesis to prepare three ShK polypeptide chain analogues, each containing either an allo-Thr or an allo-Ile residue. The three allo-Thr or allo-Ile-containing ShK polypeptides were able to fold into defined protein products, but with different folding propensities. Their relative thermal stabilities were measured and were consistent with the MD simulation data. Structures of the three ShK analogue proteins were determined by quasi-racemic X-ray crystallography and were similar to wild-type ShK. All three ShK analogues retained ion-channel blocking activity.

ShK toxin, a potassium channel blocker from the sea anemone Stichodactyla helianthus, is a 35 residue polypeptide cross-linked by three disulfide bridges: Cys3-Cys35, Cys12-Cys28, and Cys17-Cys32. To investigate the role of these disulfides in the structure and channel-blocking activity of ShK toxin, a series of analogues was synthesized by selective replacement of each pair of half-cystines with two alpha-amino-butyrate (Abu) residues. The remaining two disulfide pairs were formed unambiguously using an orthogonal protecting group strategy of Cys(Trt) or Cys(Acm) at the appropriate position. The peptides were tested in vitro for their ability to block Kv1.1 and Kv1.3 potassium channels and their ability to displace [(125)I]dendrotoxin binding to rat brain synaptosomal membranes. The monocyclic peptides showed no activity in these assays. Of the dicyclic peptides, [Abu12,28]ShK(3-35,17)(-)(32) (where the subscript indicates disulfide connectivities) had weak activity on Kv1.3 and Kv1.1. [Abu17,32]ShK(3-35,12)(-)(28) blocked Kv1.3 with low nanomolar potency, but was less effective (being comparable to [Abu12,28]ShK(3-35,17)(-)(32)) against Kv1.1. [Abu3, 35]ShK(12-28,17)(-)(32), retained high picomolar affinity against both channels. Corroborating these results, [Abu3,35]ShK(12-28, 17)(-)(32) had an IC(50) ratio relative to native toxin of 18 in the displacement assay, whereas [Abu17,32]ShK(3-35,12)(-)(28) and [Abu12, 28]ShK(3-35,17)(-)(32) had ratios of 69 and 390, respectively. Thus, the disulfide bond linking the N- and C-terminal regions is less important for activity than the internal disulfides. NMR analysis of the [Abu12,28] and [Abu17,32] analogues indicated that they had little residual structure, consistent with their significantly reduced activities. By contrast, [Abu3,35]ShK(12-28,17)(-)(32) had a moderately well-defined solution structure, with a mean pairwise root-mean-square deviation of 1.33 A over the backbone heavy atoms. This structure nevertheless showed significant differences from that of native ShK toxin. The possible interactions of this analogue with the channel and the distinction between native secondary and tertiary structure on one hand and global topology imposed by the disulfide bridges on the other are discussed.