1. Reinvestigation of the biological activity of d-allo-ShK protein
Bobo Dang, Sandeep Chhabra, Michael W Pennington, Raymond S Norton, Stephen B H Kent J Biol Chem. 2017 Jul 28;292(30):12599-12605. doi: 10.1074/jbc.M117.793943. Epub 2017 Jun 8.
ShK toxin from the sea anemone Stichodactyla helianthus is a 35-residue protein that binds to the Kv1.3 ion channel with high affinity. Recently we determined the X-ray structure of ShK toxin by racemic crystallography, in the course of which we discovered that d-ShK has a near-background IC50 value ~50,000 times lower than that of the l-ShK toxin. This lack of activity was at odds with previously reported results for an ShK diastereomer designated d-allo-ShK, for which significant biological activity had been observed in a similar receptor-blocking assay. As reported, d-allo-ShK was made up of d-amino acids, but with retention of the natural stereochemistry of the chiral side chains of the Ile and Thr residues, i.e. containing d-allo-Ile and d-allo-Thr along with d-amino acids and glycine. To understand its apparent biological activity, we set out to chemically synthesize d-allo-ShK and determine its X-ray structure by racemic crystallography. Using validated allo-Thr and allo-Ile, both l-allo-ShK and d-allo-ShK polypeptide chains were prepared by total chemical synthesis. Neither the l-allo-ShK nor the d-allo-ShK polypeptides folded, whereas both l-ShK and d-ShK folded smoothly under the same conditions. Re-examination of NMR spectra of the previously reported d-allo-ShK protein revealed that diagnostic Thr and Ile signals were the same as for authentic d-ShK. On the basis of these results, we conclude that the previously reported d-allo-ShK was in fact d-ShK, the true enantiomer of natural l-ShK toxin, and that the apparent biological activity may have arisen from inadvertent contamination with trace amounts of l-ShK toxin.
2. Conformational exchange in the potassium channel blocker ShK
Naoto Iwakawa, Nicola J Baxter, Dorothy C C Wai, Nicholas J Fowler, Rodrigo A V Morales, Kenji Sugase, Raymond S Norton, Mike P Williamson Sci Rep. 2019 Dec 17;9(1):19307. doi: 10.1038/s41598-019-55806-3.
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.
3. Substitution of a single residue in Stichodactyla helianthus peptide, ShK-Dap22, reveals a novel pharmacological profile
Richard E Middleton, et al. Biochemistry. 2003 Nov 25;42(46):13698-707. doi: 10.1021/bi035209e.
ShK, a peptide isolated from Stichodactyla helianthus venom, blocks the voltage-gated potassium channels, K(v)1.1 and K(v)1.3, with similar high affinity. ShK-Dap(22), a synthetic derivative in which a diaminopropionic acid residue has been substituted at position Lys(22), has been reported to be a selective K(v)1.3 inhibitor and to block this channel with equivalent potency as ShK [Kalman et al. (1998) J. Biol. Chem. 273, 32697-32707]. In this study, a large body of evidence is presented which indicates that the potencies of wild-type ShK peptide for both K(v)1.3 and K(v)1.1 channels have been previously underestimated. Therefore, the affinity of ShK-Dap(22) for both channels appears to be ca. 10(2)-10(4)-fold weaker than ShK. ShK-Dap(22) does display ca. 20-fold selectivity for human K(v)1.3 vs K(v)1.1 when measured by the whole-cell voltage clamp method but not in equilibrium binding assays. ShK-Dap(22) has low affinity for K(v)1.2 channels, but heteromultimeric K(v)1.1-K(v)1.2 channels form a receptor with ca. 200-fold higher affinity for ShK-Dap(22) than K(v)1.1 homomultimers. In fact, K(v)1.1-K(v)1.2 channels bind ShK-Dap(22) with only ca. 10-fold less potency than ShK and reveal a novel pharmacology not predicted from the homomultimers of K(v)1.1 or K(v)1.2. The concentrations of ShK-Dap(22) needed to inhibit human T cell activation were ca. 10(3)-fold higher than those of ShK, in good correlation with the relative affinities of these peptides for inhibiting K(v)1.3 channels. All of these data, taken together, suggest that ShK-Dap(22) will not have the same in vivo immunosuppressant efficacy of other K(v)1.3 blockers, such as margatoxin or ShK. Moreover, ShK-Dap(22) may have undesired side effects due to its interaction with heteromultimeric K(v)1.1-K(v)1.2 channels, such as those present in brain and/or peripheral tissues.
