Structure, folding and stability of a minimal homologue fromAnemonia sulcataof the sea anemone potassium channel blocker ShK

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Graphical abstractAsK132958 is a 29-residue peptide identified in a transcriptomic study of Anemonia sulcata. It has the same disulfide framework and a similar structure to ShK. AsK132958 is not active against KV1.3 channels, owing to the lack of a Lys-Tyr dyad and other functionally important amino acid residues. AsK132958 is more resistant to proteolysis than ShK. Introducing a Lys-Tyr functional dyad to the AsK132958 structural scaffold may be a useful way of developing a proteolytically stable KV1.3 blocker.HighlightsAsK132958 is one of the shortest peptides with a ShK/BgK-like cysteine framework.AsK132958 is a structural homologue of ShK.Despite having an ShK-like scaffold, AsK132958 is not active against KV1.3 or related potassium channels.AsK132958 is more resistant to proteases than ShK and is a promising scaffold for engineering other activities.AsK132958 could be an evolutionary precursor of peptides with ShK-like scaffold and activity.Peptide toxins elaborated by sea anemones target various ion-channel sub-types. Recent transcriptomic studies of sea anemones have identified several novel candidate peptides, some of which have cysteine frameworks identical to those of previously reported sequences. One such peptide is AsK132958, which was identified in a transcriptomic study of Anemonia sulcata and has a cysteine framework similar to that of ShK from Stichodactyla helianthus, but is six amino acid residues shorter. We have determined the solution structure of this novel peptide using NMR spectroscopy. The disulfide connectivities and structural scaffold of AsK132958 are very similar to those of ShK but the structure is more constrained. Toxicity assays were performed using grass shrimp (Palaemonetes sp) and Artemia nauplii, and patch-clamp electrophysiology assays were performed to assess the activity of AsK132958 against a range of voltage-gated potassium (KV) channels. AsK132958 showed no activity against grass shrimp, Artemia nauplii, or any of the KV channels tested, owing partly to the absence of a functional Lys-Tyr dyad. Three AsK132958 analogues, each containing a Tyr in the vicinity of Lys19, were therefore generated in an effort to restore binding, but none showed activity against any of KV channels tested. However, AsK132958 and its analogues are less susceptible to proteolysis than that of ShK. Our structure suggests that Lys19, which might be expected to occupy the pore of the channel, is not sufficiently accessible for binding, and therefore that AsK132958 must have a distinct functional role that does not involve KV channels.

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