Stichodactyla toxin

Stichodactyla toxin (ShK, ShkT) is a 35-residue basic peptide from the sea anemone Stichodactyla helianthus that blocks a number of potassium channels. Related peptides form a conserved family of protein domains known as the ShkT domain. Another well-studied toxin of the family is BgK from Bunodosoma granulifera. An analogue of the toxin called Dalazatide is in human trials as a therapeutic for autoimmune diseases.

History

Stichodactyla helianthus is a species of sea anemone (Phylum: Cnidaria) belonging to the family Stichodactylidae. Helianthus comes from the Greek words helios meaning sun, and anthos meaning flower, which corresponds to the species' common name "sun anemone". It is sessile and uses potent neurotoxins for defense against its primary predator, the spiny lobster. The venom contains, among other components, numerous ion channel-blocking peptides. In 1995, a group led by Olga Castaneda and Evert Karlsson isolated ShK, a potassium channel-blocking 35-residue peptide from S. helianthus. The same year, William Kem and his collaborator Michael Pennington synthesized and folded ShK, and showed it blocked neuronal and lymphocyte voltage-dependent potassium channels. In 1996, Ray Norton determined the three-dimensional structure of ShK. In 2005–2006, George Chandy, Christine Beeton and Michael Pennington developed ShK-170 and ShK-186 (ShK-L5), selective blockers of Kv1.3. ShK-186, now called Dalazatide, was advanced to human trials in 2015-2017 by Shawn Iadonato and Eric Tarcha, as the first-in-man Kv1.3 blocker for autoimmune disease.

Structure

ShK is cross-linked by three disulfide bridges: Cys3-Cys35, Cys12-Cys28, and Cys17-Cys32. The solution structure of ShK reveals two short α-helices comprising residues 14-19 and 21–24; the N-terminal eight residues adopt an extended conformation, followed by a pair of interlocking turns that resemble a 310 helix; the C-terminal Cys35 residue forms a nearly head-to-tail cyclic structure through a disulfide bond with Cys3.

Phylogenetic relationships of ShK and ShK domains

The SMART database at the EMBL, as of May 2018, lists 3345 protein domains with structural resemblance to ShK in 1797 proteins (1 to 8 domains/protein), many in the worm Caenorhabditis elegans and venomous snakes. The majority of these domains are in metallopeptidases, whereas others are in prolyl 4-hydroxylases, tyrosinases, peroxidases, oxidoreductases, or proteins containing epidermal growth factor-like domains, thrombospondin-type repeats, or trypsin-like serine protease domains. The only human proteins containing ShK-like domains are MMP-23 (matrix metalloprotease 23) and MFAP-2 (microfibril-associated glycoprotein 2).

Channel targets

The ShK peptide blocks potassium (K+) ion channels Kv1.1, Kv1.3, Kv1.6, Kv3.2 and KCa3.1 with nanomolar to picomolar potency, and has no effect on the HERG (Kv11.1) cardiac potassium channel. The neuronal Kv1.1 channel and the T lymphocyte Kv1.3 channel are most potently inhibited by ShK.

Binding configuration in K+ channels ShK and its analogues are blockers of the channel pore. They bind to all four subunits in the K+ channel tetramer by interacting with the shallow 'vestibule' at the outer entrance to the channel pore. These peptides are anchored in the external vestibule by two key interactions. The first is Lys22, which protrudes into and occludes the channel's pore like a "cork in a bottle" and blocks the passage of potassium ions through the channel pore. The second is the neighboring Tyr23, which together with Lys22 forms a "functional dyad" required for channel block. Many K+ channel-blocking peptides contain such a dyad of a lysine and a neighboring aromatic or aliphatic residue. Some K+ channel-blocking peptides lack the functional dyad, but even in these peptides a lysine physically blocks the channel, regardless of the position of the lysine in the peptide sequence. Additional interactions anchor ShK and its analogues in the external vestibule and contribute to potency and selectivity. For example, Arg11 and Arg29 in ShK interact with two Asp386 residues in adjacent subunits in the mouse Kv1.3 external vestibule (corresponds to Asp433 in human Kv1.3).

Analogues that block the Kv1.3 channel Several ShK analogues have been generated to enhance specificity for the Kv1.3 channel over the neuronal Kv1.1 channel and other closely related channels.

ShK-Dap22: This was the first analogue that showed some degree of specificity for Kv1.3. The pore-occluding lysine22 of ShK is replaced by diaminopropionic acid (Dap) in ShK-Dap22. Dap is a non-natural lysine analogue with a shorter side chain length (2.5 Å from Cα) than lysine (6.3 Å). Dap22 interacts with residues further out in the external vestibule in contrast to lysine22, which interacts with the channel's selectivity filter. As a consequence, the orientations of ShK and ShK-Dap22 in the external vestibule are significantly different. ShK-Dap22 exhibits >20-fold selectivity for Kv1.3 over closely related channels in whole-cell patch clamp experiments, but in equilibrium binding assays it binds Kv1.1-Kv1.2 heterotetramers with almost the same potency as ShK, which is not predicted from the study of homotetrameric Kv1.1 or Kv1.2 channels.ShK-F6CA: Attaching a fluorescein to the N-terminus of the peptide via a hydrophilic AEEA linker (2-aminoethoxy-2-ethoxy acetic acid; mini-PEG) resulted in a peptide, ShK-F6CA (fluorescein-6-carboxyl), with 100-fold specificity for Kv1.3 over Kv1.1 and related channels. Attachment of a tetramethylrhodamine or a biotin via the AEEA linker to ShK's N-terminus did not increase specificity for Kv1.3 over Kv1.1. The enhanced specificity of ShK-F6CA might be explained by differences in charge: F6CA is negatively charged; tetramethylrhodamine is positively charged; and biotin is neutral. Subsequent studies with other analogues suggest that the negatively charged F6CA likely interacts with residues on the turret of the Kv1.3 channel as shown for ShK-192 and ShK-EWSS.

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