Summary: ShK domain-like
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Stichodactyla toxin Edit Wikipedia article
Stichodactyla toxin (ShK) is a 35-residue basic peptide from the sea anemone Stichodactyla helianthus that blocks a number of potassium channels. An analogue of ShK called ShK-186 or Dalazatide is in human trials as a therapeutic for autoimmune diseases.
- 1 History
- 2 Structure
- 3 Phylogenetic relationships of ShK and ShK domains
- 4 Channel targets
- 5 Extending circulating half-life
- 6 Modulation of T cell function
- 7 Effects on microglia
- 8 Efficacy of analogues in animal models of human diseases
- 8.1 Experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis
- 8.2 Pristane-induced arthritis (PIA), a model for rheumatoid arthritis
- 8.3 Rat models of atopic dermatitis
- 8.4 Psoriasis
- 8.5 Diet-induced obesity and fatty liver disease
- 8.6 Arousal and anesthesia
- 8.7 Preventing brain damage following therapeutic brain radiation
- 8.8 Toxicity of ShK and its analogues
- 9 Functions of ShK-like domains in proteins
- 10 References
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, 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.
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).
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).
|'ShK-F6CA'(IC50)||'ShK-198'(IC50)||'MMP-23 ShK domain'(IC50)|
|Kv1.1||16-28 pM||7 nM||22 nM||5.4 nM||4 nM||159 pM||49 μM|
|Kv1.2||10 nM||48 nM||ND||>100 nM||>100 nM||ND||>100 μM|
|Kv1.3||10-16 pM||70 pM||140 pM||34 pM||48 pM||41 pM||2.8 μM|
|Kv1.6||200 pM||18 nM||10.6 nM||ND||ND||ND||400 nM|
|Kv3.2||5 nM||20 nM||4.2 nM||ND||ND||ND||49 μM|
|KCa3.1||30 nM||115 nM||>100 nM||>100 nM||ND||ND||>100 μM|
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 tetramethyl-rhodamine 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.
- ShK-170, ShK-186, ShK-192 and ShK-EWSS: Based on ShK-F6CA additional analogues were made. Attaching a L-phosphotyrosine to the N-terminus of ShK via an AEEA linker resulted in a peptide, ShK-170, with 100-1000-fold specificity for Kv1.3 over related channels. ShK-186 [a.k.a. SL5; a.k.a. Dalazatide] is identical to ShK-170 except the C-terminal carboxyl is replaced by an amide. ShK-186 blocks Kv1.3 with an IC50 of 69 pM and exhibits the same specificity for Kv1.3 over closely related channels as ShK-170. The L-phosphotyrosine of ShK-170 and ShK-186 rapidly gets dephosphorylated in vivo generating an analogue, ShK-198, with reduced specificity for Kv1.3. To overcome this problem, ShK-192 and ShK-EWSS were developed. In ShK-192, the N-terminal L-phosphotyrosine is replaced by a non-hydrolyzable para-phosphonophenylalanine (Ppa), and Met21 is replaced by the non-natural amino acid norleucine to avoid methionine oxidation. In ShK-EWSS, the AEEA linker and L-phosphotyrosine are replaced by the residues glutamic acid (E), tryptophan (W) and two serines (S). Both ShK-192 and ShK-EWSS are highly specific for Kv1.3 over related channels.
- ShK-K18A: Docking and molecular dynamics simulations on Kv1.3 and Kv1.1 followed by umbrella sampling simulations, paved the way to the selective Kv1.3 inhibitor ShK-K18A.
- ShK-related peptides in parasitic worms: AcK1, a 51-residue peptide from hookworms Ancylostoma caninum and Ancylostoma ceylanicum, and BmK1, the C-terminal domain of a metalloprotease from filarial worm Brugia malayi, adopt helical structures closely resembling ShK. AcK1 and BmK1 block Kv1.3 channels at nanomolar-micromolar concentrations, and they suppress rat effector memory T cells without affecting naïve and central memory T cell subsets. Further, they suppress IFN-g production by human T cells and they inhibit the delayed type hypersensitivity response caused by skin-homing effector memory T cells. Teladorsagia circumcincta is an economically-important parasite that infects sheep and goats. TcK6, a 90-residue protein with a C-terminal ShK-related domain, is upregulated during the mucosal dwelling larval stage of this parasite. TcK6 causes modest suppression of thapsigargin-triggered IFN-g production by sheep T cells, suggesting that the parasite use this protein for immune evasion by modulating mucosal T cells.
Extending circulating half-life
Due to their low molecular mass, ShK and its analogues are prone to rapid renal elimination. In rats, the half-life is ~6 min for ShK-186 and ~11 min for ShK-198, with a clearance rate of ~950 ml/kg·min. In monkeys, the half-life is ~12 min for ShK-186 and ~46 min for ShK-198, with a clearance rate of ~80 ml/kg·min.
PEGylation of ShK: Conjugation of polyethylene glycol (PEG) to ShK[Q16K], an ShK analogue, increased its molecular mass and thereby reduced renal clearance and extended plasma half-life to 15 h in mice and 64 h in cynomolgus monkeys. PEGylation can also decrease immunogenicity and protect a peptide from proteolysis and non-specific adsorption to inert surfaces. PEGylated ShK[Q16K] prevented adoptive-transfer experimental autoimmune encephalomyelitis in rats, a model for multiple sclerosis.
- Conjugation of ShK to larger proteins: The circulating half-life of peptides can be prolonged by coupling them to larger proteins or protein domains. By screening a combinatorial ShK peptide library, novel analogues were identified, which when fused to the C-termini of IgG1-Fc retained picomolar potency, effectively suppressed in vivo delayed type hypersensitivity and exhibited a prolonged circulating half-life.
- Prolonged effects despite rapid plasma clearance: SPECT/CT imaging studies with a 111In-DOTA-conjugate of ShK-186 in rats and squirrel monkeys revealed a slow release from the injection site and blood levels above the channel blocking dose for 2 and 7 days, respectively. Studies on human peripheral blood T cells showed that a brief exposure to ShK-186 was sufficient to suppress cytokine responses. These findings suggest that ShK-186, despite its short circulating half-life, may have a prolonged therapeutic effect. In rats, the peptide is effective in treating disease in animal models of autoimmune diseases when administered once a day to once in 3 days. In humans, subcutaneous injections twice a week are sufficient to ameliorate disease in patients with plaque psoriasis.
The low molecular mass of ShK and its analogues, combined with their high isoelectric points, makes it unlikely that these peptides will be absorbed from the stomach or intestine following oral administration. Sub-lingual delivery is a possibility. A fluorescent ShK analogue was absorbed into the blood stream at pharmacological concentrations following sublingual administration with a mucoadhesive chitosan-based gel, with or without the penetration enhancer cetrimide. Delivery of the peptide as an aerosol through the lung, or across the skin, or as eye drops are also possibilities.
Modulation of T cell function
During T cell-activation, calcium enters lymphocytes through store-operated CRAC channels (calcium release activated channel) formed as a complex of Orai and Stim proteins. The rise in intracellular calcium initiates a signaling cascade culminating in cytokine production and proliferation. The Kv1.3 K+ channel and the calcium-activated KCa3.1 K+ channel in T cells promote calcium entry into the cytoplasm through CRAC by providing a counterbalancing cation efflux. Blockade of Kv1.3 depolarizes the membrane potential of T cells, suppresses calcium signaling and IL-2 production, but not IL2-receptor expression. Kv1.3 blockers have no effect on activation pathways that are independent of a rise in intracellular calcium (e.g. anti-CD28, IL-2). Expression of the Kv1.3 and KCa3.1 channels varies during T cell activation and differentiation into memory T cells. When naïve T cells and central memory T cells (TCM) are activated they upregulate KCa3.1 expression to ~500 per cell without significant change in Kv1.3 numbers. In contrast, when terminally differentiated effector memory subsets (TEM, TEMRA [T effector memory re-expressing CD45RA]) are activated, they upregulate Kv1.3 to 1500 per cell without changes in KCa3.1. The Kv1.3 channel number increases and the KCa3.1 channel number decreases as T cells are chronically activated. As a result of this differential expression, blockers of KCa3.1 channels preferentially suppress the function of naïve and TCM cells, while ShK and its analogues that selectively inhibit Kv1.3 channels preferentially suppress the function of chronically-activated effector memory T cells (TEM, TEMRA).
Of special interest are the large number of ShK analogues developed at Amgen that suppressed interleukin-2 and interferon gamma production by T cells. This inhibitory effect of Kv1.3 blockers is partial and stimulation strength dependent, with reduced inhibitory efficacy on T cells under strengthened anti-CD3/CD28 stimulation. Chronically-activated CD28null effector memory T cells are implicated in autoimmune diseases (e.g. lupus, Crohn’s disease, rheumatoid arthritis, multiple sclerosis).
Blockade of Kv1.3 channels in these chronically-activated T cells suppresses calcium signaling, cytokine production (interferon gamma, interleukin-2, interleukin 17), and cell proliferation. Effector memory T cells that are CD28+ are refractory to suppression by Kv1.3 blockers when they are co-stimulated by anti-CD3 and anti-CD28 antibodies, but are sensitive to suppression when stimulated by anti-CD3 antibodies alone. In vivo, ShK-186 paralyzes effector-memory T cells at the site of an inflammatory delayed type hypersensitivity response and prevents these T cells from activating in the inflamed tissue. In contrast, ShK-186 does not affect the homing and motility of naive and TCM cells to and within lymph nodes, most likely because these cells express the KCa3.1 channel and are therefore protected from the effect of Kv1.3 blockade.
Effects on microglia
Kv1.3 plays an important role in microglial activation. ShK-223, an analogue of ShK-186, decreased lipopolysaccharide (LPS) induced focal adhesion formation by microglia, reversed LPS-induced inhibition of microglial migration, and inhibited LPS-induced upregulation of EH domain containing protein 1 (EHD1), a protein involved in microglia trafficking. Increased Kv1.3 expression was reported in microglia in Alzheimer plaques. Kv1.3 inhibitors may have use in the management of Alzheimer’s disease, as reported in a proof-of-concept study in which a small molecule Kv1.3 blocker (PAP-1) alleviated Alzheimer’s disease-like characteristics in a mouse model of AD.
Efficacy of analogues in animal models of human diseases
Experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis
ShK, ShK-Dap22, ShK-170 and PEGylated ShK-Q16K prevent adoptive-transfer EAE in Lewis rats, a model of multiple sclerosis. Since multiple sclerosis is a relapsing-remitting disease, ShK-186 and ShK-192 were evaluated in a relapsing-remitting EAE model in DA (Dark Agouti) rats. Both prevented and treated disease when administered once a day to once in three days. Thus, Kv1.3 inhibitors are effective in treating disease in rat models of multiple sclerosis when administered alone, and therapeutic effectiveness does not appear to be compromised by compensatory over-expression of KCa3.1 channels.
Pristane-induced arthritis (PIA), a model for rheumatoid arthritis
ShK-186 was effective in treating PIA when administered every day or on alternate days. A scorpion toxin inhibitor of KV1.3 was also effective in this model. In both these studies, blockade of Kv1.3 alone was sufficient to ameliorate disease and simultaneous blockade of KCa3.1 was not necessary as has been suggested.
Rat models of atopic dermatitis
Most infiltrating T-cells in skin lesions from patients with moderate-to-severe atopic dermatitis (AD) express high levels of Kv1.3, suggesting that inhibitors of Kv1.3 may be effective in treating AD. Ovalbumin-induced delayed type hypersensitivity and oxazolone-induced dermatitis are considered to be models of atopic dermatitis. ShK, ShK-170, ShK-186, ShK-192 and ShK-IgG-Fc were all effective in the ovalbumin-induced delayed type hypersensitivity model, while a topical formulation of ShK-198 was effective in treating oxazolone-induced dermatitis. Even where compensation by KCa3.1 channels was reported to over-ride KV1.3 block, ShK administered alone suppressed delayed type hypersensitivity significantly in 2 of 3 studies, albeit modestly.
Psoriasis is a severe autoimmune disease of the skin that afflicts many people worldwide. Despite the success of recent biologics in ameliorating disease, there is still a search for safe and effective drugs for psoriasis. KV1.3 inhibitors (ShK, PAP-1) have been reported to treat disease in psoriasiform (psoriasis-like) SCID (severe combined immunodeficiency) mouse model. In a Phase 1b placebo-controlled clinical study in patients with plaque psoriasis, ShK-186 administered twice a week (30 or 60 mg/dose/patient) by subcutaneous injection caused improvements with a statistically significant reduction in their PASI (Psoriasis Area and Severity Index) score between baseline and day 32. These patients also exhibited reduced plasma levels of multiple inflammation markers and decreased expression of T cell activation markers on peripheral blood memory T cells.
Diet-induced obesity and fatty liver disease
Obesity and diabetes are major healthcare problems globally. There is need for safe drugs for these metabolic diseases. In a mouse model of diet-induced obesity, ShK-186 counteracted the negative effects of increased caloric intake. It reduced weight gain, adiposity, and fatty liver; decreased blood levels of cholesterol, sugar, HbA1c, insulin, and leptin; and enhanced peripheral insulin sensitivity. Genetic deletion of the Kv1.3 gene has the same effect, indicating that ShK-186’s effect is due to Kv1.3 blockade. At least two mechanisms contribute to ShK-186’s therapeutic benefits. The high calorie diet induced Kv1.3 expression in brown fat tissues. By blocking Kv1.3, ShK-186 doubled glucose uptake and increased β-oxidation of fatty acids, glycolysis, fatty acid synthesis and uncoupling protein 1 expression by brown fat. As a consequence of brown fat activation, oxygen consumption and energy expenditure were augmented. The obesity diet also induced Kv1.3 expression in the liver, and ShK-186 caused profound alterations in energy and lipid metabolism in the liver. ShK, its analogues or other Kv1.3 blockers may have use in controlling the negative consequences of high calorie diets.
Arousal and anesthesia
The mechanisms of general anesthesia involve multiple molecular targets and pathways that are not completely understood. Sevoflurane is a common anesthetic used to induce general anesthesia during surgery. Rats continually exposed to sevoflurane lose their righting reflex as an index of loss of consciousness. In these rats, microinfusion of ShK into the central medial thalamic nucleus (CMT) reversed sevoflurane-induced anesthesia in rodents. ShK-treated rats righted themselves fully (restored consciousness) despite being continually exposed to sevoflurane. ShK-microinfusion into neighboring regions of the brain did not have this effect. Sevoflurane enhanced potassium currents in the CMT, while ShK and ShK-186 countered this effect. These studies suggest that ShK-sensitive K+ channels in the CMT are important for suppressing arousal during anesthesia.
Preventing brain damage following therapeutic brain radiation
Brain radiation is used to treat tumors of the head, neck, and brain, but this treatment carries a significant risk of neurologic injury. Injury is, in part, due to the activation of microglia and microglia-mediated damage of neurons. Neuroprotective therapies for radiation-induced brain injury are still limited. In a mouse model of brain radiation, ShK-170 reversed neurological deficits, and protected neurons from radiation-induced brain injury by suppressing microglia.
Toxicity of ShK and its analogues
ShK and ShK-Dap22
ShK peptide has a low toxicity profile in mice. ShK is effective in treating autoimmune diseases at 10 to 100 mg/kg bodyweight. It has a median paralytic dose of approximately 25 mg/kg bodyweight (250-2500 higher than the pharmacological dose). In rats the therapeutic safety index is greater than 75-fold. ShK-Dap22 displayed a lower toxicity profile. A 1.0 mg dose did not induce any hyperactivity, seizures or mortality in rats. The median paralytic dose for ShK-Dap22 is about 200 mg/kg bodyweight (2000-20000 higher than pharmacological dose). PEGylated ShK[Q16K] showed no adverse toxicity in monkeys over a period of several months.
ShK-186 also displays a low toxicity profile in rats. Daily administration of ShK-170 or ShK-186 (100 µg/kg/day) by subcutaneous injection over 4 weeks in rats does not induce any changes in blood counts, blood chemistry or histopathology. By virtue of suppressing only TEM and TEMRA cells, ShK-186 did not compromise protective immune responses to influenza virus and chlamydial infection in rats, most likely because naïve and TCM cells unaffected by Kv1.3 blockade mounted effective immune responses. ShK-186 is poorly immunogenic and did not elicit anti-ShK antibodies in rats repeatedly administered the peptide. This is possibly because the peptide’s disulfide-bonded structure hinders processing and antigen presentation by antigen-presenting cells. ShK-186 also shares sequence and structural similarity to a ShK-like domain in matrix metalloprotease 23, which may cause the immune system to assume it is a normal protein in the body. ShK-186 was safe in non-human primates. In Phase 1a and 1b trials in healthy human volunteers, ShK-186 was well tolerated, no grade 3 or 4 adverse effects or laboratory abnormalities were noted, and the predicted range of drug exposures were achieved. The most common adverse events were temporary mild (Grade 1) hypoesthesia and paresthesia involving the hands, feet, or perioral area. Mild muscle spasms, sensitivity of teeth, and injection site pain were also observed.
Functions of ShK-like domains in proteins
MMP-23 belongs to the family of zinc- and calcium-dependent matrix metalloproteases. It is anchored in the cell membrane by an N-terminal prodomain, and it contains three extracellular domains: catalytic metalloprotease domain, ShK domain and immunoglobulin-like cell adhesion molecule (Ig-CaM) domain. The prodomain traps the voltage-gated potassium channel KV1.3, but not the closely related KV1.2 channel, in the endoplasmic reticulum. Studies with chimeras suggest that the prodomain interacts with the KV1.3 region from the S5 transmembrane segment to the C terminus. NMR studies of the prodomain reveal a single trans-membrane alpha-helix, joined by a short linker to a juxta-membrane alpha-helix, which is associated with the surface of the membrane. The prodomain shares topological similarity with proteins (KCNE1, KCNE2, KCNE4) known to trap potassium channels in the secretory pathway, suggesting a shared mechanism of channel regulation. MMP-23’s catalytic domain displays structural homology with catalytic domains in other metalloproteases, and likely functions as an endopeptidase. MMP-23’s ShK domain lies immediately after the catalytic domain and is connected to the IgCAM domain by a short proline-rich linker. It shares phylogenetic relatedness to sea anemone toxins and ICR-CRISP domains, being most similar to the BgK toxin from sea anemone Bunodosoma granulifera. This ShK domain blocks voltage-gated potassium channels (KV1.6 > KV1.3 > KV1.1 = KV3.2 > Kv1.4, in decreasing potency) in the nanomolar to low micromolar range. KV1.3 is required for sustaining calcium signaling during activation of human T cells. By trapping KV1.3 in the endoplasmic reticulum via the prodomain, and by blocking the KV1.3 channel with the ShK domain, MMP-23 may serve as an immune checkpoint to reduce excessive T cell activation during an immune response. In support, increased expression of MMP-23 in melanoma cancer cells decreases tumor-infiltrating lymphocytes, and is associated with cancer recurrence and shorter periods of progression-free survival. However, in melanomas, expression of MMP-23 does not correlate with Kv1.3 expression, suggesting that MMP-23’s deleterious effect in melanomas may not be connected with its Kv1.3 channel-modulating function. MMP-23’s C-terminal IgCAM domain shares sequence similarity with IgCAM domains in proteins known to mediate protein-protein and protein-lipid interactions (e.g. CDON, human Brother of CDO, ROBO1-4, hemicentin, NCAM1 and NCAM2). In summary, the four domains of MMP-23 may work synergistically to modulate immune responses in vivo.
In male Caenorhabditis elegans worms, the absence of a protein called Mab7 results in malformed sensory rays that are required for mating. Introduction of Mab7 into these male worms restores normal development of normal sensory rays. Introduction of Mab7 proteins lacking the ShK domain does not correct the defect of sensory rays, suggesting a role for the ShK-domain of Mab7 in sensory ray development.
HMP2 and PMP1
HMP2 and PMP-1 are astacin metalloproteinases from the Cnidarian Hydra vulgaris and the jellyfish Podocoryne carnea that contain ShK-like domains at their C-termini. Both these ShK-domains contain the critical pore-occluding lysine required for K+ channel block. HMP2 plays a critical role in foot regeneration of Hydra, while PMP1 is found in the feeding organ of the jelly fish and the ShK-domain may paralyze prey after they are ingested.
The 2018 version of this article has passed academic peer review (here), has been published in WikiJournal of Science and can be cited as:
"ShK toxin: history, structure and therapeutic applications for autoimmune diseases". WikiJournal of Science. 1 (1). 2018. doi:10.15347/wjs/2018.003.
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This tab holds the annotation information that is stored in the Pfam database. As we move to using Wikipedia as our main source of annotation, the contents of this tab will be gradually replaced by the Wikipedia tab.
ShK domain-like Provide feedback
This domain of is found in several C. elegans proteins. The domain is 30 amino acids long and rich in cysteine residues. There are 6 conserved cysteine positions in the domain that form three disulphide bridges. The domain is found in the potassium channel inhibitor ShK in sea anemone .
Tudor JE, Pallaghy PK, Pennington MW, Norton RS; , Nat Struct Biol. 1996;3:317-320.: Solution structure of ShK toxin, a novel potassium channel inhibitor from a sea anemone. PUBMED:8599755 EPMC:8599755
Castaneda O, Sotolongo V, Amor AM, Stocklin R, Anderson AJ, Harvey AL, Engstrom A, Wernstedt C, Karlsson E; , Toxicon. 1995;33:603-613.: Characterization of a potassium channel toxin from the Caribbean Sea anemone Stichodactyla helianthus. PUBMED:7660365 EPMC:7660365
External database links
This tab holds annotation information from the InterPro database.
InterPro entry IPR003582
BgK, a 37-residue peptide toxin from the sea anemone Bunodosoma granulifera, and ShK, a 35-residue peptide toxin from the sea anemone Stichodactyla helianthus, are potent inhibitors of K(+) channels. There is a large superfamily of proteins that contains domains (referred to as ShKT domains) ressembling these two toxins. Many of these proteins are metallopeptidases, whereas others are prolyl-4-hydroxylases, tyrosinases, peroxidases, oxidoreductases, or proteins containing epidermal growth factor-like domains, thrombospondin-type repeats, or trypsin-like serine protease domains [PUBMED:19965868]. The ShKT domain has also been called NC6 (nematode six-cysteine) domain [PUBMED:10950959], SXC (six-cysteine) domain [PUBMED:10950959, PUBMED:11412804, PUBMED:9851921, PUBMED:14653817] and ICR (ion channel regulator) [PUBMED:19965868, PUBMED:16339766]. The ShKT domain is short (36 to 42 amino acids), with six conserved cysteines and a number of other conserved residues. The fold adopted by the ShKT domain contains two nearly perpendicular stretches of helices, with no additional canonical secondary structures [PUBMED:9020148]. The globular architecture of the ShKT domain is stabilised by three disulfides, one of them linking the two helices. In venomous creatures, the ShKT domain may have been modified to give rise to potent ion channel blockers, whereas the incorporation of this domain into plant oxidoreductases and prolyl hydroxylases and into worm astacin-like metalloproteases and trypsin-like serines protaeses produced enzymes with potential channel-modulatory activity.
Some proteins known to contain a ShKT domain are listed below:
- Caribbean sea anemone ShK, a potassium channel toxin [PUBMED:7660365].
- Sea anemone BgK, a potassium channel toxin [PUBMED:9020148].
- Toxocara canis family of secreted mucins Tc-MUC-1 to -5, which are implicated in immune evasion. They combine two evolutionarily distinct modules, the mucin and ShkT domains [PUBMED:10950959, PUBMED:11412804].
- Some Caenorhabditis elegans astacin-like proteins (nematode astacins, NAS), metalloproteases [PUBMED:14653817].
- Vertebrate cysteine-rich secretory proteins (Crisp) [PUBMED:16339766].
- Mammalian microfibrillar-associated protein 2 (MFAP2 or MAGP1), a matrix protein.
- Plant prolyl 4-hydroxylase.
Below is a listing of the unique domain organisations or architectures in which this domain is found. More...
The graphic that is shown by default represents the longest sequence with a given architecture. Each row contains the following information:
- the number of sequences which exhibit this architecture
a textual description of the architecture, e.g. Gla, EGF x 2, Trypsin.
This example describes an architecture with one
Gladomain, followed by two consecutive
EGFdomains, and finally a single
- a link to the page in the Pfam site showing information about the sequence that the graphic describes
- the UniProt description of the protein sequence
- the number of residues in the sequence
- the Pfam graphic itself.
Note that you can see the family page for a particular domain by clicking on the graphic. You can also choose to see all sequences which have a given architecture by clicking on the Show link in each row.
Finally, because some families can be found in a very large number of architectures, we load only the first fifty architectures by default. If you want to see more architectures, click the button at the bottom of the page to load the next set.
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Members of this clan include the Crisp domain which is involved in ryanodine receptor Ca2+ signalling, and the ShK domain which is named after the ShK channel inhibitor toxin. Both domains are cysteine rich and contain multiple disulphide bonds .
The clan contains the following 2 members:Crisp ShK
We store a range of different sequence alignments for families. As well as the seed alignment from which the family is built, we provide the full alignment, generated by searching the sequence database (reference proteomes) using the family HMM. We also generate alignments using four representative proteomes (RP) sets, the UniProtKB sequence database, the NCBI sequence database, and our metagenomics sequence database. More...
There are various ways to view or download the sequence alignments that we store. We provide several sequence viewers and a plain-text Stockholm-format file for download.
We make a range of alignments for each Pfam-A family:
- the curated alignment from which the HMM for the family is built
- the alignment generated by searching the sequence database using the HMM
- Representative Proteomes (RPs) at 15%, 35%, 55% and 75% co-membership thresholds
- alignment generated by searching the UniProtKB sequence database using the family HMM
- alignment generated by searching the NCBI sequence database using the family HMM
- alignment generated by searching the metagenomics sequence database using the family HMM
You can see the alignments as HTML or in three different sequence viewers:
- a Java applet developed at the University of Dundee. You will need Java installed before running jalview
- an HTML page showing the whole alignment.Please note: full Pfam alignments can be very large. These HTML views are extremely large and often cause problems for browsers. Please use either jalview or the Pfam viewer if you have trouble viewing the HTML version
- an HTML-based representation of the alignment, coloured according to the posterior-probability (PP) values from the HMM. As for the standard HTML view, heatmap alignments can also be very large and slow to render.
You can download (or view in your browser) a text representation of a Pfam alignment in various formats:
You can also change the order in which sequences are listed in the alignment, change how insertions are represented, alter the characters that are used to represent gaps in sequences and, finally, choose whether to download the alignment or to view it in your browser directly.
You may find that large alignments cause problems for the viewers and the reformatting tool, so we also provide all alignments in Stockholm format. You can download either the plain text alignment, or a gzipped version of it.
We make a range of alignments for each Pfam-A family. You can see a description of each above. You can view these alignments in various ways but please note that some types of alignment are never generated while others may not be available for all families, most commonly because the alignments are too large to handle.
1Cannot generate PP/Heatmap alignments for seeds; no PP data available
Key: available, not generated, — not available.
Format an alignment
We make all of our alignments available in Stockholm format. You can download them here as raw, plain text files or as gzip-compressed files.
You can also download a FASTA format file containing the full-length sequences for all sequences in the full alignment.
HMM logos is one way of visualising profile HMMs. Logos provide a quick overview of the properties of an HMM in a graphical form. You can see a more detailed description of HMM logos and find out how you can interpret them here. More...
If you find these logos useful in your own work, please consider citing the following article:
This page displays the phylogenetic tree for this family's seed alignment. We use FastTree to calculate neighbour join trees with a local bootstrap based on 100 resamples (shown next to the tree nodes). FastTree calculates approximately-maximum-likelihood phylogenetic trees from our seed alignment.
Note: You can also download the data file for the tree.
Curation and family details
This section shows the detailed information about the Pfam family. You can see the definitions of many of the terms in this section in the glossary and a fuller explanation of the scoring system that we use in the scores section of the help pages.
|Seed source:||Pfam-B_662 (release 4.0)|
|Author:||Bashton M , Bateman A|
|Number in seed:||137|
|Number in full:||9610|
|Average length of the domain:||36.30 aa|
|Average identity of full alignment:||28 %|
|Average coverage of the sequence by the domain:||20.22 %|
|HMM build commands:||
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 45638612 -E 1000 --cpu 4 HMM pfamseq
|Family (HMM) version:||24|
|Download:||download the raw HMM for this family|
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This visualisation provides a simple graphical representation of the distribution of this family across species. You can find the original interactive tree in the More....
This chart is a modified "sunburst" visualisation of the species tree for this family. It shows each node in the tree as a separate arc, arranged radially with the superkingdoms at the centre and the species arrayed around the outermost ring.
How the sunburst is generated
The tree is built by considering the taxonomic lineage of each sequence that has a match to this family. For each node in the resulting tree, we draw an arc in the sunburst. The radius of the arc, its distance from the root node at the centre of the sunburst, shows the taxonomic level ("superkingdom", "kingdom", etc). The length of the arc represents either the number of sequences represented at a given level, or the number of species that are found beneath the node in the tree. The weighting scheme can be changed using the sunburst controls.
In order to reduce the complexity of the representation, we reduce the number of taxonomic levels that we show. We consider only the following eight major taxonomic levels:
Colouring and labels
Segments of the tree are coloured approximately according to their superkingdom. For example, archeal branches are coloured with shades of orange, eukaryotes in shades of purple, etc. The colour assignments are shown under the sunburst controls. Where space allows, the name of the taxonomic level will be written on the arc itself.
As you move your mouse across the sunburst, the current node will be highlighted. In the top section of the controls panel we show a summary of the lineage of the currently highlighed node. If you pause over an arc, a tooltip will be shown, giving the name of the taxonomic level in the title and a summary of the number of sequences and species below that node in the tree.
Anomalies in the taxonomy tree
There are some situations that the sunburst tree cannot easily handle and for which we have work-arounds in place.
Missing taxonomic levels
Some species in the taxonomic tree may not have one or more of the main eight levels that we display. For example, Bos taurus is not assigned an order in the NCBI taxonomic tree. In such cases we mark the omitted level with, for example, "No order", in both the tooltip and the lineage summary.
Unmapped species names
The tree is built by looking at each sequence in the full alignment for the family. We take the name of the species given by UniProt and try to map that to the full taxonomic tree from NCBI. In some cases, the name chosen by UniProt does not map to any node in the NCBI tree, perhaps because the chosen name is listed as a synonym or a misspelling in the NCBI taxonomy.
So that these nodes are not simply omitted from the sunburst tree, we group them together in a separate branch (or segment of the sunburst tree). Since we cannot determine the lineage for these unmapped species, we show all levels between the superkingdom and the species as "uncategorised".
Since we reduce the species tree to only the eight main taxonomic levels, sequences that are mapped to the sub-species level in the tree would not normally be shown. Rather than leave out these species, we map them instead to their parent species. So, for example, for sequences belonging to one of the Vibrio cholerae sub-species in the NCBI taxonomy, we show them instead as belonging to the species Vibrio cholerae.
Too many species/sequences
For large species trees, you may see blank regions in the outer layers of the sunburst. These occur when there are large numbers of arcs to be drawn in a small space. If an arc is less than approximately one pixel wide, it will not be drawn and the space will be left blank. You may still be able to get some information about the species in that region by moving your mouse across the area, but since each arc will be very small, it will be difficult to accurately locate a particular species.
The tree shows the occurrence of this domain across different species. More...
We show the species tree in one of two ways. For smaller trees we try to show an interactive representation, which allows you to select specific nodes in the tree and view them as an alignment or as a set of Pfam domain graphics.
Unfortunately we have found that there are problems viewing the interactive tree when the it becomes larger than a certain limit. Furthermore, we have found that Internet Explorer can become unresponsive when viewing some trees, regardless of their size. We therefore show a text representation of the species tree when the size is above a certain limit or if you are using Internet Explorer to view the site.
If you are using IE you can still load the interactive tree by clicking the "Generate interactive tree" button, but please be aware of the potential problems that the interactive species tree can cause.
For all of the domain matches in a full alignment, we count the number that are found on all sequences in the alignment. This total is shown in the purple box.
We also count the number of unique sequences on which each domain is found, which is shown in green. Note that a domain may appear multiple times on the same sequence, leading to the difference between these two numbers.
Finally, we group sequences from the same organism according to the NCBI code that is assigned by UniProt, allowing us to count the number of distinct sequences on which the domain is found. This value is shown in the pink boxes.
We use the NCBI species tree to group organisms according to their taxonomy and this forms the structure of the displayed tree. Note that in some cases the trees are too large (have too many nodes) to allow us to build an interactive tree, but in most cases you can still view the tree in a plain text, non-interactive representation. Those species which are represented in the seed alignment for this domain are highlighted.
You can use the tree controls to manipulate how the interactive tree is displayed:
- show/hide the summary boxes
- highlight species that are represented in the seed alignment
- expand/collapse the tree or expand it to a given depth
- select a sub-tree or a set of species within the tree and view them graphically or as an alignment
- save a plain text representation of the tree
Please note: for large trees this can take some time. While the tree is loading, you can safely switch away from this tab but if you browse away from the family page entirely, the tree will not be loaded.
For those sequences which have a structure in the Protein DataBank, we use the mapping between UniProt, PDB and Pfam coordinate systems from the PDBe group, to allow us to map Pfam domains onto UniProt sequences and three-dimensional protein structures. The table below shows the structures on which the ShK domain has been found. There are 5 instances of this domain found in the PDB. Note that there may be multiple copies of the domain in a single PDB structure, since many structures contain multiple copies of the same protein sequence.
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