The voltage-gated potassium channel Kv1.3 is an important target for the treatment of autoimmune diseases and asthma. Blockade of Kv1.3 by the sea anemone peptide ShK inhibits the proliferation of effector memory T lymphocytes and ameliorates autoimmune diseases in animal models. However, the lack of selectivity of ShK for Kv1.3 over the Kv1.1 subtype has driven a search for Kv1.3-selective analogues. Here we describe N-terminally extended analogues of ShK that contain a negatively-charged Glu, designed to mimic the phosphonate adduct in earlier Kv1.3-selective analogues, and that consist entirely of common protein amino acids. Molecular dynamic simulations indicated that a Trp residue at position [-3] of the tetrapeptide extension could form stable interactions with Pro377 of Kv1.3 and best discriminates between Kv1.3 and Kv1.1. This led to the development of [EWSS]ShK, which inhibits Kv1.3 with an IC50 of 34 pM and is 158-fold selective for Kv1.3 over Kv1.1. In addition, [EWSS]ShK is more than 2900-fold more selective for Kv1.3 over Kv1.2 and KCa3.1 channels. As a highly Kv1.3-selective analogue of ShK based entirely on protein amino acids, which can be produced by recombinant expression, this peptide is a valuable addition to the complement of therapeutic candidates for the treatment of autoimmune diseases.
ShK; potassium channels; electrophysiology; molecular dynamics; N-terminal extension
MSP2 is an intrinsically disordered protein that is abundant on the merozoite surface and essential to the parasite Plasmodium falciparum. Naturally-acquired antibody responses to MSP2 are biased towards dimorphic sequences within the central variable region of MSP2 and have been linked to naturally-acquired protection from malaria. In a phase IIb study, an MSP2-containing vaccine induced an immune response that reduced parasitemias in a strain-specific manner. A subsequent phase I study of a vaccine that contained both dimorphic forms of MSP2 induced antibodies that exhibited functional activity in vitro. We have assessed the contribution of the conserved and variable regions of MSP2 to the generation of a strain-transcending antibody response by generating MSP2 chimeras that included conserved and variable regions of the 3D7 and FC27 alleles. Robust anti-MSP2 antibody responses targeting both conserved and variable regions were generated in mice, although the fine specificity and the balance of responses to these regions differed amongst the constructs tested. We observed significant differences in antibody subclass distribution in the responses to these chimeras. Our results suggest that chimeric MSP2 antigens can elicit a broad immune response suitable for protection against different strains of P. falciparum.
In this study, a proteogenomic annotation strategy was used to identify a novel bioactive peptide from the venom of the predatory marine snail Conus victoriae. The peptide, conorfamide-Vc1 (CNF-Vc1), defines a new gene family. The encoded mature peptide was unusual for conotoxins in that it was cysteine-free and, despite low overall sequence similarity, contained two short motifs common to known neuropeptides/hormones. One of these was the C-terminal RF-amide motif, commonly observed in neuropeptides from a range of organisms, including humans. The mature venom peptide was synthesized and characterized structurally and functionally. The peptide was bioactive upon injection into mice, and calcium imaging of mouse dorsal root ganglion (DRG) cells revealed that the peptide elicits an increase in intracellular calcium levels in a subset of DRG neurons. Unusually for most Conus venom peptides, it also elicited an increase in intracellular calcium levels in a subset of non-neuronal cells.
calcium imaging; conotoxin; neuropeptide; proteogenomics; RF-amide; venom
ShK is a 35-residue peptide that binds with high affinity to human voltage-gated potassium channels through a conserved K-Y dyad. Here we employ NMR measurements of backbone amide 15N spin relaxation rates to investigate motions of the ShK backbone. Although ShK is rigid on the ps-ns timescale, increased linewidths observed for eleven backbone amide 15N resonances identify chemical or conformational exchange contributions to spin relaxation. Relaxation dispersion profiles indicate that exchange between major and minor conformers occurs on the sub-millisecond timescale. Affected residues are mostly clustered around the central helix-kink-helix structure and the critical K22-Y23 motif. We suggest that the less structured minor conformer increases the exposure of Y23, known to contribute to binding affinity and selectivity, thereby facilitating its interaction with potassium channels. These findings have potential implications for the design of new channel blockers based on ShK.
NMR spectroscopy; potassium channels blockers; structural biology; protein dynamics; relaxation dispersion
Malaria remains a significant global health burden. The development of an effective malaria vaccine remains as a major challenge with the potential to significantly reduce morbidity and mortality. While Plasmodium spp. have been shown to contain a large number of intrinsically disordered proteins (IDPs) or disordered protein regions, the relationship of protein structure to subcellular localisation and adaptive immune responses remains unclear. In this study, we employed several computational prediction algorithms to identify IDPs at the proteome level of six Plasmodium spp. and to investigate the potential impact of protein disorder on adaptive immunity against P. falciparum parasites. IDPs were shown to be particularly enriched within nuclear proteins, apical proteins, exported proteins and proteins localised to the parasitophorous vacuole. Furthermore, several leading vaccine candidates, and proteins with known roles in host-cell invasion, have extensive regions of disorder. Presentation of peptides by MHC molecules plays an important role in adaptive immune responses, and we show that IDP regions are predicted to contain relatively few MHC class I and II binding peptides owing to inherent differences in amino acid composition compared to structured domains. In contrast, linear B-cell epitopes were predicted to be enriched in IDPs. Tandem repeat regions and non-synonymous single nucleotide polymorphisms were found to be strongly associated with regions of disorder. In summary, immune responses against IDPs appear to have characteristics distinct from those against structured protein domains, with increased antibody recognition of linear epitopes but some constraints for MHC presentation and issues of polymorphisms. These findings have major implications for vaccine design, and understanding immunity to malaria.
Peptide toxins provide valuable therapeutic leads for many diseases. As they bind to their targets with high affinity, potency is usually ensured. However, toxins also bind to off-target receptors, causing potential side effects. Thus, a major challenge in generating drugs from peptide toxins is ensuring their specificity for their intended targets. Computational methods can play an important role in solving such design problems through construction of accurate models of receptor–toxin complexes and calculation of binding free energies. Here we review the computational methods used for this purpose and their application to toxins targeting ion channels. We describe ShK and HsTX1 toxins, high-affinity blockers of the voltage-gated potassium channel Kv1.3, which could be developed as therapeutic agents for autoimmune diseases.
μ-Conotoxins block voltage-gated sodium channels (VGSCs) and compete with tetrodotoxin for binding to the sodium conductance pore. Early efforts identified μ-conotoxins that preferentially blocked the skeletal muscle subtype (NaV1.4). However, the last decade witnessed a significant increase in the number of μ-conotoxins and the range of VGSC subtypes inhibited (NaV1.2, NaV1.3 or NaV1.7). Twenty μ-conotoxin sequences have been identified to date and structure–activity relationship studies of several of these identified key residues responsible for interactions with VGSC subtypes. Efforts to engineer-in subtype specificity are driven by in vivo analgesic and neuromuscular blocking activities. This review summarizes structural and pharmacological studies of μ-conotoxins, which show promise for development of selective blockers of NaV1.2, and perhaps also NaV1.1,1.3 or 1.7.
Inhibitors of the neuronal voltage-gated sodium channel subtype NaV1.3 are of interest as pharmacological tools for the study of neuropathic pain associated with spinal cord injury and have potential therapeutic applications. The recently described μ-conotoxin BuIIIB from Conus bullatus (μ-BuIIIB) was shown to block NaV1.3 with sub-micromolar potency (Kd = 0.2 μM), making it one of the most potent peptidic inhibitors of this subtype described to date. However, oxidative folding of μ-BuIIIB results in numerous folding isoforms, making it difficult to obtain sufficient quantities of the active form of the peptide for detailed structure-activity studies. Here we report the synthesis and characterization of μ-BuIIIB analogs incorporating a disulfide-deficient, diselenide-containing scaffold designed to simplify synthesis and facilitate structure-activity studies directed at identifying amino acid residues involved in NaV1.3 blockade. Our results indicate that, like other μ-conotoxins, the C-terminal residues (Trp16, Arg18 and His20) are most crucial for NaV1 block. At the N-terminus, replacement of Glu3 by Ala resulted in an analog with increased potency for NaV1.3 (Kd = 0.07 μM), implicating this position as a potential site for modification for increased potency and/or selectivity. Further examination of this position showed that increased negative charge, through γ-carboxyglutamate replacement, decreased potency (Kd = 0.33 μM), while replacement with positively-charged 2,4-diamonobutyric acid increased potency (Kd = 0.036 μM). These results provide a foundation for the design and synthesis of μ-BuIIIB-based analogs with increased potency against NaV1.3.
Conotoxin; disulfide; neuropathic pain; selenocysteine; voltage-gated sodium channel
Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the surface of the merozoite stage of Plasmodium falciparum, is a potential component of a malaria vaccine, having shown some efficacy in a clinical trial in Papua New Guinea. MSP2 is a GPI-anchored protein consisting of conserved N- and C-terminal domains and a variable central region. Previous studies have shown that it is an intrinsically unstructured protein with a high propensity for fibril formation, in which the conserved N-terminal domain plays a key role. Secondary structure predictions suggest that MSP2 contains long stretches of random coil with very little α-helix or β-strand. Circular dichroism spectroscopy confirms this prediction under physiological conditions (pH 7.4) and in more acidic solutions (pH 6.2 and 3.4). Pulsed field gradient NMR diffusion measurements showed that MSP2 under physiological conditions has a large effective hydrodynamic radius consistent with an intrinsic pre-molten globule state, as defined by Uversky. This was supported by sedimentation velocity studies in the analytical ultracentrifuge. NMR resonance assignments have been obtained for FC27 MSP2, allowing the residual secondary structure and backbone dynamics to be defined. There is some motional restriction in the conserved C-terminal region in the vicinity of an intramolecular disulfide bond. Two other regions show motional restrictions, both of which display helical structure propensities. One of these helical regions is within the conserved N-terminal domain, which adopts essentially the same conformation in full-length MSP2 as in corresponding peptide fragments. We see no evidence of long-range interactions in the full-length protein. MSP2 associates with lipid micelles, but predominantly through the N-terminal region rather than the C-terminus, which is GPI-anchored to the membrane in the parasite.
intrinsically unstructured protein; merozoite surface protein 2; NMR; backbone dynamics; lipid interactions
Merozoite surface protein 2 (MSP2) is an intrinsically disordered, membrane-anchored antigen of the malaria parasite Plasmodium falciparum. MSP2 can elicit a protective, albeit strain-specific, antibody response in humans. Antibodies are generated to the conserved N- and C-terminal regions but many of these react poorly with the native antigen on the parasite surface. Here we demonstrate that recognition of a conserved N-terminal epitope by mAb 6D8 is incompatible with the membrane-bound conformation of that region, suggesting a mechanism by which native MSP2 escapes antibody recognition. Furthermore, crystal structures and NMR spectroscopy identify transient, strain-specific interactions between the 6D8 antibody and regions of MSP2 beyond the conserved epitope. These interactions account for the differential affinity of 6D8 for the two allelic families of MSP2, even though 6D8 binds to a fully conserved epitope. These results highlight unappreciated mechanisms that may modulate the specificity and efficacy of immune responses towards disordered antigens.
Merozoite surface protein 2 (MSP2) of Plasmodium falciparum is an abundant, intrinsically disordered protein that is GPI-anchored to the surface of the invasive blood stage of the malaria parasite. Recombinant MSP2 has been trialled as a component of a malaria vaccine, and is one of several disordered proteins that are candidates for inclusion in vaccines for malaria and other diseases. Nonetheless, little is known about the implications of protein disorder for the development of an effective antibody response. We have therefore undertaken a detailed analysis of the conformational dynamics of the two allelic forms of MSP2 (3D7 and FC27) using NMR spectroscopy. Chemical shifts and NMR relaxation data indicate that conformational and dynamic properties of the N- and C-terminal conserved regions in the two forms of MSP2 are essentially identical, but significant variation exists between and within the central variable regions. We observe a strong relationship between the conformational dynamics and the antigenicity of MSP2, as assessed with antisera to recombinant MSP2. Regions of increased conformational order in MSP2, including those in the conserved regions, are more strongly antigenic, while the most flexible regions are minimally antigenic. This suggests that modifications that increase conformational order may offer a means to tune the antigenicity of MSP2 and other disordered antigens, with implications for vaccine design.
ShK, from the sea anemone Stichodactyla helianthus, is a 35-residue disulfide-rich peptide that blocks the voltage-gated potassium channel Kv1.3 at ca. 10 pM and the related channel Kv1.1 at ca. 16 pM. We developed an analog of this peptide, ShK-186, which is currently in Phase 1b-2a clinical trials for the treatment of autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. While ShK-186 displays a >100-fold improvement in selectivity for Kv1.3 over Kv1.1 compared with ShK, there is considerable interest in developing peptides with an even greater selectivity ratio. In this report, we describe several variants of ShK that incorporate p-phophono-phenylalanine at the N-terminus coupled with internal substitutions at Gln16 and Met21. In addition, we also explored the combinatorial effects of these internal substitutions with an alanine extension at the C-terminus. Their selectivity was determined by patch-clamp electrophysiology on Kv1.3 and Kv1.1 channels stably expressed in mouse fibroblasts. The peptides with an alanine extension blocked Kv1.3 at low pM concentrations and exhibited up to 2250-fold selectivity for Kv1.3 over Kv1.1. Analogs that incorporates p-phosphono-phenylalanine at the N-terminus blocked Kv1.3 with IC50s in the low pM range and did not affect Kv1.1 at concentrations up to 100 nM, displaying a selectivity enhancement of >10,000-fold for Kv1.3 over Kv1.1. Other potentially important Kv channels such as Kv1.4 and Kv1.6 were only partially blocked at 100 nM concentrations of each of the ShK analogs.
immunomodulator; T lymphocyte; potassium channel; disulfide-rich peptide; sea anemone toxin; K+ channel blocker
Conotoxins are the peptidic components of the venoms of marine cone snails (genus Conus). They are remarkably diverse in terms of structure and function. Unique potency and selectivity profiles for a range of neuronal targets have made several conotoxins valuable as research tools, drug leads and even therapeutics, and has resulted in a concerted and increasing drive to identify and characterise new conotoxins. Conotoxins are translated from mRNA as peptide precursors, and cDNA sequencing is now the primary method for identification of new conotoxin sequences. As a result, gene superfamily, a classification based on precursor signal peptide identity, has become the most convenient method of conotoxin classification. Here we review each of the described conotoxin gene superfamilies, with a focus on the structural and functional diversity present in each. This review is intended to serve as a practical guide to conotoxin superfamilies and to facilitate interpretation of the increasing number of conotoxin precursor sequences being identified by targeted-cDNA sequencing and more recently high-throughput transcriptome sequencing.
conotoxin; gene superfamily; conopeptide; Conus; venom; toxin
Apical membrane antigen 1 (AMA1) of the human malaria parasite Plasmodium falciparum has been implicated in invasion of the host erythrocyte. It interacts with malarial rhoptry neck (RON) proteins in the moving junction that forms between the host cell and the invading parasite. Agents that block this interaction inhibit invasion and may serve as promising leads for anti-malarial drug development. The invasion-inhibitory peptide R1 binds to a hydrophobic cleft on AMA1, which is an attractive target site for small molecules that block parasite invasion. In this work, truncation and mutational analyses show that Phe5-Phe9, Phe12 and Arg15 in R1 are the most important residues for high affinity binding to AMA1. These residues interact with two well-defined binding hot spots on AMA1. Computational solvent mapping reveals that one of these hot spots is suitable for small molecule targeting. We also confirm that R1 in solution binds to AMA1 with 1∶1 stoichiometry and adopts a secondary structure consistent with the major form of R1 observed in the crystal structure of the complex. Our results provide a basis for designing high affinity inhibitors of the AMA1-RON2 interaction.
µ-SIIIA, a novel µ-conotoxin from Conus striatus, appeared to be a selective blocker of tetrodotoxin-sensitive sodium channels in frog preparations. It also exhibited potent analgesic activity in mice, although its selectivity profile against mammalian sodium channels remained unknown. We have determined the structure of µ-SIIIA in aqueous solution and characterized its backbone dynamics by NMR and its functional properties electrophysiologically. Consistent with the absence of hydroxyprolines, µ-SIIIA adopts a single conformation with all peptide bonds in the trans conformation. The C-terminal region contains a well-defined helix encompassing residues 11–16, while residues 3–5 in the N-terminal region form a helix-like turn resembling 310 helix. The Trp12 and His16 side chains are in close proximity, as in the related conotoxin µ-SmIIIA, but Asn2 is further away. Dynamics measurements show that the N-terminus and Ser9 have larger magnitude motions on the sub-ns timescale, while the C-terminus is more rigid. Cys4, Trp12 and Cys13 undergo significant conformational exchange on µs - ms timescales. µ-SIIIA is a potent, nearly irreversible blocker of NaV1.2, but also blocks NaV1.4 and NaV1.6 with submicromolar potency. The selectivity profile of µ-SIIIA, including poor activity against the cardiac sodium channel, NaV1.5, is similar to that of the closely related µ-KIIIA, suggesting that the C-terminal regions of both are critical for blocking neuronal NaV1.2. The structural and functional characterization described in this paper of an analgesic µ-conotoxin that targets neuronal subtypes of mammalian sodium channels provides a basis for the design of novel analogues with an improved selectivity profile.
Among the μ-conotoxins that block vertebrate voltage-gated sodium channels (VGSCs), some have been shown to be potent analgesics following systemic administration in mice. We have determined the solution structure of a new representative of this family, μ-BuIIIB, and established its disulfide connectivities by direct mass spectrometric collision induced dissociation fragmentation of the peptide with disulfides intact. The major oxidative folding product adopts a 1-4/2-5/3-6 pattern with the following disulfide bridges: Cys5-Cys17, Cys6-Cys23 and Cys13-Cys24. The solution structure reveals that the unique N-terminal extension in μ-BuIIIB, which is also present in μ-BuIIIA and μ-BuIIIC but absent in other μ-conotoxins, forms part of a short α-helix encompassing Glu3 to Asn8. This helix is packed against the rest of the toxin and stabilized by the Cys5-Cys17 and Cys6-Cys23 disulfide bonds. As such, the side chain of Val1 is located close to the aromatic rings of Trp16 and His20, which are located on the canonical helix that displays several residues found to be essential for VGSC blockade in related μ-conotoxins. Mutations of residues 2 and 3 in the N-terminal extension enhanced the potency of μ-BuIIIB for NaV1.3. One analog, [d-Ala2]BuIIIB, showed a 40-fold increase, making it the most potent peptide blocker of this channel characterized to date and thus a useful new tool with which to characterize this channel. Based on previous results for related μ-conotoxins, the dramatic effects of mutations at the N-terminus were unanticipated, and suggest that further gains in potency might be achieved by additional modifications of this region.
The μ-conotoxin μ-KIIIA, from Conus kinoshitai, blocks mammalian neuronal voltage-gated sodium channels (VGSCs) and is a potent analgesic following systemic administration in mice. We have determined its solution structure using NMR spectroscopy. Key residues identified previously as being important for activity against VGSCs (Lys7, Trp8, Arg10, Asp11, His12 and Arg14) all reside on an α-helix with the exception of Arg14. To further probe structure-activity relationships of this toxin against VGSC subtypes, we have characterised the analogue μ-KIIIA[C1A,C9A], in which the Cys residues involved in one of the three disulfides in μ-KIIIA were replaced with Ala. Its structure is quite similar to that of μ-KIIIA, indicating that the Cys1-Cys9 disulfide bond could be removed without any significant distortion of the α-helix bearing the key residues. Consistent with this, μ-KIIIA[C1A,C9A] retained activity against VGSCs, with its rank order of potency being essentially the same as that of μ-KIIIA, namely, NaV1.2 > NaV1.4 > NaV1.7 ≥ NaV1.1 > NaV1.3 > NaV1.5. Kinetics of block were obtained for NaV1.2, NaV1.4 and NaV1.7, and in each case both kon and koff values of μ-KIIIA[C1A,C9A] were larger than those of μ-KIIIA. Our results show that the key residues for VGSC binding lie mostly on an α-helix and that the first disulfide bond can be removed without significantly affecting the structure of this helix, although the modification accelerates the on- and off-rates of the peptide against all tested VGSC subtypes. These findings lay the groundwork for the design of minimized peptides and helical mimetics as novel analgesics.
In the preparation of synthetic conotoxins containing multiple disulfide bonds, oxidative folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of μ-conotoxin KIIIA, which was predicted originally to have a [C1-C9,C2-C15,C4-C16] disulfide pattern based on homology with closely-related μ-conotoxins. The two major isomers of synthetic μ-KIIIA formed during oxidative folding were purified and their disulfide connectivities mapped by direct mass spectrometric CID fragmentation of the disulfide-bonded polypeptides. Our results show that the major oxidative folding product adopts a [C1-C15,C2-C9,C4-C16] disulfide connectivity, while the minor product adopts a [C1-C16,C2-C9,C4-C15] connectivity. Both of these peptides were potent blockers of NaV1.2 (Kd 5 and 230 nM, respectively). The solution structure for μ-KIIIA based on NMR data was recalculated with the [C1-C15,C2-C9,C4-C16] disulfide pattern; its structure was very similar to the μ-KIIIA structure calculated with the incorrect [C1-C9,C2-C15,C4-C16] disulfide pattern, with an α-helix spanning residues 7–12. In addition, the major folding isomers of μ-KIIIB, an N-terminally extended isoform of μ-KIIIA identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as for μ-KIIIA, and both blocked NaV1.2 (Kd 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic μ-KIIIA/μ-KIIIB folded in vitro is 1-5/2-4/3-6 but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of μ-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means to further expand the snail's repertoire of active peptides.
HsTX1 toxin, from the scorpion Heterometrus spinnifer, is a 34-residue, C-terminally amidated peptide cross-linked by four disulfide bridges. Here we describe new HsTX1 analogues with an Ala, Phe, Val or Abu substitution at position 14. Complexes of HsTX1 with the voltage-gated potassium channels Kv1.3 and Kv1.1 were created using docking and molecular dynamics simulations, then umbrella sampling simulations were performed to construct the potential of mean force (PMF) of the ligand and calculate the corresponding binding free energy for the most stable configuration. The PMF method predicted that the R14A mutation in HsTX1 would yield a > 2 kcal/mol gain for the Kv1.3/Kv1.1 selectivity free energy relative to the wild-type peptide. Functional assays confirmed the predicted selectivity gain for HsTX1[R14A] and HsTX1[R14Abu], with an affinity for Kv1.3 in the low picomolar range and a selectivity of more than 2,000-fold for Kv1.3 over Kv1.1. This remarkable potency and selectivity for Kv1.3, which is significantly up-regulated in activated effector memory cells in humans, suggest that these analogues represent valuable leads in the development of therapeutics for autoimmune diseases.
Animal venoms represent a vast library of bioactive peptides and proteins with proven potential, not only as research tools but also as drug leads and therapeutics. This is illustrated clearly by marine cone snails (genus Conus), whose venoms consist of mixtures of hundreds of peptides (conotoxins) with a diverse array of molecular targets, including voltage- and ligand-gated ion channels, G-protein coupled receptors and neurotransmitter transporters. Several conotoxins have found applications as research tools, with some being used or developed as therapeutics. The primary objective of this study was the large-scale discovery of conotoxin sequences from the venom gland of an Australian cone snail species, Conus victoriae. Using cDNA library normalization, high-throughput 454 sequencing, de novo transcriptome assembly and annotation with BLASTX and profile hidden Markov models, we discovered over 100 unique conotoxin sequences from 20 gene superfamilies, the highest diversity of conotoxins so far reported in a single study. Many of the sequences identified are new members of known conotoxin superfamilies, some help to redefine these superfamilies and others represent altogether new classes of conotoxins. In addition, we have demonstrated an efficient combination of methods to mine an animal venom gland and generate a library of sequences encoding bioactive peptides.
ShK, a 35-residue peptide from a sea anemone, is a potent blocker of potassium channels. Here we describe a new ShK analogue with an additional C-terminus Lys residue and amide. ShK-K-amide is a potent blocker of Kv1.3 and, in contrast to ShK and ShK-amide, is selective for Kv1.3. To understand this selectivity, we created complexes of ShK-K-amide with Kv1.3 and Kv1.1 using docking and molecular dynamics simulations, then performed umbrella sampling simulations to construct the potential of mean force of the ligand and calculate the corresponding binding free energy for the most stable configuration. The results agree well with experimental data.
ShK; C-terminal amide; potassium channel; electrophysiology; potential of mean force; umbrella sampling
The voltage-gated potassium channel Kv1.3 is a well-established target for treatment of autoimmune diseases. ShK peptide from a sea anemone is one of the most potent blockers of Kv1.3 but its application as a therapeutic agent for autoimmune diseases is limited by its lack of selectivity against other Kv channels, in particular Kv1.1. Accurate models of Kv1.x-ShK complexes suggest that specific charge mutations on ShK could considerably enhance its specificity for Kv1.3. Here we evaluate the K18A mutation on ShK, and calculate the change in binding free energy associated with this mutation using the path-independent free energy perturbation and thermodynamic integration methods, with a novel implementation that avoids convergence problems. To check the accuracy of the results, the binding free energy differences were also determined from path-dependent potential of mean force calculations. The two methods yield consistent results for the K18A mutation in ShK and predict a 2 kcal/mol gain in Kv1.3/Kv1.1 selectivity free energy relative to wild-type peptide. Functional assays confirm the predicted selectivity gain for ShK[K18A] and suggest that it will be a valuable lead in the development of therapeutics for autoimmune diseases.
The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which play a crucial role in the activation of human effector memory T-cells (TEM). Selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by TEM cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. We have established a recombinant peptide expression system in order to generate isotopically-labelled ShK and various ShK analogues for in-depth biophysical and pharmacological studies. ShK was expressed as a thioredoxin fusion protein in Escherichia coli BL21 (DE3) cells and purified initially by Ni2+ iminodiacetic acid affinity chromatography. The fusion protein was cleaved with enterokinase and purified to homogeneity by reverse-phase HPLC. NMR spectra of 15N-labelled ShK were similar to those reported previously for the unlabelled synthetic peptide, confirming that recombinant ShK was correctly folded. Recombinant ShK blocked Kv1.3 channels with a Kd of 25 pM and inhibited the proliferation of human and rat T lymphocytes with a preference for TEM cells, with similar potency to synthetic ShK in all assays. This expression system also enables the efficient production of 15N-labelled ShK for NMR studies of peptide dynamics and of the interaction of ShK with Kv1.3 channels.
ShK; potassium channels; protein expression; isotopic labelling; NMR
Suppressor of Cytokine Signaling (SOCS)5 is thought to act as a tumour suppressor through negative regulation of JAK/STAT and epidermal growth factor (EGF) signaling. However, the mechanism/s by which SOCS5 acts on these two distinct pathways is unclear. We show for the first time that SOCS5 can interact directly with JAK via a unique, conserved region in its N-terminus, which we have termed the JAK interaction region (JIR). Co-expression of SOCS5 was able to specifically reduce JAK1 and JAK2 (but not JAK3 or TYK2) autophosphorylation and this function required both the conserved JIR and additional sequences within the long SOCS5 N-terminal region. We further demonstrate that SOCS5 can directly inhibit JAK1 kinase activity, although its mechanism of action appears distinct from that of SOCS1 and SOCS3. In addition, we identify phosphoTyr317 in Shc-1 as a high-affinity substrate for the SOCS5-SH2 domain and suggest that SOCS5 may negatively regulate EGF and growth factor-driven Shc-1 signaling by binding to this site. These findings suggest that different domains in SOCS5 contribute to two distinct mechanisms for regulation of cytokine and growth factor signaling.
Peptide toxins typically bind to their target ion channels or receptors with high potency and selectivity, making them attractive leads for therapeutic development. In some cases the native peptide as it is found in the venom from which it originates can be used directly, but in many instances it is desirable to truncate and/or stabilize the peptide to improve its therapeutic properties. A complementary strategy is to display the key residues that make up the pharmacophore of the peptide toxin on a non-peptidic scaffold, thereby creating a peptidomimetic. This review exemplifies these approaches with peptide toxins from marine organisms, with a particular focus on conotoxins.
peptide toxin; peptidomimetic; ion channel; pain; cone snail