Kalium 3.0 is a comprehensive depository of natural, artificial, and labeled polypeptides acting on potassium channels.

Here, we introduce the third release of Kalium database (http://kaliumdb.org/), a manually curated comprehensive depository that accumulates data on polypeptide ligands of potassium channels. The major goal of this amplitudinous update is to summarize findings for natural polypeptide ligands of K+ channels, as well as data for the artificial derivatives of these substances obtained over the decades of exploration. We manually analyzed more than 700 original manuscripts and systematized the information on mutagenesis, production of radio- and fluorescently labeled derivatives, and the molecular pharmacology of K+ channel ligands. As a result, data on more than 1200 substances were processed and added enriching the database content fivefold. We also included the electrophysiological data obtained on the understudied and neglected K+ channels including the heteromeric and concatenated channels. We associated target channels in Kalium with corresponding entries in the official database of the International Union of Basic and Clinical Pharmacology. Kalium was supplemented with an adaptive Statistics page, where users are able to obtain actual data output. Several other improvements were introduced, such as a color code to distinguish the range of ligand activity concentrations and advanced tools for filtration and sorting. Kalium is a fully open-access database, crosslinked to other databases of interest. It can be utilized as a convenient resource containing ample up-to-date information about polypeptide ligands of K+ channels.

Artificial pore blocker acts specifically on voltage-gated potassium channel isoform KV1.6.

Among voltage-gated potassium channel (KV) isoforms, KV1.6 is one of the most widespread in the nervous system. However, there are little data concerning its physiological significance, in part due to the scarcity of specific ligands. The known high-affinity ligands of KV1.6 lack selectivity, and conversely, its selective ligands show low affinity. Here, we present a designer peptide with both high affinity and selectivity to KV1.6. Previously, we have demonstrated that KV isoform-selective peptides can be constructed based on the simplistic α-hairpinin scaffold, and we obtained a number of artificial Tk-hefu peptides showing selective blockage of KV1.3 in the submicromolar range. We have now proposed amino acid substitutions to enhance their activity. As a result, we have been able to produce Tk-hefu-11 that shows an EC50 of ≈70 nM against KV1.3. Quite surprisingly, Tk-hefu-11 turns out to block KV1.6 with even higher potency, presenting an EC50 of ≈10 nM. Furthermore, we have solved the peptide structure and used molecular dynamics to investigate the determinants of selective interactions between artificial α-hairpinins and KV channels to explain the dramatic increase in KV1.6 affinity. Since KV1.3 is not highly expressed in the nervous system, we hope that Tk-hefu-11 will be useful in studies of KV1.6 and its functions.

Differences in Medium-Induced Conformational Plasticity Presumably Underlie Different Cytotoxic Activity of Ricin and Viscumin.

Structurally similar catalytic subunits A of ricin (RTA) and viscumin (MLA) exhibit cytotoxic activity through ribosome inactivation. Ricin is more cytotoxic than viscumin, although the molecular mechanisms behind this difference are still poorly understood. To shed more light on this problem, we used a combined biochemical/molecular modeling approach to assess possible relationships between the activity of toxins and their structural/dynamic properties. Based on bioassay measurements, it was suggested that the differences in activity are associated with the ability of RTA and MLA to undergo structural/hydrophobic rearrangements during trafficking through the endoplasmic reticulum (ER) membrane. Molecular dynamics simulations and surface hydrophobicity mapping of both proteins in different media showed that RTA rearranges its structure in a membrane-like environment much more efficiently than MLA. Their refolded states also drastically differ in terms of hydrophobic organization. We assume that the higher conformational plasticity of RTA is favorable for the ER-mediated translocation pathway, which leads to a higher rate of toxin penetration into the cytoplasm.

Potassium channel blocker crafted by α-hairpinin scaffold engineering.

The α-Hairpinins are a family of plant defense peptides with a common fold presenting two short α-helices stabilized by two invariant S-S-bridges. We have shown previously that substitution of just two amino acid residues in a wheat α-hairpinin Tk-AMP-X2 leads to Tk-hefu-2 that features specific affinity to voltage-gated potassium channels KV1.3. Here, we utilize a combined molecular modeling approach based on molecular dynamics simulations and protein surface topography technique to improve the affinity of Tk-hefu-2 to KV1.3 while preserving its specificity. An important advance of this work compared with our previous studies is transition from the analysis of various physicochemical properties of an isolated toxin molecule to its consideration in complex with its target, a membrane-bound ion channel. As a result, a panel of computationally designed Tk-hefu-2 derivatives was synthesized and tested against KV1.3. The most active mutant Tk-hefu-10 showed a half-maximal inhibitory concentration of ∼150 nM being >10 times more active than Tk-hefu-2 and >200 times more active than the original Tk-hefu. We conclude that α-hairpinins provide an attractive disulfide-stabilized scaffold for the rational design of ion channel inhibitors. Furthermore, the success rate can be considerably increased by the proposed "target-based" iterative strategy of molecular design.

Sea Anemone Kunitz-Type Peptides Demonstrate Neuroprotective Activity in the 6-Hydroxydopamine Induced Neurotoxicity Model.

Kunitz-type peptides from venomous animals have been known to inhibit different proteinases and also to modulate ion channels and receptors, demonstrating analgesic, anti-inflammatory, anti-histamine and many other biological activities. At present, there is evidence of their neuroprotective effects. We have studied eight Kunitz-type peptides of the sea anemone Heteractis crispa to find molecules with cytoprotective activity in the 6-OHDA-induced neurotoxicity model on neuroblastoma Neuro-2a cells. It has been shown that only five peptides significantly increase the viability of neuronal cells treated with 6-OHDA. The TRPV1 channel blocker, HCRG21, has revealed the neuroprotective effect that could be indirect evidence of TRPV1 involvement in the disorders associated with neurodegeneration. The pre-incubation of Neuro-2a cells with HCRG21 followed by 6-OHDA treatment has resulted in a prominent reduction in ROS production compared the untreated cells. It is possible that the observed effect is due to the ability of the peptide act as an efficient free-radical scavenger. One more leader peptide, InhVJ, has shown a neuroprotective activity and has been studied at concentrations of 0.01-10.0 µM. The target of InhVJ is still unknown, but it was the best of all eight homologous peptides in an absolute cell viability increment on 38% of the control in the 6-OHDA-induced neurotoxicity model. The targets of the other three active peptides remain unknown.

Tuning Scorpion Toxin Selectivity: Switching From KV1.1 to KV1.3.

Voltage-gated potassium channels (KVs) perform vital physiological functions and are targets in different disorders ranging from ataxia and arrhythmia to autoimmune diseases. An important issue is the search for and production of selective ligands of these channels. Peptide toxins found in scorpion venom named KTx excel in both potency and selectivity with respect to some potassium channel isoforms, which may present only minute differences in their structure. Despite several decades of research the molecular determinants of KTx selectivity are still poorly understood. Here we analyze MeKTx13-3 (Kalium ID: α-KTx 3.19) from the lesser Asian scorpion Mesobuthus eupeus, a high-affinity KV1.1 blocker (IC50 ~2 nM); it also affects KV1.2 (IC50 ~100 nM), 1.3 (~10 nM) and 1.6 (~60 nM). By constructing computer models of its complex with KV1.1-1.3 channels we identify specific contacts between the toxin and the three isoforms. We then perform mutagenesis to disturb the identified contacts with KV1.1 and 1.2 and produce recombinant MeKTx13-3_AAAR, which differs by four amino acid residues from the parent toxin. As predicted by the modeling, this derivative shows decreased activity on KV1.1 (IC50 ~550 nM) and 1.2 (~200 nM). It also has diminished activity on KV1.6 (~1500 nM) but preserves KV1.3 affinity as measured using the voltage-clamp technique on mammalian channels expressed in Xenopus oocytes. In effect, we convert a selective KV1.1 ligand into a new specific KV1.3 ligand. MeKTx13-3 and its derivatives are attractive tools to study the structure-function relationship in potassium channel blockers.

Impact of external amino acids on fluorescent protein chromophore biosynthesis revealed by molecular dynamics and mutagenesis studies.

The precise positioning of catalytic amino acids against the substrate in an enzyme active site is a crucial factor in biocatalysis. Biosynthesis of the chromophores of fluorescent proteins (FPs) is an autocatalytic process that must conform to these requirements. Here, we show that, in addition to the internal amino acid residues in the proximity of the chromophore, chromophore biosynthesis is influenced by the remote amino acids exposed on the outer surface of the ß-barrel structure of the FP. It has been shown earlier that chromophore biosynthesis of the red FP from Zoanthus sp. (zoan2RFP) proceeds via an immature green state. At the same time, the green state is the final stage of chromophore biosynthesis of green FP (zoanGFP), which is highly homologous to zoan2RFP. It was also shown that a single N66D substitution in the chromophore-forming sequence of zoanGFP might trigger the synthesis of the red chromophore. However, in this case, the synthesis of the red chromophore is incomplete and occurs only at elevated temperatures. Here, we tried to uncover additional structural determinants that govern the biosynthesis of the red chromophore. A comparison of zoanGFP and zoan2RFP revealed intrabarrel amino acid differences at five positions. Exhaustive substitutions of these five positions in zoanGFP-N66D gave rise to zoanGFPmut with the same intrabarrel amino acid composition as zoan2RFP. zoanGFPmut showed only partial green-to-red chromophore transformation at elevated temperatures. To elucidate the extra factors that can affect red chromophore biosynthesis, we performed comparative molecular dynamics simulations of zoan2RFP and zoanGFPmut. The simulations revealed several external amino acids that might influence the arrangement and flexibility of the chromophore-surrounding amino acid residues in these proteins. Mutagenesis experiments confirmed the crucial role of these residues in red chromophore biosynthesis. The obtained zoanGFPmut2 exhibited complete green-to-red transformation, suggesting that the mutated amino acids exposed on the surface of the ß-barrel contribute to red chromophore biosynthesis.

Complex approach for analysis of snake venom α-neurotoxins binding to HAP, the high-affinity peptide.

Snake venom α-neurotoxins, invaluable pharmacological tools, bind with high affinity to distinct subtypes of nicotinic acetylcholine receptor. The combinatorial high-affinity peptide (HAP), homologous to the C-loop of α1 and α7 nAChR subunits, binds biotinylated α-bungarotoxin (αBgt) with nanomolar affinity and might be a protection against snake-bites. Since there are no data on HAP interaction with other toxins, we checked its binding of α-cobratoxin (αCtx), similar to αBgt in action on nAChRs. Using radioiodinated αBgt, we confirmed a high affinity of HAP for αBgt, the complex formation is supported by mass spectrometry and gel chromatography, but only weak binding was registered with αCtx. A combination of protein intrinsic fluorescence measurements with the principal component analysis of the spectra allowed us to measure the HAP-αBgt binding constant directly (29 nM). These methods also confirmed weak HAP interaction with αCtx (>10000 nM). We attempted to enhance it by modification of HAP structure relying on the known structures of α-neurotoxins with various targets and applying molecular dynamics. A series of HAP analogues have been synthesized, HAP[L9E] analogue being considerably more potent than HAP in αCtx binding (7000 nM). The proposed combination of experimental and computational approaches appears promising for analysis of various peptide-protein interactions.

Protein surface topography as a tool to enhance the selective activity of a potassium channel blocker.

Tk-hefu is an artificial peptide designed based on the α-hairpinin scaffold, which selectively blocks voltage-gated potassium channels Kv1.3. Here we present its spatial structure resolved by NMR spectroscopy and analyze its interaction with channels using computer modeling. We apply protein surface topography to suggest mutations and increase Tk-hefu affinity to the Kv1.3 channel isoform. We redesign the functional surface of Tk-hefu to better match the respective surface of the channel pore vestibule. The resulting peptide Tk-hefu-2 retains Kv1.3 selectivity and displays ∼15 times greater activity compared with Tk-hefu. We verify the mode of Tk-hefu-2 binding to the channel outer vestibule experimentally by site-directed mutagenesis. We argue that scaffold engineering aided by protein surface topography represents a reliable tool for design and optimization of specific ion channel ligands.

Kalium 2.0, a comprehensive database of polypeptide ligands of potassium channels.

Potassium channels are the most diverse group of ion channels in humans. They take vital parts in numerous physiological processes and their malfunction gives rise to a range of pathologies. In addition to small molecules, there is a wide selection of several hundred polypeptide ligands binding to potassium channels, the majority of which have been isolated from animal venoms. Until recently, only scorpion toxins received focused attention being systematically assembled in the manually curated Kalium database, but there is a diversity of well-characterized potassium channel ligands originating from other sources. To address this issue, here we present the updated and improved Kalium 2.0 that covers virtually all known polypeptide ligands of potassium channels and reviews all available pharmacological data. In addition to an expansion, we have introduced several new features to the database including posttranslational modification annotation, indication of ligand mode of action, BLAST search, and possibility of data export.

CARD domain of rat RIP2 kinase: Refolding, solution structure, pH-dependent behavior and protein-protein interactions.

RIP2, one of the RIP kinases, interacts with p75 neurotrophin receptor, regulating the neuron survival, and with NOD1 and NOD2 proteins, causing the innate immune response against gram-negative and gram-positive bacteria via its caspase recruitment domain (CARD). This makes RIP2 a prospective target for novel therapies, aimed to modulate the inflammatory diseases and neurogenesis/neurodegeneration. Several studies report the problems with the stability of human RIP2 CARD and its production in bacterial hosts, which is a prerequisite for the structural investigation with solution NMR spectroscopy. In the present work, we report the high yield production and refolding protocols and resolve the structure of rat RIP2 CARD. The structure reveals the important differences to the previously published conformation of the homologous human protein. Using solution NMR, we characterized the intramolecular mobility and pH-dependent behavior of RIP2 CARD, and found the propensity of the protein to form high-order oligomers at physiological pH while being monomeric under acidic conditions. The oligomerization of protein may be explained, based on the electrostatic properties of its surface. Analysis of the structure and sequences of homologous proteins reveals the residues which are significant for the unusual fold of RIP2 CARD domains from different species. The high-throughput protein production/refolding protocols and proposed explanation for the protein oligomerization, provide an opportunity to design the stabilized variants of RIP2 CARD, which could be used to study the structural details of RIP2/NOD1/NOD2 interaction and perform the rational drug design.

KV1.2 channel-specific blocker from Mesobuthus eupeus scorpion venom: Structural basis of selectivity.

Scorpion venom is an unmatched source of selective high-affinity ligands of potassium channels. There is a high demand for such compounds to identify and manipulate the activity of particular channel isoforms. The objective of this study was to obtain and characterize a specific ligand of voltage-gated potassium channel KV1.2. As a result, we report the remarkable selectivity of the peptide MeKTx11-1 (α-KTx 1.16) from Mesobuthus eupeus scorpion venom to this channel isoform. MeKTx11-1 is a high-affinity blocker of KV1.2 (IC50 ∼0.2 nM), while its activity against KV1.1, KV1.3, and KV1.6 is 10 000, 330 and 45 000 fold lower, respectively, as measured using the voltage-clamp technique on mammalian channels expressed in Xenopus oocytes. Two substitutions, G9V and P37S, convert MeKTx11-1 to its natural analog MeKTx11-3 (α-KTx 1.17) having 15 times lower activity and reduced selectivity to KV1.2. We produced MeKTx11-1 and MeKTx11-3 as well as their mutants MeKTx11-1(G9V) and MeKTx11-1(P37S) recombinantly and demonstrated that point mutations provide an intermediate effect on selectivity. Key structural elements that explain MeKTx11-1 specificity were identified by molecular modeling of the toxin-channel complexes. Confirming our molecular modeling predictions, site-directed transfer of these elements from the pore region of KV1.2 to KV1.3 resulted in the enhanced sensitivity of mutant KV1.3 channels to MeKTx11-1. We conclude that MeKTx11-1 may be used as a selective tool in neurobiology.

C-Terminal residues in small potassium channel blockers OdK1 and OSK3 from scorpion venom fine-tune the selectivity.

We report isolation, sequencing, and electrophysiological characterization of OSK3 (α-KTx 8.8 in Kalium and Uniprot databases), a potassium channel blocker from the scorpion Orthochirus scrobiculosus venom. Using the voltage clamp technique, OSK3 was tested on a wide panel of 11 voltage-gated potassium channels expressed in Xenopus oocytes, and was found to potently inhibit Kv1.2 and Kv1.3 with IC50 values of ~331nM and ~503nM, respectively. OdK1 produced by the scorpion Odontobuthus doriae differs by just two C-terminal residues from OSK3, but shows marked preference to Kv1.2. Based on the charybdotoxin-potassium channel complex crystal structure, a model was built to explain the role of the variable residues in OdK1 and OSK3 selectivity.

High-Affinity α-Conotoxin PnIA Analogs Designed on the Basis of the Protein Surface Topography Method.

Despite some success for small molecules, elucidating structure-function relationships for biologically active peptides - the ligands for various targets in the organism - remains a great challenge and calls for the development of novel approaches. Some of us recently proposed the Protein Surface Topography (PST) approach, which benefits from a simplified representation of biomolecules' surface as projection maps, which enables the exposure of the structure-function dependencies. Here, we use PST to uncover the "activity pattern" in α-conotoxins - neuroactive peptides that effectively target nicotinic acetylcholine receptors (nAChRs). PST was applied in order to design several variants of the α-conotoxin PnIA, which were synthesized and thoroughly studied. Among the best was PnIA[R9, L10], which exhibits nanomolar affinity for the α7 nAChR, selectivity and a slow wash-out from this target. Importantly, these mutations could hardly be delineated by "standard" structure-based drug design. The proposed combination of PST with a set of experiments proved very efficient for the rational construction of new bioactive molecules.

New Kunitz-Type HCRG Polypeptides from the Sea Anemone Heteractis crispa.

Sea anemones are a rich source of Kunitz-type polypeptides that possess not only protease inhibitor activity, but also Kv channels toxicity, analgesic, antihistamine, and anti-inflammatory activities. Two Kunitz-type inhibitors belonging to a new Heteractis crispa RG (HCRG) polypeptide subfamily have been isolated from the sea anemone Heteractis crispa. The amino acid sequences of HCRG1 and HCRG2 identified using the Edman degradation method share up to 95% of their identity with the representatives of the HCGS polypeptide multigene subfamily derived from H. crispa cDNA. Polypeptides are characterized by positively charged Arg at the N-terminus as well as P1 Lys residue at their canonical binding loop, identical to those of bovine pancreatic trypsin inhibitor (BPTI). These polypeptides are shown by our current evidence to be more potent inhibitors of trypsin than the known representatives of the HCGS subfamily with P1Thr. The kinetic and thermodynamic characteristics of the intermolecular interactions between inhibitors and serine proteases were determined by the surface plasmon resonance (SPR) method. Residues functionally important for polypeptide binding to trypsin were revealed using molecular modeling methods. Furthermore, HCRG1 and HCRG2 possess anti-inflammatory activity, reducing tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) secretions, as well as proIL-1ß expression in lipopolysaccharide (LPS)-activated macrophages. However, there was no effect on nitric oxide (NO) generation.

A new multigene superfamily of Kunitz-type protease inhibitors from sea anemone Heteractis crispa.

Despite a considerable number of publications devoted to isolation and physicochemical properties of protease inhibitors from sea anemones, virtually nothing is known about the structure of the genes, and the nature of their isoforms diversity. Using the PCR-based cloning approach we discovered the Kunitz-type multigene superfamily composed of distinct gene families (GS-, RG-, GG-, and GN-gene families). It has been identified only three full-length GS-transcripts indicating a much greater variety of Kunitz homologs in Heteractis crispa. We have examined an exon-intron structure of GS-genes; an open reading frame is interrupted by a single intron located at the middle of the signal peptide. 33 deduced mature GS-polypeptides have been categorized into three groups according to the nature of a P1 residue. Some of them corresponded to native Kunitz-type protease inhibitors earlier isolated from H. crispa. The deduced GS-polypeptide sequences demonstrated diverse charge distribution ranging from the local point charges forms to the overall positive ones. We have suggested that the GS-gene family has evolved through gene tandem duplication followed by adaptive divergence of the P1 residue in the reactive site selected for divergent functions in paralogs. The expansion of this Kunitz-type multigene superfamily during evolution is lineage-specific, providing the tropical sea anemone H. crispa with the ability to interact an increasing diversity of the preys and predators. Our results show that the Kunitz-type polypeptides are encoded by a multigene superfamily and realized via a combinatory Kunitz-type library in the H. crispa tentacles venom.