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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., (© 2023 The Protein Society.)

Among voltage-gated potassium channel (K V ) isoforms, K V 1.6 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 K V 1.6 lack selectivity, and conversely, its selective ligands show low affinity. Here, we present a designer peptide with both high affinity and selectivity to K V 1.6. Previously, we have demonstrated that K V 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 K V 1.3 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 EC 50 of ≈70 nM against K V 1.3. Quite surprisingly, Tk-hefu-11 turns out to block K V 1.6 with even higher potency, presenting an EC 50 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 K V channels to explain the dramatic increase in K V 1.6 affinity. Since K V 1.3 is not highly expressed in the nervous system, we hope that Tk-hefu-11 will be useful in studies of K V 1.6 and its functions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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 K V 1.3. 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 K V 1.3 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 K V 1.3. 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., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Voltage-gated potassium channels (K V s) 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 K V 1.1 blocker (IC 50 ~2 nM); it also affects K V 1.2 (IC 50 ~100 nM), 1.3 (~10 nM) and 1.6 (~60 nM). By constructing computer models of its complex with K V 1.1-1.3 channels we identify specific contacts between the toxin and the three isoforms. We then perform mutagenesis to disturb the identified contacts with K V 1.1 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 K V 1.1 (IC 50 ~550 nM) and 1.2 (~200 nM). It also has diminished activity on K V 1.6 (~1500 nM) but preserves K V 1.3 affinity as measured using the voltage-clamp technique on mammalian channels expressed in Xenopus oocytes. In effect, we convert a selective K V 1.1 ligand into a new specific K V 1.3 ligand. MeKTx13-3 and its derivatives are attractive tools to study the structure-function relationship in potassium channel blockers., (Copyright © 2020 Gigolaev, Kuzmenkov, Peigneur, Tabakmakher, Pinheiro-Junior, Chugunov, Efremov, Tytgat and Vassilevski.)

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 β-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 β-barrel contribute to red chromophore biosynthesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Tk-hefu is an artificial peptide designed based on the α-hairpinin scaffold, which selectively blocks voltage-gated potassium channels K v 1.3. 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 K v 1.3 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 K v 1.3 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., (© 2019 Berkut et al.)

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.

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 K V 1.2. 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 K V 1.2 (IC 50 ∼0.2 nM), while its activity against K V 1.1, K V 1.3, and K V 1.6 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 K V 1.2. 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 K V 1.2 to K V 1.3 resulted in the enhanced sensitivity of mutant K V 1.3 channels to MeKTx11-1. We conclude that MeKTx11-1 may be used as a selective tool in neurobiology., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

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 IC 50 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

Using methods of molecular biology we defined the structures of the 31 sea anemone Heteractis crispa genes encoding polypeptides which are structurally homologous to the Kunitz proteinase inhibitor family. Identified amino acid sequences have point residue substitutions, high degree of homology with sequences of known H. crispa Kunitz family members, and represent a combinatorial library of polypeptides. We generated their three-dimensional structures by homologous modeling methods. Analysis of their molecular electrostatic potential enabled us to divide given polypeptides into three clusters. One of them includes polypeptides APHC1, APHC2 and APHC3, which were earlier shown to possess a unique property of inhibiting of the pain vanilloid receptor TRPV1 in vitro and providing the analgesic effects in vivo in addition to their trypsin inhibitory activity. Molecular docking made possible establishing the spatial structure of the complexes, the nature of the polypeptides binding with TRPV1, as well as functionally important structural elements involved in the complex formation. Structural models have enabled us to propose a hypothesis contributing to understanding the APHC1-3 impact mechanism for the pain signals transduction by TRPV1: apparently, there is an increase of the receptor relaxation time resulted in binding of its two chains with the polypeptide molecule, which disrupt the functioning of the TRPV1 and leads to partial inhibition of signal transduction in electrophysiological experiments.

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)