Valentin M. Tabakmakher

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.

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-1β expression in lipopolysaccharide (LPS)-activated macrophages. However, there was no effect on nitric oxide (NO) generation.

Interaction of sea anemone Heteractis crispa Kunitz type polypeptides with pain vanilloid receptor TRPV1: In silico investigation

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 protease inhibitor family. The identified sequences have single-point amino acid substitutions, a high degree of homology with sequences of known Kunitz family members from H. crispa, and represent a combinatorial library of polypeptides. We generated their three-dimensional structures by methods of homology modeling. Analysis of their molecular electrostatic potential allowed the division of the polypeptides into three clusters. One of them includes polypeptides APHC1, APHC2, and APHC3 which have been shown to possess, in addition to their trypsin inhibitory activity, a unique property of inhibiting the pain vanilloid receptor TRPV1 in vitro and providing the analgesic effects in vivo. The spatial structure of the polypeptide complexes with TRPV1, the nature of the interactions, as well as functionally important structural elements involved in the complex formation, were established by molecular docking technique. The designed models allowed us to propose a hypothesis contributing to the understanding of how APHC1-APHC3 affect the pain signals transduction by TRPV1: apparently, relaxation time of the receptor increases due to binding of its two chains with a polypeptide molecule which disrupts functioning of TRPV1 and leads to partial inhibition of the signal transduction in electrophysiological experiments.

Analgesic effect of novel Kunitz-type polypeptides of the sea anemone Heteractis crispa

80 To date, ample experimental data have been accu mulated demonstrating the role of cellular receptors and ion channels of excitable and nonexcitable mem branes of the peripheral and central nervous system in perception and transmission of nociceptive stimuli [1]. The disruption of the functioning of thee membrane components can lead to the development of various channelopathies and chronic pain. In view of this, the search for and study of the molecules that specifically and selectively interact with ionotropic receptors and ion channels is one of the most important tasks of the complex of sciences that investigate the molecular organization and mechanisms of functioning of living systems. The stability of the peptide components of animal venoms in combination with their biological activity, selectivity, and specificity of action on the molecular targets allows the compounds to be used as unique bio medical tools and models for the development of ther apeutics [2]. That is why the polypeptides of the Kunitz structural family, found in the venoms of dif ferent species of sea anemones, which modulate and block ion channels [3], draw an increasing interest of researchers. Earlier, as a result of the study of serine proteinase inhibitors of the sea anemone Heteractis crispa, it was found that the APHC1 polypeptide has a unique abil ity to inhibit the nociceptive vanilloid receptor TRPV1 [4] and exert analgesic effects in vivo [4, 5]. Later, it was shown that the homologous polypeptides APHC2 and APHC3 exhibit similar properties [6]. Electrophysiological studies on TRPV1 channels expressed in frog oocytes and on thermal pain stimu lation models showed that, despite the partial (35%) inhibition of the channel function, the analgesic polypeptides APHC1–APHC3 block the pain signal transmission and that the analgesic effect reached in this case is hundreds of times greater than the effect of morphine [4, 6]. Recent studies in vivo showed that, unlike the majority of low molecular weight TRPV1 antagonists, polypeptides APHC1 and APHC3 exert analgesic effect accompanied by hypothermia [7], probably due to the combined effect on the vanilloid receptor and inflammatory proteases [8].

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.

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.

Rational design of new ligands for nicotinic receptors on the basis of α-conotoxin PnIA

106 αConotoxins, comparatively short peptides of the venom of predatory marine mollusks of the genus Conus, are effective blockers of nicotinic acetylcholine receptors (nAChRs), which are actively used in studies of various nAChR types [1]. A significant advantage of conotoxins as compared to other known nAChR blockers—the polypeptide αneurotoxins of snake venom—is an initially higher specificity with respect to certain subtypes of nicotinic receptors and possibill ity to produce their various analogs by peptide synthee sis. Such analogs are actively synthesized aiming to obtain highly specific ligands for each individual nAChR subtype mainly via introducing or replacing various amino acid residues in the structure of a selected αconotoxin. For example, just a single mutation, [Ala10Leu], in αconotoxin PnIA changes its specificity from nAChR subtype α3β2 to α7 [2], which is involved in pathogeneses of several diseases (Alzheimers disease, Parkinsons disease, schizoo phrenia, etc.). Thus, the possibility to rationally design selective and potent α7 nAChR ligands is not only theoretically, but also practically important problem with a potential to become a breakthrough in molecular medicine. Numerous αconotoxin PnIA analogs have been proo duced to date, in particular, by Alaascanning mutagenesis [3] or by introduction of various charged amino acid residues to different positions in the pepp tide molecule to obtain an analog more efficient and selective with respect to the α7 receptor subtype [4]. In the last work, we selected mutations by a classical computer modeling (docking of flexible ligand to rigid receptor) using the known crystal structures of muss cleetype nAChR from the ray electric organ [5] and the complexes of acetylcholineebinding proteins (AChBPs) with several αconotoxins [6, 7] or αneurotoxin [8]. These waterrsoluble proteins, consisting of five identii cal subunits, are structural homologs of the ligandd binding Nterminal domains in all nAChRs, being the closest to the homooligomeric α7 subtype. This approach has allowed us to produce several αconoo toxin PnIA analogs with a high affinity and selectivity for the AChBPs from Lymnaea stagnalis or Aplysia call ifornica as well as an increased affinity for the human α7 nAChR. However, the best analog in the last case exhibited an affinity of approximately several hunn dreds of nanomoles per liter. This work continues the previous study and is aimed at designing a more efficient ligand for α7 nAChR using αconotoxin PnIA. Here, we applied the computational protein surface topography (PST) technique, developed at the Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian …

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

ABSTRACT 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 (&agr;‐KTx 1.16) from Mesobuthus eupeus scorpion venom to this channel isoform. MeKTx11–1 is a high‐affinity blocker of KV1.2 (IC50 ˜0.2nM), while its activity against KV1.1, KV1.3, and KV1.6 is 10000, 330 and 45000 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 (&agr;‐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. HIGHLIGHTSMeKTx11–1 from Mesobuthus eupeus is a selective high‐affinity ligand of KV1.2.MeKTx11–3 is a natural analog of MeKTx11–1 that has lower activity and selectivity.Molecular modeling identified key structural elements underlying the selectivity.Mutant KV1.3 channels with residues from KV1.2 presented enhanced sensitivity.The P‐S6 loop is the most important channel region for selective toxin activity.

Biologically active polypeptides of sea anemones: Structure, function, and prospects for application

This paper presents the results of a structure-functional study of neurotoxins, actinoporins, and proteinase inhibitors from the tropical sea anemone Heteractis crispa (= Radianthus macrodactylus) and the low boreal sea anemone Oulactis orientalis. The new data significantly contribute to the knowledge of the mechanisms of action of polypeptides from marine coelenterates and can be useful for solving practical problems of the biotechnological development of medicines.