Igor E. Kasheverov

Crystal Structure of Nicotinic Acetylcholine Receptor Homolog Achbp in Complex with an Alpha-Conotoxin Pnia Variant

Conotoxins (Ctx) form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. The subgroup α-Ctx specifically and selectively binds to subtypes of nicotinic acetylcholine receptors (nAChRs), which are targets for treatment of several neurological disorders. Here we present the structure at a resolution of 2.4 Å of α-Ctx PnIA (A10L D14K), a potent blocker of the α7-nAChR, bound with high affinity to acetylcholine binding protein (AChBP), the prototype for the ligand-binding domains of the nAChR superfamily. α-Ctx is buried deep within the ligand-binding site and interacts with residues on both faces of adjacent subunits. The toxin itself does not change conformation, but displaces the C loop of AChBP and induces a rigid-body subunit movement. Knowledge of these contacts could facilitate the rational design of drug leads using the Ctx framework and may lead to compounds with increased receptor subtype selectivity.

Assembly of nicotinic and other Cys-loop receptors

J. Neurochem. (2011) 116, 734–741.

Polypeptide and peptide toxins, magnifying lenses for binding sites in nicotinic acetylcholine receptors.

At present the cryo-electron microscopy structure at 4A resolution is known for the Torpedo marmorata nicotinic acetylcholine receptor (nAChR), and high-resolution X-ray structures have been recently determined for bacterial ligand-gated ion channels which have the same type of spatial organization. Together all these structures provide the basis for better understanding functioning of muscle-type and neuronal nAChRs, as well as of other Cys-loop receptors: 5HT3-, glycine-, GABA-A and some other. Detailed information about the ligand-binding sites in nAChRs, necessary both for understanding the receptor functioning and for rational drug design, became available when the X-ray structures were solved for the acetylcholine-binding proteins (AChBP), excellent models for the ligand-binding domains of all Cys-loop receptors. Of special value in this respect are the X-ray structures of AChBP complexes with agonists and antagonists. Among the latter are the complexes with polypeptide and peptide antagonists, that is with protein neurotoxins from snake venoms and peptide neurotoxins (alpha-conotoxins) from poisonous marine snails of Conus genus. The role of a bridge between the AChBP and nAChRs is played by the X-ray structure of the ligand-binding domain of alpha1 subunit of nAChR in the complex with alpha-bungarotoxin. The purpose of this review is to show the role of well-known and new polypeptide and peptide neurotoxins, from the earlier days of nAChRs research until present time, in identification of different nAChR subtypes and mapping their binding sites.

Naturally Occurring Disulfide-bound Dimers of Three-fingered Toxins A PARADIGM FOR BIOLOGICAL ACTIVITY DIVERSIFICATION

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of α-cobratoxin (a long-chain α-neurotoxin) and heterodimers formed by α-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26–30 in polypeptide loop II of α-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the α-cobratoxin capacity to compete with α-bungarotoxin for binding to Torpedo and α7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that α-cobratoxin dimer not only interacts with α7 nAChR but, in contrast to α-cobratoxin monomer, also blocks α3β2 nAChR. In the latter activity it resembles κ-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric α3β2 nAChRs.

NMR Structure and Action on Nicotinic Acetylcholine Receptors of Water-soluble Domain of Human LYNX1

Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake α-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and three-finger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking a GPI anchor (ws-LYNX1) and its concentration-dependent activity on nicotinic acetylcholine receptors (nAChRs). At 5–30 μm, ws-LYNX1 competed with 125I-α-bungarotoxin for binding to the acetylcholine-binding proteins (AChBPs) and to Torpedo nAChR. Exposure of Xenopus oocytes expressing α7 nAChRs to 1 μm ws-LYNX1 enhanced the response to acetylcholine, but no effect was detected on α4β2 and α3β2 nAChRs. Increasing ws-LYNX1 concentration to 10 μm caused a modest inhibition of these three nAChR subtypes. A common feature for ws-LYNX1 and LYNX1 is a decrease of nAChR sensitivity to high concentrations of acetylcholine. NMR and functional analysis both demonstrate that ws-LYNX1 is an appropriate model to shed light on the mechanism of LYNX1 action. Computer modeling, based on ws-LYNX1 NMR structure and AChBP x-ray structure, revealed a possible mode of ws-LYNX1 binding.

Naturally Occurring and Synthetic Peptides Acting on Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors (nAChRs) are pentameric membrane-bound proteins belonging to the large family of ligand-gated ion channels. nAChRs possess various binding sites which interact with compounds of different chemical nature, including peptides. Historically first peptides found to act on nAChR were synthetic fragments of snake alpha-neurotoxins, competitive receptor antagonists. Later it was shown that fragments of glycoprotein from rabies virus, having homology to alpha-neurotoxins, and polypeptide neurotoxins waglerins from the venom of Waglers pit viper Trimeresurus (Tropidolaemus) wagleri bind in a similar way, waglerins being efficient blockers of muscle-type nAChRs. Neuropeptide substance P appears to interact with the channel moiety of nAChR. beta-Amyloid, a peptide forming senile plaques in Alzheimers disease, also can bind to nAChR, although the mode of binding is still unclear. However, the most well-studied peptides interacting with the ligand-binding sites of nAChRs are so-called alpha-conotoxins, peptide neurotoxins from marine snails of Conus genus. First alpha-conotoxins were discovered in the late 1970s, and now it is a rapidly growing family due to isolation of peptides from multiple Conus species, as well as to cloning, and chemical synthesis of new analogues. Because of their unique selectivity towards distinct nAChR subtypes, alpha-conotoxins became valuable tools in nAChR research. Recent X-ray structures of alpha-conotoxin complexes with acetylcholine-binding protein, a model of nAChR ligand-binding domains, revealed the details of the nAChR ligand-binding sites and provided the basis for design of novel ligands.

Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor

SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,—non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to ‘metabotropic’ signaling pathway through α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.

Azemiopsin from Azemiops feae Viper Venom, a Novel Polypeptide Ligand of Nicotinic Acetylcholine Receptor

Background: Venoms from rare snake species may contain toxins of new structural or/and pharmacological types. Results: Amino acid sequence of the new polypeptide azemiopsin isolated from Azemiops feae viper venom was established, and its biological activity was determined. Conclusion: Azemiopsin is the first natural toxin that blocks nicotinic acetylcholine receptors and does not contain disulfide bridges. Significance: Azemiopsin is the first member of a new toxin group. Azemiopsin, a novel polypeptide, was isolated from the Azemiops feae viper venom by combination of gel filtration and reverse-phase HPLC. Its amino acid sequence (DNWWPKPPHQGPRPPRPRPKP) was determined by means of Edman degradation and mass spectrometry. It consists of 21 residues and, unlike similar venom isolates, does not contain cysteine residues. According to circular dichroism measurements, this peptide adopts a β-structure. Peptide synthesis was used to verify the determined sequence and to prepare peptide in sufficient amounts to study its biological activity. Azemiopsin efficiently competed with α-bungarotoxin for binding to Torpedo nicotinic acetylcholine receptor (nAChR) (IC50 0.18 ± 0.03 μm) and with lower efficiency to human α7 nAChR (IC50 22 ± 2 μm). It dose-dependently blocked acetylcholine-induced currents in Xenopus oocytes heterologously expressing human muscle-type nAChR and was more potent against the adult form (α1β1ϵδ) than the fetal form (α1β1γδ), EC50 being 0.44 ± 0.1 μm and 1.56 ± 0.37 μm, respectively. The peptide had no effect on GABAA (α1β3γ2 or α2β3γ2) receptors at a concentration up to 100 μm or on 5-HT3 receptors at a concentration up to 10 μm. Ala scanning showed that amino acid residues at positions 3–6, 8–11, and 13–14 are essential for binding to Torpedo nAChR. In biological activity azemiopsin resembles waglerin, a disulfide-containing peptide from the Tropidechis wagleri venom, shares with it a homologous C-terminal hexapeptide, but is the first natural toxin that blocks nAChRs and does not possess disulfide bridges.

α‐Conotoxin analogs with additional positive charge show increased selectivity towards Torpedo californica and some neuronal subtypes of nicotinic acetylcholine receptors

α‐Conotoxins from Conus snails are indispensable tools for distinguishing various subtypes of nicotinic acetylcholine receptors (nAChRs), and synthesis of α‐conotoxin analogs may yield novel antagonists of higher potency and selectivity. We incorporated additional positive charges into α‐conotoxins and analyzed their binding to nAChRs. Introduction of Arg or Lys residues instead of Ser12 in α‐conotoxins GI and SI, or D12K substitution in α‐conotoxin SIA increased the affinity for both the high‐ and low‐affinity sites in membrane‐bound Torpedo californica nAChR. The effect was most pronounced for [D12K]SIA with 30‐ and 200‐fold enhancement for the respective sites, resulting in the most potent α‐conotoxin blocker of the Torpedo nAChR among those tested. Similarly, D14K substitution in α‐conotoxin [A10L]PnIA, a blocker of neuronal α7 nAChR, was previously shown to increase the affinity for this receptor and endowed [A10L,D14K]PnIA with the capacity to distinguish between acetylcholine‐binding proteins from the mollusks Lymnaea stagnalis and Aplysia californica. We found that [A10L,D14K]PnIA also distinguishes two α7‐like anion‐selective nAChR subtypes present on identified neurons of L. stagnalis: [D14K] mutation affected only slightly the potency of [A10L]PnIA to block nAChRs on neurons with low sensitivity to α‐conotoxin ImI, but gave a 50‐fold enhancement of blocking activity in cells with high sensitivity to ImI. Therefore, the introduction of an additional positive charge in the C‐terminus of α‐conotoxins targeting some muscle or neuronal nAChRs made them more discriminative towards the respective nAChR subtypes. In the case of muscle‐type α‐conotoxin [D12K]SIA, the contribution of the Lys12 positive charge to enhanced affinity towards Torpedo nAChR was rationalized with the aid of computer modeling.

Diversity of nicotinic receptors mediating Cl- current in Lymnaea neurons distinguished with specific agonists and antagonist.

Diversity of nicotinic acetylcholine receptors (nAChRs) mediating Cl- current in voltage-clamped identifiable Lymnaea stagnalis neurons was studied using acetylcholine (ACh), three agonists and alpha-conotoxin ImI (ImI). Cytisine, nicotine, and choline, full agonists at alpha7 subunit-containing nAChRs of vertebrates, were found to evoke at saturating concentration 84-92% of the maximal current elicited by ACh. ImI, known to block selectively alpha7 and alpha9 nAChRs, markedly diminished the responses to ACh. The average maximal ImI-induced block was 80%, leaving a residual current which had very slow kinetics. The choline-, cytisine-, and nicotine-induced currents were blocked by ImI almost completely, suggesting that they activate only ImI-sensitive receptors. Two groups of cells which differ in desensitization kinetics and in sensitivity to ImI were revealed. IC50 values for ImI against ACh were 10.3 and 288 nM, respectively, with the rapidly desensitizing current being the more sensitive to ImI. The data obtained suggest the existence of at least three pharmacologically distinct subtypes of nicotinic receptors in Lymnaea neurons. Two of the subtypes are similar to alpha7 nAChRs of vertebrates, but differ from each other in their affinity for ImI and in their desensitization kinetics. The third subtype is quite distinct, in that it is resistant to ImI, is not activated by nicotine, cytisine or choline, and mediates a very slowly developing current.