Maxim N. Zhmak

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.

NMR spatial structure of α-conotoxin ImI reveals a common scaffold in snail and snake toxins recognizing neuronal nicotinic acetylcholine receptors1

A 600 MHz NMR study of α‐conotoxin ImI from Conus imperialis, targeting the α7 neuronal nicotinic acetylcholine receptor (nAChR), is presented. ImI backbone spatial structure is well defined basing on the NOEs, spin‐spin coupling constants, and amide protons hydrogen‐deuterium exchange data: rmsd of the backbone atom coordinates at the 2–12 region is 0.28 Å in the 20 best structures. The structure is described as a type I β‐turn (positions 2–5) followed bya distorted helix (positions 5–11). Similar structural psattern can be found in all neuronal‐specific α‐conotoxins. Highly mobile side chains of the Asp‐5, Arg‐7 and Trp‐10 residues form a single site for ImI binding to the α7 receptor. When depicted with opposite directions of the polypeptide chains, the ImI helix and the tip of the central loop of long chain snake neurotoxins demonstrate a common scaffold and similar positioning of the functional side chains, both of these structural elements appearing essential for binding to the neuronal nAChRs.

Mitochondria express several nicotinic acetylcholine receptor subtypes to control various pathways of apoptosis induction

Nicotinic acetylcholine receptors control survival, proliferation and cytokine release in non-excitable cells. Previously we reported that α7 nicotinic receptors were present in the outer membranes of mouse liver mitochondria to regulate mitochondrial pore formation and cytochrome c release. Here we used a wide spectrum of nicotinic receptor subunit-specific antibodies to show that mitochondria express several nicotinic receptor subtypes in a tissue-specific manner: brain and liver mitochondria contain α7β2, α4β2 and less α3β2 nicotinic receptors, while mitochondria from the lung express preferentially α3β4 receptor subtype; all of them are non-covalently connected to voltage-dependent anion channels and control cytochrome c release. By using selective ligands of different nicotinic receptor subtypes (acetylcholine (1 μM) or dihydro-β-erythroidine (1 μM) for α4β2), conotoxin MII (1 nM) for α3β2, MLA (50 nM) for α7β2 and acetylcholine (10 μM) for all subtypes) and apoptogenic agents triggering different mitochondrial signaling pathways (1 μM wortmannin, 90 μM Ca(2+) or 0.5 mM H₂O₂) it was found that α7β2 receptors affect mainly PI₃K/Akt pathway, while α3β2 and α4β2 nAChRs also significantly influence CaKMII- and Src-dependent pathways. It is concluded that cholinergic regulation in mitochondria is realized through multiple nicotinic receptor subtypes, which control various pathways inducing mitochondrial type of apoptosis.

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.

Interaction of alpha-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor.

α‐Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine‐binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α‐Conotoxins blocking muscle‐type or α7 nAChRs compete with α‐bungarotoxin. However, α‐conotoxin ImII, a close homolog of the α7 nAChR‐targeting α‐conotoxin ImI, blocked α7 and muscle nAChRs without displacing α‐bungarotoxin ( Ellison et al. 2003, 2004 ), suggesting binding at a different site. We synthesized α‐conotoxin ImII, its ribbon isomer (ImIIiso), ‘mutant’ ImII(W10Y) and found similar potencies in blocking human α7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [125I]‐α‐bungarotoxin from human α7 nAChRs in the cell line GH4C1 (IC50 17 and 23 μM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC50 2.0–9.0 μM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized α‐bungarotoxin (Kd and IC50 2.5–8.2 μM). On Torpedo nAChR, α‐conotoxin [125I]‐ImII(W10Y) revealed specific binding (Kd 1.5–6.1 μM) and could be displaced by α‐conotoxin ImII, ImIIiso and ImII(W10Y) with IC50 2.7, 2.2 and 3.1 μM, respectively. As α‐cobratoxin and α‐conotoxin ImI displaced [125I]‐ImII(W10Y) only at higher concentrations (IC50≥ 90 μM), our results indicate that α‐conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.

Design of new α-conotoxins: from computer modeling to synthesis of potent cholinergic compounds.

A series of 14 new analogs of α-conotoxin PnIA Conus pennaceus was synthesized and tested for binding to the human α7 nicotinic acetylcholine receptor (nAChR) and acetylcholine-binding proteins (AChBP) Lymnaea stagnalis and Aplysia californica. Based on computer modeling and the X-ray structure of the A. californica AChBP complex with the PnIA[A10L, D14K] analog [1], single and multiple amino acid substitutions were introduced in α-conotoxin PnIA aimed at compounds of higher affinity and selectivity. Three analogs, PnIA[L5H], PnIA[A10L, D14K] and PnIA[L5R, A10L, D14R], have high affinities for AChBPs or α7 nAChR, as found in competition with radioiodinated α-bungarotoxin. That is why we prepared radioiodinated derivatives of these α-conotoxins, demonstrated their specific binding and found that among the tested synthetic analogs, most had almost 10-fold higher affinity in competition with radioactive α-conotoxins as compared to competition with radioactive α-bungarotoxin. Thus, radioiodinated α-conotoxins are a more sensitive tool for checking the activity of novel α-conotoxins and other compounds quickly dissociating from the receptor complexes.

The β-subunit composition of nicotinic acetylcholine receptors in the neurons of the guinea pig inferior mesenteric ganglion

The antibodies against synthetic (183-192) fragments of beta2- and beta4-subunits of rat neuronal nicotinic acetylcholine receptor were used to study a beta-subunit composition of nicotinic receptors in the inferior mesenteric ganglion of the guinea pig by both immunocytochemical staining and blocking of excitatory postsynaptic potentials induced by electric stimulation of the pre-ganglionic nerve (intermesenteric trunk). The beta4-specific antibody stained 59.8 +/- 7.5% of neurons and inhibited the synaptic responses in all (n = 10) neurons studied by 25.5 +/- 1.8%. The beta2-specific antibody did not stain ganglionic neurons and did not affect the synaptic transmission. Taking into account the previously obtained data on the alpha-subunits found in this ganglion, it is concluded that the neurons of inferior mesenteric ganglion contain nicotinic receptors of alpha3(alpha5)beta4 subtypes involved in synaptic transmission through the intermesenteric tract.

Structure and Alignment of the Membrane-Associated Antimicrobial Peptide Arenicin by Oriented Solid-State NMR Spectroscopy

The antimicrobial arenicin peptides are cationic amphipathic sequences that strongly interact with membranes. Through a cystine ring closure a cyclic β-sheet structure is formed in aqueous solution, which persists when interacting with model membranes. In order to investigate the conformation, interactions, dynamics, and topology of their bilayer-associated states, arenicin 1 and 2 were prepared by chemical solid-phase peptide synthesis or by bacterial overexpression, labeled selectively or uniformly with (15)N, reconstituted into oriented membranes, and investigated by proton-decoupled (31)P and (15)N solid-state NMR spectroscopy. Whereas the (31)P NMR spectra indicate that the peptide induces orientational disorder at the level of the phospholipid head groups, the (15)N chemical shift spectra agree well with a regular β-sheet conformation such as the one observed in micellar environments. In contrast, the data do not fit the twisted β-sheet structure found in aqueous buffer. Furthermore, the chemical shift distribution is indicative of considerable conformational and/or topological heterogeneity when at the same time the (15)N NMR spectra exclude alignments of the peptide where the β-sheet lies side ways on the membrane surface. The ensemble of experimental constraints, the amphipathic character of the peptide, and in particular the distribution of the six arginine residues are in agreement with a boatlike dimer structure, similar or related to the one observed in micellar solution, that floats on the membrane surface with the possibility to oligomerize into higher order structures and/or to insert in a transmembrane fashion.

Natural Compounds Interacting with Nicotinic Acetylcholine Receptors: From Low-Molecular Weight Ones to Peptides and Proteins

Nicotinic acetylcholine receptors (nAChRs) fulfill a variety of functions making identification and analysis of nAChR subtypes a challenging task. Traditional instruments for nAChR research are d-tubocurarine, snake venom protein α-bungarotoxin (α-Bgt), and α-conotoxins, neurotoxic peptides from Conus snails. Various new compounds of different structural classes also interacting with nAChRs have been recently identified. Among the low-molecular weight compounds are alkaloids pibocin, varacin and makaluvamines C and G. 6-Bromohypaphorine from the mollusk Hermissenda crassicornis does not bind to Torpedo nAChR but behaves as an agonist on human α7 nAChR. To get more selective α-conotoxins, computer modeling of their complexes with acetylcholine-binding proteins and distinct nAChRs was used. Several novel three-finger neurotoxins targeting nAChRs were described and α-Bgt inhibition of GABA-A receptors was discovered. Information on the mechanisms of nAChR interactions with the three-finger proteins of the Ly6 family was found. Snake venom phospholipases A2 were recently found to inhibit different nAChR subtypes. Blocking of nAChRs in Lymnaea stagnalis neurons was shown for venom C-type lectin-like proteins, appearing to be the largest molecules capable to interact with the receptor. A huge nAChR molecule sensible to conformational rearrangements accommodates diverse binding sites recognizable by structurally very different compounds.