Lymnaea Stagnalis Acetylcholine Binding Protein Research Articles

Spatial Structure and Activity of Synthetic Fragments of Lynx1 and of Nicotinic Receptor Loop C Models.

Lynx1, membrane-bound protein co-localized with the nicotinic acetylcholine receptors (nAChRs) and regulates their function, is a three-finger protein (TFP) made of three β-structural loops, similarly to snake venom α-neurotoxin TFPs. Since the central loop II of α-neurotoxins is involved in binding to nAChRs, we have recently synthesized the fragments of Lynx1 central loop, including those with the disulfide between Cys residues introduced at N- and C-termini, some of them inhibiting muscle-type nAChR similarly to the whole-size water-soluble Lynx1 (ws-Lynx1). Literature shows that the main fragment interacting with TFPs is the C-loop of both nAChRs and acetylcholine binding proteins (AChBPs) while some ligand-binding capacity is preserved by analogs of this loop, for example, by high-affinity peptide HAP. Here we analyzed the structural organization of these peptide models of ligands and receptors and its role in binding. Thus, fragments of Lynx1 loop II, loop C from the Lymnaea stagnalis AChBP and HAP were synthesized in linear and Cys-cyclized forms and structurally (CD and NMR) and functionally (radioligand assay on Torpedo nAChR) characterized. Connecting the C- and N-termini by disulfide in the ws-Lynx1 fragment stabilized its conformation which became similar to the loop II within the 1H-NMR structure of ws-Lynx1, the activity being higher than for starting linear fragment but lower than for peptide with free cysteines. Introduced disulfides did not considerably change the structure of HAP and of loop C fragments, the former preserving high affinity for α-bungarotoxin, while, surprisingly, no binding was detected with loop C and its analogs.

Arachidonoylcholine and Other Unsaturated Long-Chain Acylcholines Are Endogenous Modulators of the Acetylcholine Signaling System.

Cholines acylated with unsaturated fatty acids are a recently discovered family of endogenous lipids. However, the data on the biological activity of acylcholines remain very limited. We hypothesized that acylcholines containing residues of arachidonic (AA-CHOL), oleic (Ol-CHOL), linoleic (Ln-CHOL), and docosahexaenoic (DHA-CHOL) acids act as modulators of the acetylcholine signaling system. In the radioligand binding assay, acylcholines showed inhibition in the micromolar range of both α7 neuronal nAChR overexpressed in GH4C1 cells and muscle type nAChR from Torpedo californica, as well as Lymnaea stagnalis acetylcholine binding protein. Functional response was checked in two cell lines endogenously expressing α7 nAChR. In SH-SY5Y cells, these compounds did not induce Ca2+ rise, but inhibited the acetylcholine-evoked Ca2+ rise with IC50 9 to 12 μM. In the A549 lung cancer cells, where α7 nAChR activation stimulates proliferation, Ol-CHOL, Ln-CHOL, and AA-CHOL dose-dependently decreased cell viability by up to 45%. AA-CHOL inhibited human erythrocyte acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE) by a mixed type mechanism with Ki= 16.7 ± 1.5 μM and αKi= 51.4 ± 4.1 μM for AChE and Ki= 70.5 ± 6.3 μM and αKi= 214 ± 17 μM for BChE, being a weak substrate of the last enzyme only, agrees with molecular docking results. Thus, long-chain unsaturated acylcholines could be viewed as endogenous modulators of the acetylcholine signaling system.

Molecular Dynamics Simulations Study on the Resistant Mechanism of Insects to Imidacloprid due to Y151-S and R81T Mutations in nAChRs.

Imidacloprid (IMI) is the first widely used neonicotinoid insecticide due to its high insecticidal activity and low toxicity. However, as its extensive use in crop protection, many insects are resistant to IMI. One of the main resistance mechanisms of insects to IMI is Y151-S and R81T mutations in nicotinic acetylcholine receptor (nAChR). However, how these two mutations affect the interaction of IMI with nAChR is unknown. Here, to uncover the resistant mechanism of nAChR to IMI due to Y151-S and R81T mutations, molecular dynamics simulations and molecular mechanics/generalized Born surface area (MM-GBSA) calculation, residue interaction network (RIN) analysis were performed. Due that the structure of nAChR is still unkonwn, the crystal structure of lymnaea stagnalis acetylcholine binding protein (Ls-AChBP) was used here to simulate nAChR. Y151 and R81 in nAChR correspond to H145 and Q55 in Ls-AChBP, respectively. The calculated binding free energy indicated that two mutations reduced the binding ability of IMI with Ls-AChBP. Q55T mutation reduced the contribution of several key residues, such as W53, T55, Y113, T144 and C187. As for H145-S mutation, the contribution of W53, Q55 and Y113 residues also decreased. RIN analysis showed that two mutants changed the binding pocket by changing the conformation of residues that interact directly with the mutated residues. The obtained resistance mechanism of IMI will be helpful for the design of potent insecticides.

Structural mechanisms for \u03b1-conotoxin activity at the human \u03b13\u03b24 nicotinic acetylcholine receptor

Nicotinic acetylcholine receptors (nAChR) are therapeutic targets for a range of human diseases. α-Conotoxins are naturally occurring peptide antagonists of nAChRs that have been used as pharmacological probes and investigated as drug leads for nAChR related disorders. However, α-conotoxin interactions have been mostly characterised at the α7 and α3β2 nAChRs, with interactions at other subtypes poorly understood. This study provides novel structural insights into the molecular basis for α-conotoxin activity at α3β4 nAChR, a therapeutic target where subtype specific antagonists have potential to treat nicotine addiction and lung cancer. A co-crystal structure of α-conotoxin LsIA with Lymnaea stagnalis acetylcholine binding protein guided the design and functional characterisations of LsIA analogues that identified the minimum pharmacophore regulating α3β4 antagonism. Interactions of the LsIA R10F with β4 K57 and the conserved –NN– α-conotoxin motif with β4 I77 and I109 conferred α3β4 activity to the otherwise inactive LsIA. Using these structural insights, we designed LsIA analogues with α3β4 activity. This new understanding of the structural basis of protein-protein interactions between α-conotoxins and α3β4 may help rationally guide the development of α3β4 selective antagonists with therapeutic potential.

Conformationally restrained carbamoylcholine homologues. Synthesis, pharmacology at neuronal nicotinic acetylcholine receptors and biostructural considerations

Exploration of small selective ligands for the nicotinic acetylcholine receptors (nAChRs) based on acetylcholine (ACh) has led to the development of potent agonists with clear preference for the α4β2 nAChR, the most prevalent nAChR subtype in the central nervous system. In this work we present the continuation of these efforts aimed at increasing this subtype selectivity by introduction of conformational restriction in the carbamoylcholine homologue, 3-(dimethylaminobutyl) dimethylcarbamate (DMABC). Our results highlight the importance of the N-carbamoyl substitution in α4β2-subtype selectivity. Moreover, we have confirmed the non-linear conformation of DMABC bound to nAChRs suggested by recent crystal structures of the compound in complex with the Lymnaea stagnalis ACh binding protein.

Studies on an acetylcholine binding protein identify a basic residue in loop G on the β1 strand as a new structural determinant of neonicotinoid actions.

Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs). Their widespread use and possible risks to pollinators make it extremely urgent to understand the mechanisms underlying their actions on insect nAChRs. We therefore elucidated X-ray crystal structures of the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP) and its Gln55Arg mutant, more closely resembling insect nAChRs, in complex with a nitromethylene imidacloprid analog (CH-IMI) and desnitro-imidacloprid metabolite (DN-IMI) as well as commercial neonicotinoids, imidacloprid, clothianidin, and thiacloprid. Unlike imidacloprid, clothianidin, and CH-IMI, thiacloprid did not stack with Tyr185 in the wild-type Ls-AChBP, but did in the Gln55Arg mutant, interacting electrostatically with Arg55. In contrast, DN-IMI lacking the NO2 group was directed away from Lys34 and Arg55 to form hydrogen bonds with Tyr89 in loop A and the main chain carbonyl of Trp143 in loop B. Unexpectedly, we found that several neonicotinoids interacted with Lys34 in loop G on the β1 strand in the crystal structure of the Gln55Arg mutant. Basic residues introduced into the α7 nAChR at positions equivalent to AChBP Lys34 and Arg55 enhanced agonist actions of neonicotinoids, while reducing the actions of acetylcholine, (-)-nicotine, and DN-IMI. Thus, not only the basic residues in loop D, but also those in loop G determine the actions of neonicotinoids. These novel findings provide new insights into the modes of action of neonicotinoids and emerging derivatives.

Development of a solid-phase receptor-based assay for the detection of cyclic imines using a microsphere-flow cytometry system.

Biologically active macrocycles containing a cyclic imine were isolated for the first time from aquaculture sites in Nova Scotia, Canada, during the 1990s. These compounds display a "fast-acting" toxicity in the traditional mouse bioassay for lipophilic marine toxins. Our work aimed at developing a receptor-based detection method for spirolides using a microsphere/flow cytometry Luminex system. For the assay, two alternatives were considered as binding proteins, the Torpedo marmorata nicotinic acetylcholine receptor (nAChR) and the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP). A receptor-based inhibition assay was developed using the immobilization of nAChR or Ls-AChBP on the surface of carboxylated microspheres and the competition of cyclic imines with biotin-α-bungarotoxin (α-BTX) for binding to these proteins. The amount of biotin-α-BTX bound to the surface of the microspheres was quantified using phycoerythrin (PE)-labeled streptavidin, and the fluorescence was analyzed in a Luminex 200 system. AChBP and nAChR bound to 13-desmethyl spirolide C efficiently; however, the cross-reactivity profile of the nAChR for spirolides and gymnodimine more closely matched the relative toxic potencies reported for these toxins. The nAChR was selected for further assay development. A simple sample preparation protocol consisting of an extraction with acetone yielded a final extract with no matrix interference on the nAChR/microsphere-based assay for mussels, scallops, and clams. This cyclic imine detection method allowed the detection of 13-desmethyl spirolide C in the range of 10-6000 μg/kg of shellfish meat, displaying a higher sensitivity and wider dynamic range than other receptor-based assays previously published. This microsphere-based assay provides a rapid, sensitive, and easily performed screening method that could be multiplexed for the simultaneous detection of several marine toxins.

Generation of selective ligands for nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are members of the cys‐loop receptor family located in synapses of the CNS, neuromuscular junctions, and autonomic ganglia. The α4β2 and α7 subtypes are the most abundant isoforms in the brain and associated with a range of disorders including; smoking addiction, schizophrenia, and Alzheimer's disease making them attractive pharmaceutical targets. Subtype selective ligands have been difficult to design due to the conserved nature of the binding site. The Aplysia californica and Lymnaea stagnalis acetylcholine binding proteins (AChBPs) are structural surrogates for the extracellular domain of nAChRs. We mutated regions of the binding pocket with the Aplysia AChBP towards α7 to obtain pentameric chimeras. We employed a structure‐guided strategy utilizing wildtype and α7‐chimera AChBPs to catalyze in situ azide‐alkyne triazole cycloaddition reactions generating compounds targeted for the α7 nAChR. Responses for α7 and α4β2 nAChRs were characterized using an in vitro fluorescent functional assay using a genetically encoded Ca2+ indicator. The majority of compounds were α7 agonists with some showing selectivity over α4β2. All compounds with α4β2 activity were identified as antagonists. These results illustrate the utility of in situ click chemistry and its potential expansion into membrane protein templates for generating selective allosteric modulators. Support: R37‐GM18360, UO1‐DA019372, NSF GK12 0742551

Ligand-binding domain of an α7-nicotinic receptor chimera and its complex with agonist

The α(7) acetylcholine receptor (AChR) mediates pre- and postsynaptic neurotransmission in the central nervous system and is a potential therapeutic target in neurodegenerative, neuropsychiatric and inflammatory disorders. We determined the crystal structure of the extracellular domain of a receptor chimera constructed from the human α(7) AChR and Lymnaea stagnalis acetylcholine binding protein (AChBP), which shares 64% sequence identity and 71% similarity with native α(7). We also determined the structure with bound epibatidine, a potent AChR agonist. Comparison of the structures revealed molecular rearrangements and interactions that mediate agonist recognition and early steps in signal transduction in α(7) AChRs. The structures further revealed a ring of negative charge within the central vestibule, poised to contribute to cation selectivity. Structure-guided mutational studies disclosed distinctive contributions to agonist recognition and signal transduction in α(7) AChRs. The structures provide a realistic template for structure-aided drug design and for defining structure-function relationships of α(7) AChRs.

In silico characterization of cytisinoids docked into an acetylcholine binding protein

Homology models of nicotinic acetylcholine receptors (nAChRs) suggest that subtype specificity is due to non-conserved residues in the complementary subunit of the ligand-binding pocket. Cytisine and its derivatives generally show a strong preference for heteromeric α4β2∗ nAChRs over the homomeric α7 subtype, and the structural modifications studied do not cause large changes in their nAChR subtype selectivity. In the present work we docked cytisine, N-methylcytisine, and several pyridone ring-substituted cytisinoids into the crystallographic structure of the Lymnaea stagnalis acetylcholine binding protein (AChBP) co-crystallized with nicotine (1UW6). The graphical analysis of the best poses showed that cytisinoids have weak interactions with the side chains of the non-conserved amino acids in the complementary subunit justifying the use of PDB 1UWB as a surrogate for nAChR. Furthermore, we found a high correlation (R2=0.96) between the experimental pIC50 values at α4β2∗ nAChR and docking energy (S) of the best cytisinoid poses within the AChBP. Due to the quality of the correlation we suggest that this equation might be used as a predictive model to propose new cytisine-derived nAChRs ligands. Our docking results also suggest that further structural modifications of these cytisinoids will not greatly alter their α4β2∗/α7 selectivity.

Ligand design for human nicotinic acetylcholine receptors using in situ freeze‐frame click chemistry

Nicotinic acetylcholine receptors (nAChRs) are ionotropic receptors activated by acetylcholine in synapses of the CNS, neuromuscular junctions, and autonomic ganglia. The α4β2 and α7 nAChR subtypes are therapeutic targets for smoking cessation and treatment of schizophrenia. We developed a structure‐guided design strategy utilizing the Aplysia californica and Lymnaea stagnalis acetylcholine binding proteins (AChBPs) as homologous structural surrogates for the nicotinic receptor extracellular domain. We show that in situ click chemistry can be performed at subunit interfaces to generate selective nAChR agonists and antagonists. Lead compounds can then be refined through catalytic synthesis of close triazole congeners on a submilligram scale to assay AChBP template affinity and pharmacological activity on α4β2 and α7 nAChRs. Responses to other receptors that are potential complementary or “off targets” for the compounds were measured including α1 nAChR, 5‐HT3A and M1 muscarinic receptors. Crystallography of AChBP with the compounds was implemented to identify determinants involved in agonist or antagonist responses. The results of the functional and binding responses suggest that in situ freeze‐frame click chemistry can be a powerful tool in the future for developing specific ligands of various receptors. (Support from R37‐GM18360, UO1‐DA019372, NSF GK12 0742551)

Studies on the mode of action and the selectivity of insecticides acting on ligand-gated ion channels in the nervous system

The mode of action and the mechanisms for the selectivity of neurotoxic insecticides were investigated using computational chemistry, molecular biology, homology modeling of the target site and X-ray crystallography. Whole-cell, patch-clamp electrophysiology has been used to show that non-competitive antagonists (NCAs), such as 4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) and γ-hexachlorocyclohexane (γ-HCH), are more effective blockers of native cockroach (Periplaneta americana) and recombinant house fly (Musca domestica) γ-aminobutyric acid receptors (GABARs) than glutamate-gated chloride channels (GluCls). Site-directed mutagenesis studies have been used to show that that Ser278 in the second transmembrane region is important for determining the less potent actions of these NCAs on GluCls. The structure-activity relationship and the mechanism of the selectivity of neonicotinoids targeting nicotinic acetylcholine receptors (nAChRs) have also been investigated. Electrophysiological studies show that loops C and D of insect nAChRs are critical in determining the selectivity of neonicotinoids, results consistent with the crystal structures of Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP) in complex with imidacloprid. Neonicotinoids show diverse actions on nAChRs as measured using voltage-clamp electrophysiology. Single channel recording and X-ray crystallography are helping to elucidate the mechanism of super agonist actions of clothianidin and an analog.

Molecular modeling of the α9α10 nicotinic acetylcholine receptor subtype

This study reports the comparative molecular modeling, docking and dynamic simulations of human α9α10 nicotinic acetylcholine receptors complexed with acetylcholine, nicotine and α-conotoxin RgIA, using as templates the crystal structures of Aplysia californica and Lymnaea stagnalis acetylcholine binding proteins. The molecular dynamics simulations showed that Arg112 in the complementary α10(−) subunit, is a determinant for recognition in the site that binds small ligands. However, Glu195 in the principal α9(+), and Asp114 in the complementary α10(−) subunit, might confer the potency and selectivity to α-conotoxin RgIA when interacting with Arg7 and Arg9 of this ligand.

A Molecular Basis for Agonist and Antagonist Actions at GABAC Receptors

We modelled the N-terminal ligand-binding domain of the rho1 GABA(C) receptor based on the Lymnaea stagnalis acetylcholine-binding protein (L-AChBP) crystal structure using comparative modelling and validated using flexible docking guided by known mutagenesis studies. A range of known rho1 GABA(C) receptor ligands comprising seven full agonists, 10 partial agonists, 43 antagonists and 12 inactive molecules were used to evaluate and validate the models. Of the 50 models identified, six models that allowed flexible ligand docking in accordance with the experimental data were selected and used to study detailed receptor-ligand interactions. The most refined model to accommodate all known active ligands featured a cavity comprising of a volume of 488 A(3). A detailed analysis of the interaction between the rho1 GABA(C) receptor model and the docked ligands revealed possible H-bonds and cation-pi interactions between the different ligands and binding site residues. Based on quantum mechanical/molecular mechanical (QM/MM) calculations, the model showed distinctive conformations of loop C that provided a molecular basis for agonist and antagonist actions. Agonists elicit loop C closure, while a more open loop C was observed upon antagonist binding. The model differentiates the role for key residues known to be involved in either binding and/or gating.

Channel Opening Motion of α7 Nicotinic Acetylcholine Receptor as Suggested by Normal Mode Analysis

The gating motion of the human nicotinic acetylcholine receptor (nAChR) alpha7 was investigated with normal mode analysis (NMA) of two homology models. The first model, referred to as model I, was built from both the Lymnaea stagnalis acetylcholine binding protein (AChBP) and the transmembrane (TM) domain of the Torpedo marmorata nAChR. The second model, referred to as model C, was based solely on the recent electron microscopy structure of the T. marmorata nAChR. Despite structural differences, both models exhibit nearly identical patterns of flexibility and correlated motions. In addition, both models show a similar global twisting motion that may represent channel gating. The similar results obtained for the two models indicate that NMA is most sensitive to the contact topology of the structure rather than its finer detail. The major difference between the low-frequency motions sampled for the two models is that a symmetrical pore-breathing motion, favoring channel opening, is present as the second most dominant motion in model I, whilst largely absent from model C. The absence of this mode in model C can be attributed to its less symmetrical architecture. Finally, as a further goal of the present study, an approximate open channel model, consistent with many experimental findings, has been produced.