Structure Of Acetylcholine Binding Protein Research Articles

Ligand-gated ion channels as targets of neuroactive insecticides.

The Cys-loop superfamily of ligand-gated ion channels (Cys-loop receptors) is one of the most ubiquitous ion channel families in vertebrates and invertebrates. Despite their ubiquity, they are targeted by several classes of pesticides, including neonicotinoids, phenylpyrazols, and macrolides such as ivermectins. The current commercialized compounds have high target site selectivity, which contributes to the safety of insecticide use. Structural analyses have accelerated progress in this field; notably, the X-ray crystal structures of acetylcholine binding protein and glutamate-gated Cl channels revealed the details of the molecular interactions between insecticides and their targets. Recently, the functional expression of the insect nicotinic acetylcholine receptor (nAChR) has been described, and detailed evaluations using the insect nAChR have emerged. This review discusses the basic concepts and the current insights into the molecular mechanisms of neuroactive insecticides targeting the ligand-gated ion channels, particularly Cys-loop receptors, and presents insights into target-based selectivity, resistance, and future drug design.

A Novel Structural Landscape for Ligand Binding to the α7 Nicotinic Acetylcholine Receptor

In our previous study we identified a series of 4,6‐disubstituted 2‐aminopyrimidines interacting with Acetylcholine Binding Proteins (AChBP) in a cooperative fashion [Kaczanowska et al., Proc. Natl. Acad. Sci. USA 111, 10749 (2014)]. Based on X‐ray crystal structures of AChBP in complex with the cooperative ligands, we designed a new subset of these molecules that target the α7‐nicotinic acetylcholine receptor (nAChR). Seventeen compounds corresponding to this design were synthesized and characterized in cell based functional assays and radioligand binding assay using HEK cell lines overexpressing transfected cDNAs of human α7‐nAChRs, α4β2‐nAChRs and mouse 5HT3A receptors and Lymnaea (Ls) AChBP, respectively. We found a subset of structures that show Kd values in radioligand binding for AChBP between 51–68 nM and elicited responses for α7‐nAChRs in cell based assays (EC50's) as low as 60–70 nM, reflecting high affinity for α7‐nAChRs. Differential selectivity was characterized using α4β2‐nAChRs and 5HT3A receptors as references. Selected Ls‐AChBP and α7‐nAChRs ligands (AC‐171A, AC‐171A2, AC‐171C, AC‐171D) were then crystallized with the Ls‐AChBP revealing a unique binding pose at the five subunit interfaces. These 4,6‐disubsituted 2‐aminopyrimidines may hold potential in treatment of various CNS developmental disorders. For example, in schizophrenia nicotine addiction and resistance to abstinence prevails and may be modulated by these α7‐nAChRs ligands.Support or Funding InformationSupported by NIH grant GM18360 and the Tobacco‐related Disease Research Program (TRDRP).

Exploration of the molecular architecture of the orthosteric binding site in the α4β2 nicotinic acetylcholine receptor with analogs of 3-(dimethylamino)butyl dimethylcarbamate (DMABC) and 1-(pyridin-3-yl)-1,4-diazepane

X-ray crystal structures of acetylcholine binding proteins (AChBPs) have revealed two different possible extensions to the classical ligand binding pocket known to accommodate various nicotinic agonists. One of the pockets is limited in size while the other is of considerable dimensions and protrudes along the interfacial cleft between subunits. To probe these putative extensions in functional nicotinic acetylcholine receptors (nAChRs), elongated analogs of 3-(dimethylamino)butyl dimethylcarbamate (DMABC) and 1-(pyridine-3-yl)-1,4-diazepane were prepared and characterized pharmacologically at neuronal heteromeric nAChRs. Although the new analogs, relative to parent compounds, displayed lower binding affinities, functional characterization of selected compounds revealed that they had retained partial α4β2 nAChR agonist activity. The structure–activity relationship data did not indicate an upper limit to the size of substituents as would have been expected if the ligand was bound in the smaller pocket. The data were better in agreement with a binding mode in which substituents protrude along the interfacial cleft of the receptor. This was further supported by docking into a homology model of the α4-β2 nAChR interface and by surface plasmon resonance biosensor analysis of binding of the compounds to acetylcholine-binding proteins, where they exhibit preference for Lymnaea stagnalis ACh binding protein (Ls-AChBP) over the Aplysia california ACh binding protein (Ac-AChBP). These results suggest new opportunities for expanding chemical space in the development of partial agonist and may be of interest in relation to development of novel smoking cessation aids.

From Shellfish Poisoning to Neuroscience

In this issue of Structure, Bourne etal. (2015) report X-ray structures of acetylcholine binding protein with two fast-acting phycotoxins from the pinnatoxin family. The results may pave the way for development of new CNS-penetrant and subtype-selective nAChR antagonists.

Crystal Structure of Lymnaea stagnalis AChBP Complexed with the Potent nAChR Antagonist DHβE Suggests a Unique Mode of Antagonism

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that belong to the Cys-loop receptor superfamily. These receptors are allosteric proteins that exist in different conformational states, including resting (closed), activated (open), and desensitized (closed) states. The acetylcholine binding protein (AChBP) is a structural homologue of the extracellular ligand-binding domain of nAChRs. In previous studies, the degree of the C-loop radial extension of AChBP has been assigned to different conformational states of nAChRs. It has been suggested that a closed C-loop is preferred for the active conformation of nAChRs in complex with agonists whereas an open C-loop reflects an antagonist-bound (closed) state. In this work, we have determined the crystal structure of AChBP from the water snail Lymnaea stagnalis (Ls) in complex with dihydro-β-erythroidine (DHβE), which is a potent competitive antagonist of nAChRs. The structure reveals that binding of DHβE to AChBP imposes closure of the C-loop as agonists, but also a shift perpendicular to previously observed C-loop movements. These observations suggest that DHβE may antagonize the receptor via a different mechanism compared to prototypical antagonists and toxins.

Lobeline Docking on AChBP and Nicotinic Receptors: Discriminating Importance of the Pocket Geometry and of the Ligand Configuration

Docking of lobeline, a partial agonist of nicotinic acetylcholine receptors (nAChRs), was investigated at once into crystallographic structures of acetylcholine binding proteins (AChBP) and into 7 and 42 nAChRs homology models, and compared to behavior of full agonists, nicotine and epibatidine. The homology models were built using as templates the different pocket geometries established in crystallographic AChBP structures. Systematic cross-docking of each ligand into binding pockets of the two other ligands as well as its self-docking into its own pocket were performed in order to better understand the structural features determining the binding of these three ligands chosen for their molecular diversity. In AChBPs, epibatidine and nicotine display similar docking scores in their own pocket and in other ligands pockets: in particular, they also dock favorably into the lobeline pocket. In opposite, lobeline displays different features: it only binds favorably to its own pocket in AChBPs. Furthermore, the docking poses observed starting from lobeline stereoisomers support the importance of the intramolecular hydrogen bond between the alcohol function of the-phenyl-hydroxyethyl arm and the piperidinium proton for the lobeline binding to AChBP. For homology models, cross-dockings are still discriminating and the specificity of lobeline for its binding pocket is conserved.

Structural Principles of Serotonin and Granisetron Recognition in a 5-HT3 / Binding Protein Chimera

The 5-HT3 serotonin receptor belongs to the family of pentameric ligand-gated ion channels (LGIC). A wealth of structural information on molecular recognition of nicotinic ligands has emerged from high-resolution co-crystal structures of acetylcholine binding proteins (AChBPs), which are homologous to the extracellular domain of the nicotinic acetylcholine receptor. Such information is currently missing for other members of the LGIC family, including the 5-HT3 receptor. To overcome this limitation, we used a rational approach to engineer a binding protein with ligand recognition properties similar to the 5-HT3 receptor. Amino acids contributing to binding site loops A-F in AChBP were substituted to their corresponding residues in the 5-HT3 receptor and the effect of these substitutions were characterized with a competitive binding assay using 3H-granisetron. We present crystal structures of a binding protein chimera in complex with agonists and antagonists of the 5-HT3 receptor. Together, this information provides a structural framework for understanding ligand recognition in the 5-HT3 receptor.

Spiroimine Toxins in Complex with Nicotinic Acetylcholine Receptors: Structure and Dynamics

Macrocyclic spiroimine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. [1] Representative members of this family are gymnodimines, spirolides, pinnatoxins, pteriatoxins, and spiro-prorocentrimine. Gymnodimine A and 13-desmethyl spirolide C have been shown to be potent antagonists of nicotinic acetylcholine receptor (nAChR), and have prompted the development of new tools to detect these spiroimine toxins in shellfish and for the design of novel drugs. [2,3] Very recently, crystal structures of Acetylcholine Binding Protein complexed with gymnodimine A and 13-desmethyl spirolide C were solved, thus providing insight into the orientation and the conformation adopted by these ligands in the binding pocket and their interactions with neighbouring amino-acid residues. [3]In this work we have explored the interaction of several members of spiroimine toxins family with different nAChR subtypes. Given the lack of general methods for flexible docking of macrocyclic ligands, we have developed a two-step, general approach to circumvent this problem, and applied it specifically to the docking of macrocyclic spiroimines on nAChRs. Subsequent molecular dynamics studies of the resulting receptor-ligand complexes allowed us to identify the key interactions responsible for the high affinity and target selectivity experimentally determined for some of these phycotoxins.[1] J. Molgo, E. Girard, E. Benoit, “The cyclic imines: an insight into this emerging group of bioactive marine toxins” in: Phycotoxins: Chemistry and Biochemistry (Eds.: Y. H. Hui, L. M. Botana), Blackwell Publishing, 2007, pp. 319-355.[2] R. Kharrat, D. Servent, E. Girard, G. Ouanounou, M. Amar, R. Marrouchi, E. Benoit, J. Molgo, J. Neurochem., 2008 107, 952-963.[3] Y. Bourne, Z. Radic, R. Araoz, T. T. Talley, E. Benoit, D. Servent, P. Taylor, J. Molgo, P. Marchot, Proc. Natl. Acad. Sci. USA, 2010, 107, 6076-6081.

Mechanisms of imidacloprid resistance in Nilaparvata lugens by molecular modeling

Homology models of the ligand binding domain of the wild-type and Y151S mutant brown planthopper ( Nilaparvata lugens) α1 and rat ( Rattus norvegicus) β2 nicotinic acetylcholine receptor (nAChR) subunits were generated based on the crystal structure of acetylcholine binding protein of Lymnaea stagnalis. Neonicotinoid insecticide imidacloprid was docked into the putative binding site of wild-type and mutant α1β2 dimeric receptors by Surflex-docking, and the calculated docking energies were in agreement with experimental results. The resistance mechanisms and corresponding binding modes of imidacloprid on nAChRs containing the Y151S target-site mutation were discussed.

Virtual Screening Against α-Cobratoxin

alpha-Cobratoxin (Cbtx), the neurotoxin isolated from the venom of the Thai cobra Naja kaouthia , causes paralysis by preventing acetylcholine (ACh) binding to nicotinic acetylcholine receptors (nAChRs). In the current study, the region of the Cbtx molecule that is directly involved in binding to nAChRs is used as the target for anticobratoxin drug design. The crystal structure (1YI5) of Cbtx in complex with the acetylcholine binding protein (AChBP), a soluble homolog of the extracellular binding domain of nAChRs, was selected to prepare an alpha-cobratoxin active binding site for docking. The amino acid residues (Ser182-Tyr192) of the AChBP structure, the binding site of Cbtx, were used as the positive control to validate the prepared Cbtx active binding site (root mean square deviation < 1.2 A). Virtual screening of the National Cancer Institute diversity set, a library of 1990 compounds with nonredundant pharmacophore profiles, using AutoDock against the Cbtx active site, revealed 39 potential inhibitor candidates. The adapted in vitro radioligand competition assays using [(3)H]epibatidine and [(125)I]bungarotoxin against the AChBPs from the marine species, Aplysia californica (Ac), and from the freshwater snails, Lymnaea stagnalis (Ls) and Bolinus truncates (Bt), revealed 4 compounds from the list of inhibitor candidates that had micromolar to nanomolar interferences for the toxin binding to AChBPs. Three hits (NSC42258, NSC121865, and NSC134754) can prolong the survival time of the mice if administered 30 min before injection with Cbtx, but only NSC121865 and NSC134754 can prolong the survival time if injected immediately after injection with Cbtx. These inhibitors serve as novel templates/scaffolds for the development of more potent and specific anticobratoxin.

3H]Epibatidine Photolabels Non-equivalent Amino Acids in the Agonist Binding Site of Torpedo and α4β2 Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo alpha(2)betagammadelta and expressed human alpha4beta2 nAChRs with [(3)H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of alphaTyr(198) in agonist binding site Segment C of the principal (+) face in both alpha subunits and of gammaLeu(109) and gammaTyr(117) in Segment E of the complementary (-) face, with no labeling detected in the delta subunit. For affinity-purified alpha4beta2 nAChRs, [(3)H]epibatidine photolabeled alpha4Tyr(195) (equivalent to Torpedo alphaTyr(190)) in Segment C as well as beta2Val(111) and beta2Ser(113) in Segment E (equivalent to Torpedo gammaLeu(109) and gammaTyr(111), respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation within the alpha-gamma transmitter binding site, whereas it binds in two distinct orientations in the alpha4beta2 nAChR.

Cell-free Production of the Extracellular Domain of the Nicotinic Acetylcholine Receptor

he nicotinic acetylcholine receptor (nAchr) is a li-gand-gated ion channel which is incorporated into the postsynaptic membrane of neurons [1]. nAchr is composed of five homologous subunits, whose transmem-brane domains form an ion pore and the n-terminal do-mains contain the binding sites for the ligand (Fig.1). One of the most common subtypes of nAchr receptor in mam-malian nervous systems is a homopentameric, α7 nAchr (α7nAchr), and several neurodegenerative disorders are associated with its dysfunction [2]. An effective sys-tem for the production of the individual subunits and oth-er domains of nAchr is a prerequisite for studies focused on the receptor itself, and for the design of biomedical drugs to be used in the treatment of the disease. the ten-dency of these proteins to form insoluble aggregates in so-lution makes the development of such systems difficult [3] .recently, a research group from the uSA published the x-ray structure of acetylcholine binding protein (AchBP) from Lymnaea stagnalis [4]. AchBP is a water-soluble protein com-posed of five identical subunits. this protein shares 25% se-quence homology with the extracellular domain of α7nAchrr (α7eD), and it is capable of interacting with some of the nAchr ligands (for example, acetylcholine, α-conotixins, and α-neurotoxins) [4]. It has been shown that replacing the frag-ment of α7eD which is located between cys128 and cys142 (the so-called cys-loop) with a homologous loop from AchBP increases the solubility of the domain [5].During the past 10 years, cell-free systems, especially continuous-z exchange cell-free systems, have been success-fully used in the production of recombinant proteins [7, 8]. these systems have some advantages over host-based gene expression systems: (i) the direct addition of special agents or co-factors into the reaction mixture can prevent the ag-gregation of the target protein, and (ii) the method permits the synthesis of selectively labeled proteins which can then be used in structural studies. the main objective of the present study was the develop-ment of an effective cell-free system for the production of

Understanding of nicotinic acetylcholine receptors

Understanding of nicotinic acetylcholine receptors

Use of Acetylcholine Binding Protein in the Search for Novel α7 Nicotinic Receptor Ligands. In Silico Docking, Pharmacological Screening, and X-ray Analysis

Acetylcholine binding protein (AChBP) is widely considered as a functional and structural homologue of the ligand binding domain of Cys-loop receptors. We report the use of AChBP as template to identify ligands for the nicotinic receptors (nAChRs). An in silico screening protocol was set up and applied to crystal structures of AChBP. Several ligands containing a dibenzosuberyl moiety were identified and shown to bind with high affinity to AChBP and alpha7 nAChRs. Two high affinity ligands were cocrystallized with AChBP, revealing the binding mode in the orthosteric site. Functional studies revealed that these two ligands caused inhibition of the alpha7, alpha4beta2, and 5HT(3) receptors. The noncompetive blockade of the receptors suggests that these compounds act by steric hindrance of the channel. The analysis of the dual binding mode of these dibenzosuberyl-containing compounds can lead to better understanding of the complex mode of action of similar tricyclic ligands on Cys-loop receptors.

Structural answers and persistent questions about how nicotinic receptors work.

The electron diffraction structure of nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata and the X-ray crystallographic structure of acetylcholine binding protein (AChBP) are providing new answers to persistent questions about how nAChRs function as biophysical machines and as participants in cellular and systems physiology. New high-resolution information about nAChR structures might come from advances in crystallography and NMR, from extracellular domain nAChRs as high fidelity models, and from prokaryotic nicotinoid proteins. At the level of biophysics, structures of different nAChRs with different pharmacological profiles and kinetics will help describe how agonists and antagonists bind to orthosteric binding sites, how allosteric modulators affect function by binding outside these sites, how nAChRs control ion flow, and how large cytoplasmic domains affect function. At the level of cellular and systems physiology, structures of nAChRs will help characterize interactions with other cellular components, including lipids and trafficking and signaling proteins, and contribute to understanding the roles of nAChRs in addiction, neurodegeneration, and mental illness. Understanding nAChRs at an atomic level will be important for designing interventions for these pathologies.

Defining Nicotinic Agonist Binding Surfaces through Photoaffinity Labeling

Nicotinic acetylcholine (ACh) receptor (nAChR) agonists are potential therapeutic agents for neurological dysfunction. In the present study, the homopentameric mollusk ACh binding protein (AChBP), used as a surrogate for the extracellular ligand-binding domain of the nAChR, was specifically derivatized by the highly potent agonist azidoepibatidine (AzEPI) prepared as a photoaffinity probe and radioligand. One EPI-nitrene photoactivated molecule was incorporated in each subunit interface binding site based on analysis of the intact derivatized protein. Tryptic fragments of the modified AChBP were analyzed by collision-induced dissociation and Edman sequencing of radiolabeled peptides. Each specific EPI-nitrene-modified site involved either Tyr195 of loop C on the principal or (+)-face or Met116 of loop E on the complementary or (-)-face. The two derivatization sites were observed in similar frequency, providing evidence of the reactivity of the azido/nitrene probe substituent and close proximity to both residues. [3H]AzEPI binds to the alpha4beta2 nAChR at a single high-affinity site and photoaffinity-labels only the alpha4 subunit, presumably modifying Tyr225 spatially corresponding to Tyr195 of AChBP. Phe137 of the beta2 nAChR subunit, equivalent to Met116 of AChBP, conceivably lacks sufficient reactivity with the nitrene generated from the probe. The present photoaffinity labeling in a physiologically relevant condition combined with the crystal structure of AChBP allows development of precise structural models for the AzEPI interactions with AChBP and alpha4beta2 nAChR. These findings enabled us to use AChBP as a structural surrogate to define the nAChR agonist site.

Docking of α-cobratoxin suggests a basal conformation of the nicotinic receptor

We investigate the interactions between the long chain α-cobratoxin (Cbtx) and the nicotinic acetylcholine receptor using a rigid body docking procedure. The method, (i) reproduces the binding of Cbtx to Lymnea acetylcholine-binding protein (AChBP); (ii) shows that most of the structures of AChBP obtained in the presence of antagonists are compatible with Cbtx binding; and (iii) reveals a complex between Cbtx and muscle nAChR that corresponds to the basal “resting” state conformation. The structures are made available for further understanding of the allosteric transitions of the nAChR as well as for drug design.

Structural and Conformational Studies on 5-[1’-Methylpyrrolidin-2’-yl]-1,3-oxazolidin-2-one Free Base and Hydrochloride Form

The molecular structures of (5R,2'S)-5-[l'-methylpyrrolidin-2'-yl]-1,3-oxazolidin-2-one free base (1) and its enantiomeric hydrochloride salt (2) have been determined in order to understand their interaction at neuronal acetylcholine receptor. The molecules are in a bent conformation with the pyrrolidine and the oxazolidinone rings nearly at 60° to each other. The molecular assembly is characterized by the formation of chains joined via hydrogen bonds N-H...N in 1 and N-H...Cl in 2. The solid state structures have been compared with the theoretical conformations and docked into the crystal structure of Acetylcholine Binding Protein (AChBP), homolog of the ligand binding domain of nAChR. A closer analogy between the receptor bound conformation and the solid state has been found in the hydrochoride form with respect to the free base. This latter (1) forms an hydrogen bond with Trp 6702, while 2 beside two additional interactions with Trp 6702 is linked also to Ile 118.

Molecular Determinants for G Protein βγ Modulation of Ionotropic Glycine Receptors

The ligand-gated ion channel superfamily plays a critical role in neuronal excitability. The functions of glycine receptor (GlyR) and nicotinic acetylcholine receptor are modulated by G protein betagamma subunits. The molecular determinants for this functional modulation, however, are still unknown. Studying mutant receptors, we identified two basic amino acid motifs within the large intracellular loop of the GlyR alpha(1) subunit that are critical for binding and functional modulation by Gbetagamma. Mutations within these sequences demonstrated that all of the residues detected are important for Gbetagamma modulation, although both motifs are necessary for full binding. Molecular modeling predicts that these sites are alpha-helixes near transmembrane domains 3 and 4, near to the lipid bilayer and highly electropositive. Our results demonstrate for the first time the sites for G protein betagamma subunit modulation on GlyRs and provide a new framework regarding the ligand-gated ion channel superfamily regulation by intracellular signaling.

Targeted Molecular Dynamics Study of C-Loop Closure and Channel Gating in Nicotinic Receptors

The initial coupling between ligand binding and channel gating in the human α7 nicotinic acetylcholine receptor (nAChR) has been investigated with targeted molecular dynamics (TMD) simulation. During the simulation, eight residues at the tip of the C-loop in two alternating subunits were forced to move toward a ligand-bound conformation as captured in the crystallographic structure of acetylcholine binding protein (AChBP) in complex with carbamoylcholine. Comparison of apo- and ligand-bound AChBP structures shows only minor rearrangements distal from the ligand-binding site. In contrast, comparison of apo and TMD simulation structures of the nAChR reveals significant changes toward the bottom of the ligand-binding domain. These structural rearrangements are subsequently translated to the pore domain, leading to a partly open channel within 4 ns of TMD simulation. Furthermore, we confirmed that two highly conserved residue pairs, one located near the ligand-binding pocket (Lys145 and Tyr188), and the other located toward the bottom of the ligand-binding domain (Arg206 and Glu45), are likely to play important roles in coupling agonist binding to channel gating. Overall, our simulations suggest that gating movements of the α7 receptor may involve relatively small structural changes within the ligand-binding domain, implying that the gating transition is energy-efficient and can be easily modulated by agonist binding/unbinding.