Crystal Structure of the Extracellular Domain of a Bacterial Ligand-Gated Ion Channel

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Pentameric ligand-gated ion channels mediate fast chemical transmission of nerve signals. The structure of a bacterial proton-gated homolog has been established in its open and locally closed conformations at acidic pH. Here we report its crystal structure at neutral pH, thereby providing the X-ray structures of the two end-points of the gating mechanism in the same pentameric ligand-gated ion channel. The large structural variability in the neutral pH structure observed in the four copies of the pentamer present in the asymmetric unit has been used to analyze the intrinsic fluctuations in this state, which are found to prefigure the transition to the open state. In the extracellular domain (ECD), a marked quaternary change is observed, involving both a twist and a blooming motion, and the pore in the transmembrane domain (TMD) is closed by an upper bend of helix M2 (as in locally closed form) and a kink of helix M1, both helices no longer interacting across adjacent subunits. On the tertiary level, detachment of inner and outer β sheets in the ECD reshapes two essential cavities at the ECD-ECD and ECD-TMD interfaces. The first one is the ligand-binding cavity; the other is close to a known divalent cation binding site in other pentameric ligand-gated ion channels. In addition, a different crystal form reveals that the locally closed and open conformations coexist as discrete ones at acidic pH. These structural results, together with site-directed mutagenesis, physiological recordings, and coarse-grained modeling, have been integrated to propose a model of the gating transition pathway.X-ray crystallography | allostery | signal transduction | cys-loop receptor P entameric ligand-gated ion channels (pLGICs) are a superfamily of membrane receptors that mediate fast chemical transmission of nerve signals in the central and peripheral nervous system (1). These allosteric receptors couple neurotransmitter (agonist) binding in the extracellular domain (ECD) to the opening of the ionic pore located in the transmembrane domain (TMD). There are two classes in pLGIC, with either cationic channels (acetylcholine -nAChR -and 5HT3 receptors) or anionic ones (glycine and GABA receptors). The molecular understanding of their allosteric transitions is a central issue in the pharmacology of pLGICs, as it would allow the rational design of novel orthosteric and allosteric ligands. Recently reported full-length structures of several prokaryotic and eukaryotic members of the family have provided significant insights into the conserved architecture of these receptors (2-5). Nevertheless, little is known about the structural events that link the binding/unbinding of agonist to the opening/closure of the channel gate. To understand the gating mechanism, the structures of the same receptor in different allosteric states are needed at atomic resolution. Here we report two crystal structures of wildtype GLIC, a proton-gated bacterial ion channel from Gloeobacter violaceus (6) at two different pHs, above and below pH 50 .GLIC structure wa...

Section: Resultsmentioning

confidence: 69%

Crystal structures of a pentameric ligand-gated ion channel provide a mechanism for activation

Sauguet

Shahsavar

Poitevin

et al. 2013

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188

338

“…This supports a location of the main binding sites for allosteric modulation within the TMD. This idea is already supported by numerous mutational (25) and affinity labeling (26) analyses performed on the glycine/GABA A receptors (27) and by the recent X-ray crystallographic data on GLIC (28). These studies stress the contribution of two cavities in the allosteric modulation, both located in the upper part of the TMD: one at the center of the four helices bundle in each subunit, and one at the interface between subunits.…”

Section: Discussionmentioning

confidence: 81%

Functional prokaryotic–eukaryotic chimera from the pentameric ligand-gated ion channel family

Duret

Renterghem

Weng

et al. 2011

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114

Pentameric ligand-gated ion channels (pLGICs), which mediate chemo-electric signal transduction in animals, have been recently found in bacteria. Despite clear sequence and 3D structure homology, the phylogenetic distance between prokaryotic and eukaryotic homologs suggests significant structural divergences, especially at the interface between the extracellular (ECD) and the transmembrane (TMD) domains. To challenge this possibility, we constructed a chimera in which the ECD of the bacterial protein GLIC is fused to the TMD of the human α1 glycine receptor (α1GlyR). Electrophysiology in Xenopus oocytes shows that it functions as a proton-gated ion channel, thereby locating the proton activation site(s) of GLIC in its ECD. Patch-clamp experiments in BHK cells show that the ion channel displays an anionic selectivity with a unitary conductance identical to that of the α1GlyR. In addition, pharmacological investigations result in transmembrane allosteric modulation similar to the one observed on α1GlyR. Indeed, the clinically active drugs propofol, four volatile general anesthetics, alcohols, and ivermectin all potentiate the chimera while they inhibit GLIC. Collectively, this work shows the compatibility between GLIC and α1GlyR domains and points to conservation of the ion channel and transmembrane allosteric regulatory sites in the chimera. This provides evidence that GLIC and α1GlyR share a highly homologous 3D structure. GLIC is thus a relevant model of eukaryotic pLGICs, at least from the anionic type. In addition, the chimera is a good candidate for mass production in Escherichia coli, opening the way for investigations of "druggable" eukaryotic allosteric sites by X-ray crystallography.Gloeobacter violaceus | allosteric effector | evolution | fusion protein | membrane protein P entameric ligand-gated ion channels (pLGICs) mediate chemo-electric signal transduction in animals, thereby fulfilling key physiological functions, including neurotransmission. They are composed of five identical or homologous subunits and carry one to five agonist binding sites on their extracellular domain (ECD) that govern the opening/closing motion of the ion channel within the transmembrane domain (TMD, composed of four helices labeled M1-M4). In human, ≈40 genes code for pLGIC subunits that are organized into two phylogenetic subclasses: cationic excitatory channels, such as nicotinic acetylcholine (nAChR) and 5HT 3 receptors, and anionic inhibitory channels, such as glycine and GABA A receptors (1). More recently, several new members of the family have been discovered in prokaryotes (2), such as the homologous protein from Gloeobacter violaceus (GLIC), which forms a homopentamer functioning as a proton-gated ion channel (3).Eukaryotic and prokaryotic receptors clearly display homologous structures, characterized by a highly conserved β-sandwichfolded ECD coupled to an all-helix TMD, as described by electron microscopy observation of the Torpedo nAChR (TnAChR) (4), and the X-ray structures of the acetylcholine binding protein (...

“…Interestingly, the two domains of Lily, when expressed alone, were previously found to self-assemble. First, we showed that the isolated ECD of GLIC folds as a soluble monomer and its X-ray structure confirmed it retains the usual β-sandwich fold (31). Second, an ensemble of 15 models of the α 1 GlyR TMD expressed alone and refolded in lipidic micelles has been solved by a combination of electron microscopy (EM) and NMR (32).…”

Section: Discussionmentioning

confidence: 88%

Allosteric and hyperekplexic mutant phenotypes investigated on an α ₁ glycine receptor transmembrane structure

Moraga-Cid

Sauguet

Huon

et al. 2015

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The glycine receptor (GlyR) is a pentameric ligand-gated ion channel (pLGIC) mediating inhibitory transmission in the nervous system. Its transmembrane domain (TMD) is the target of allosteric modulators such as general anesthetics and ethanol and is a major locus for hyperekplexic congenital mutations altering the allosteric transitions of activation or desensitization. We previously showed that the TMD of the human α 1 GlyR could be fused to the extracellular domain of GLIC, a bacterial pLGIC, to form a functional chimera called Lily. Here, we overexpress Lily in Schneider 2 insect cells and solve its structure by X-ray crystallography at 3.5 Å resolution. The TMD of the α 1 GlyR adopts a closed-channel conformation involving a single ring of hydrophobic residues at the center of the pore. Electrophysiological recordings show that the phenotypes of key allosteric mutations of the α 1 GlyR, scattered all along the pore, are qualitatively preserved in this chimera, including those that confer decreased sensitivity to agonists, constitutive activity, decreased activation kinetics, or increased desensitization kinetics. Combined structural and functional data indicate a poreopening mechanism for the α 1 GlyR, suggesting a structural explanation for the effect of some key hyperekplexic allosteric mutations. The first X-ray structure of the TMD of the α 1 GlyR solved here using GLIC as a scaffold paves the way for mechanistic investigation and design of allosteric modulators of a human receptor.glycine receptor | hyperekplexia | allostery