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Co-crystal structures of GLIC, a bacterial ligand-gated ion channel, in complex with monocarboxylate and dicarboxylate derivatives are reported. It is shown that binding occurs at two pharmacological sites in the extracellular domain, which is in agreement with the reported effect of some carboxylates as allosteric modulators of GLIC., GLIC is a bacterial homologue of the pentameric ligand-gated ion channels (pLGICs) that mediate the fast chemical neurotransmission of nerve signalling in eukaryotes. Because the activation and allosteric modulation features are conserved among prokaryotic and eukaryotic pLGICs, GLIC is commonly used as a model to study the allosteric transition and structural pharmacology of pLGICs. It has previously been shown that GLIC is inhibited by some carboxylic acid derivatives. Here, experimental evidence for carboxylate binding to GLIC is provided by solving its X-ray structures with a series of monocarboxylate and dicarboxylate derivatives, and two carboxylate-binding sites are described: (i) the ‘intersubunit’ site that partially overlaps the canonical pLGIC orthosteric site and (ii) the ‘intrasubunit’ vestibular site, which is only occupied by a subset of the described derivatives. While the intersubunit site is widely conserved in all pLGICs, the intrasubunit site is only conserved in cationic eukaryotic pLGICs. This study sheds light on the importance of these two extracellular modulation sites as potential drug targets in pLGICs.

The pentameric ligand-gated ion channel (pLGIC) from Gloeobacter violaceus (GLIC) has provided insightful structure–function views on the permeation process and the allosteric regulation of the pLGICs family. However, GLIC is activated by pH instead of a neurotransmitter and a clear picture for the gating transition driven by protons is still lacking. We used an electrostatics-based (finite difference Poisson–Boltzmann/Debye–Hückel) method to predict the acidities of all aspartic and glutamic residues in GLIC, both in its active and closed-channel states. Those residues with a predicted pK a close to the experimental pH 50 were individually replaced by alanine and the resulting variant receptors were titrated by ATR/FTIR spectroscopy. E35, located in front of loop F far away from the orthosteric site, appears as the key proton sensor with a measured individual pK a at 5.8. In the GLIC open conformation, E35 is connected through a water-mediated hydrogen-bond network first to the highly conserved electrostatic triad R192-D122-D32 and then to Y197-Y119-K248, both located at the extracellular domain–transmembrane domain interface. The second triad controls a cluster of hydrophobic side chains from the M2-M3 loop that is remodeled during the gating transition. We solved 12 crystal structures of GLIC mutants, 6 of them being trapped in an agonist-bound but nonconductive conformation. Combined with previous data, this reveals two branches of a continuous network originating from E35 that reach, independently, the middle transmembrane region of two adjacent subunits. We conclude that GLIC’s gating proceeds by making use of loop F, already known as an allosteric site in other pLGICs, instead of the classic orthosteric site.

International audience; Pentameric ligand-gated ion channels (pLGICs) constitute a widespread class of ion channels, present in archaea, bacteria, and eukaryotes. Upon binding of their agonists in the extracellular domain, the transmembrane pore opens, allowing ions to go through, via a gating mechanism that can be modulated by a number of drugs. Even though high-resolution structural information on pLGICs has increased in a spectacular way in recent years, both in bacterial and in eukaryotic systems, the structure of the open channel conformation of some intensively studied receptors whose structures are known in a nonactive (closed) form, such as Erwinia chrysanthemi pLGIC (ELIC), is still lacking. Here we describe a gammaproteobacterial pLGIC from an endo-symbiont of Tevnia jerichonana (sTeLIC), whose sequence is closely related to the pLGIC from ELIC with 28% identity. We provide an X-ray crystallographic structure at 2.3 Å in an active conformation, where the pore is found to be more open than any current conformation found for pLGICs. In addition, two charged restriction rings are present in the vestibule. Functional characterization shows sTeLIC to be a cationic channel activated at alkaline pH. It is inhibited by divalent cations, but not by quaternary ammonium ions, such as tetramethylammonium. Additionally, we found that sTeLIC is allosterically potentiated by aromatic amino acids Phe and Trp, as well as their derivatives, such as 4-bromo-cinnamate, whose cocrystal structure reveals a vestibular binding site equivalent to, but more deeply buried than, the one already described for benzodiazepines in ELIC.

Summary: Ion channel modulation by general anesthetics is a vital pharmacological process with implications for receptor biophysics and drug development. Functional studies have implicated conserved sites of both potentiation and inhibition in pentameric ligand-gated ion channels, but a detailed structural mechanism for these bimodal effects is lacking. The prokaryotic model protein GLIC recapitulates anesthetic modulation of human ion channels, and it is accessible to structure determination in both apparent open and closed states. Here, we report ten X-ray structures and electrophysiological characterization of GLIC variants in the presence and absence of general anesthetics, including the surgical agent propofol. We show that general anesthetics can allosterically favor closed channels by binding in the pore or favor open channels via various subsites in the transmembrane domain. Our results support an integrated, multi-site mechanism for allosteric modulation, and they provide atomic details of both potentiation and inhibition by one of the most common general anesthetics. : General anesthetics can both potentiate and inhibit pentameric ligand-gated ion channels, yet the structural basis for their effects remains unclear. Ten crystal structures and associated electrophysiology data by Fourati et al. point to a unified allosteric model for positive and negative modulation, providing a framework for structure-inspired drug design. Keywords: ion channels, general anesthetics, x-ray crystallography, electrophysiology, allostery

The Gloeobacter violaceus ligand-gated ion channel (GLIC) has been extensively studied by X-ray crystallography and other biophysical techniques. This provided key insights into the general gating mechanism of pentameric ligand-gated ion channel (pLGIC) signal transduction. However, the GLIC is activated by lowering the pH and the location of its putative proton activation site(s) still remain(s) unknown. To this end, every Asp, Glu, and His residue was mutated individually or in combination and investigated by electrophysiology. In addition to the mutational analysis, key mutations were structurally resolved to address whether particular residues contribute to proton sensing, or alternatively to GLIC-gating, independently of the side chain protonation. The data show that multiple residues located below the orthosteric site, notably E26, D32, E35, and D122 in the lower part of the extracellular domain (ECD), along with E222, H235, E243, and H277 in the transmembrane domain (TMD), alter GLIC activation. D122 and H235 were found to also alter GLIC expression. E35 is identified as a key proton-sensing residue, whereby neutralization of its side chain carboxylate stabilizes the active state. Thus, proton activation occurs allosterically to the orthosteric site, at the level of multiple loci with a key contribution of the coupling interface between the ECD and TMD., Author summary Pentameric ligand-gated ion channels are an important class of receptors that are involved in many neurological diseases. They have been extensively studied but a full understanding of their mechanism of action has yet to be achieved. In an effort to bypass obstacles in the research of human receptors, bacterial versions have been used to characterize the family’s structure-function relationship. One key bacterial receptor, known as GLIC, has lead the way in structural resolution of various mechanistic states along the gating pathway, yet its activation by protons is significantly less understood than its human counterparts. To define the site(s) involved in proton gating, we systematically mutated all titratable residues near the pH50 of activation: Asp, Glu, and His. We determined that a previously established His residue in the transmembrane domain is structurally important but likely plays little or no role in proton gating. We instead found that proton activation is a complex multiple loci mechanism, with the key contribution stemming from the coupling interface between the extracellular and transmembrane domain, with E35 acting as a key proton-sensing residue.

International audience; The Pat1 protein is a central player of eukaryotic mRNA decay that has also been implicated in translational control. It is commonly considered a central platform responsible for the recruitment of several RNA decay factors. We demonstrate here that a yeast-specific C-terminal region from Pat1 interacts with several short motifs, named helical leucine-rich motifs (HLMs), spread in the long C-terminal region of yeast Dcp2 decapping enzyme. Structures of Pat1-HLM complexes reveal the basis for HLM recognition by Pat1. We also identify a HLM present in yeast Xrn1, the main 5'-3' exonuclease involved in mRNA decay. We show further that the ability of yeast Pat1 to bind HLMs is required for efficient growth and normal mRNA decay. Overall, our analyses indicate that yeast Pat1 uses a single binding surface to successively recruit several mRNA decay factors and show that interaction between those factors is highly polymorphic between species.

Pentameric ligand-gated ion channels (pLGICs) mediate fast chemical signaling through global allosteric transitions. Despite the existence of several high-resolution structures of pLGICs, their dynamical properties remain elusive. Using the proton-gated channel GLIC, we engineered multiple fluorescent reporters, each incorporating a bimane and a tryptophan/tyrosine, whose close distance causes fluorescence quenching. We show that proton application causes a global compaction of the extracellular subunit interface, coupled to an outward motion of the M2-M3 loop near the channel gate. These movements are highly similar in lipid vesicles and detergent micelles. These reorganizations are essentially completed within 2 ms and occur without channel opening at low proton concentration, indicating that they report a pre-active intermediate state in the transition pathway toward activation. This provides a template to investigate the gating of eukaryotic neurotransmitter receptors, for which intermediate states also participate in activation. DOI: http://dx.doi.org/10.7554/eLife.23955.001, eLife digest In the nervous system, proteins of the pLGIC family are found in the membrane that surrounds each neuron. These proteins have channels that can allow ions to pass through the membrane and are responsible for transmitting electrical signals from one neuron to the next. Small molecules called neurotransmitters interact with the pLGICs to open or close the ion channel. If the ability of the pLGIC channels to open is altered, it can lead to behavioral changes like addiction, or diseases such as schizophrenia or epilepsy. For a pLGIC channel to switch between the “open” and “closed” states, specific parts of the protein need to move in relation to each other. However, to study these transitions researchers have previously relied on comparing the three-dimensional structures of open and closed pLGICs extracted out of the cell membrane. Different techniques are needed to directly follow these movements within membranes. Bacteria also have proteins belonging to the pLGIC family, and Menny et al. have now investigated one such bacterial protein to understand how pLGICs open. First, a small fluorescent molecule that glows differently if the environment around it changes was attached to various parts of the bacterial channel. These fluorescent markers revealed how several parts of the protein move and they also made it possible to measure how quickly these movements take place. Some of these movements happen before the channel opens, suggesting that the activation of this pLGIC protein happens in stages and involves the protein adopting a temporary intermediate state. The next step will be to better understand the structure of the intermediate state, which could help us to understand how pLGICs work in the nervous systems of animals. In future this may aid the design of new drugs that can modify the activity of these channels in patients with neurological conditions or addictions. DOI: http://dx.doi.org/10.7554/eLife.23955.002

International audience; Barbiturates induce anesthesia by modulating the activity of anionic and cationic pentameric ligand-gated ion channels (pLGICs). Despite more than a century of use in clinical practice, the prototypic binding site for this class of drugs within pLGICs is yet to be described. In this study, we present the first X-ray structures of barbiturates bound to GLIC, a cationic prokaryotic pLGIC with excellent structural homology to other relevant channels sensitive to general anesthetics and, as shown here, to barbiturates, at clinically relevant concentrations. Several derivatives of barbiturates containing anomalous scatterers were synthesized, and these derivatives helped us unambiguously identify a unique barbiturate binding site within the central ion channel pore in a closed conformation. In addition, docking calculations around the observed binding site for all three states of the receptor, including a model of the desensitized state, showed that barbiturates preferentially stabilize the closed state. The identification of this pore binding site sheds light on the mechanism of barbiturate inhibition of cationic pLGICs and allows the rationalization of several structural and functional features previously observed for barbiturates.

© 2014 Elsevier Ltd. All rights reserved. Upf1, Upf2, and Upf3 are the principal regulators of nonsense-mediated mRNA decay (NMD), a cytoplasmic surveillance pathway that accelerates the degradation of mRNAs undergoing premature translation termination. These three proteins interact with each other, the ribosome, the translation termination machinery, and multiple mRNA decay factors, but the precise mechanism allowing the selective detection and degradation of nonsense-containing transcripts remains elusive. Here, we have determined the crystal structure of the N-terminal mIF4G domain from Saccharomyces cerevisiae Upf2 and identified a highly conserved region in this domain that is essential for NMD and independent of Upf2's binding sites for Upf1 and Upf3. Mutations within this conserved region not only inactivate NMD but also disrupt Upf2 binding to specific proteins, including Dbp6, a DEAD-box helicase. Although current models indicate that Upf2 functions principally as an activator of Upf1 and a bridge between Upf1 and Upf3, our data suggest that it may also serve as a platform for the association of additional factors that play roles in premature translation termination and NMD., This work was supported by the Centre National pour la Recherche Scientifique ATIP-AVENIR program (to M.G.), the Agence Nationale pour la Recherche (grant ANR-06-BLAN-0075-02 to H.v.T., S.K., and M.G.), the Association Française Contre les Myopathies (to H.v.T., S.K., and M.G.), the Fondation pour la Recherche Médicale (to Z.F.), the European Union Sixth Framework program “3D-Repertoire” (LSHG-CT-2005-512028, to H.v.T. and M.G.), the National Institutes of Health (R37 GM27757 to A.J.), the Ministerio de Economía y Competitividad (BFU2012-35367 to I.U.), the Centro para el Desarrollo Tecnológico Industrial (IDC-2010-1173 to I.U.), and the Human Frontiers Science Program (grant RGP0018, to H.v.T., S.K., A.J., and M.G.)

INTRODUCTION: Conjugation is an excellent natural mode of DNA transfer in vivo between bacteria, particularly when these conjugative elements carry technological traits such as bacteriocin encoding genes. In the present work, the bacteriocinogenic plasmid pIBF4 from Bacillus thuringiensis responsible of Bacthuricin F4 synthesis was isolated and characterized. METHODOLOGY: To isolate pIBF4, the total plasmid DNA from a non-bacteriocin transposant carrying the mini-Tn10 spectinomycin selective marker was extracted and used to transform Escherichia coli strain Top10. PIBF4 was extracted from the obtained transformant and then subjected to restriction enzyme analysis. Plasmid curing experiments were conducted to test the stability of pIBF4 at a stringent temperature of 42°C. Conjugative behavior of pIBF4 was assessed by mating experiments using the non-bacteriocin transposant mutant as a donor strain and several Bacillus thuringiensis strains as recipients. RESULTS: The pIBF4 plasmid was isolated and had a molecular weight of 19.1 kb. Ninety-five percent of cells retained the pIBF4 plasmid after 200 generations, demonstrating its high stability. PIBF4 was successfully transferred to Bacillus thuringiensis HD1CryB strain with a transfer frequency of 1x10(-8) transconjugants per donor cell. The study of the recipient host range revealed that pIBF4 is specifically transferable to Bacillus thuringiensis strains with variable transfer frequencies depending on the recipient host strain. CONCLUSION: Our results show that pIBF4 is a 19.1 kb highly stable plasmid transferable by conjugation to Bacillus thuringiensis strains with deferent transfer frequencies. Tunisian Ministry of Higher Education, Scientific Research, and Technology

Structural Details of an Allosteric Mechanism for Bimodal Anesthetic Modulation of Pentameric Ligand-Gated Ion Channels

International audience; Pentameric ligand-gated ion channels (pLGICs) mediate fast chemical neurotransmission of nerve signalling in the central and peripheral nervous systems. GLIC is a bacterial homologue of eukaryotic pLGIC, the X-ray structure of which has been determined in three different conformations. GLIC is thus widely used as a model to study the activation and the allosteric transition of this family of receptors. The recently solved high-resolution structure of GLIC (2.4 Å resolution) in the active state revealed two bound acetate molecules in the extracellular domain (ECD). Here, it is shown that these two acetates exactly overlap with known sites of pharmacological importance in pLGICs, and their potential influence on the structure of the open state is studied in detail. Firstly, experimental evidence is presented for the correct assignment of these acetate molecules by using the anomalous dispersion signal of bromoacetate. Secondly, the crystal structure of GLIC in the absence of acetate was solved and it is shown that acetate binding induces local conformational changes that occur in strategic sites of the ECD. It is expected that this acetate-free structure will be useful in future computational studies of the gating transition in GLIC and other pLGICs.

International audience; During mRNA synthesis and maturation, the introduction of errors can strongly influence the expression of certain genes and/or the activity of the proteins for which they encode. To minimise these defects, eukaryotic cells have evolved several cytoplasmic and translation-dependent quality control pathways aimed at detecting and degrading mRNAs that would lead to the production of aberrant proteins. The nonsense-mediated mRNA decay pathway (NMD) clears cells from mRNAs harbouring premature in-frame stop codons. Two other pathways (NSD for nonstop decay and NGD for No-Go decay) degrade mRNAs on which ribosomes have stalled during elongation. In this chapter, we describe the current knowledge on the biological roles and molecular mechanisms of these surveillance pathways, which were mainly unravelled using baker’s yeast as model system.

International audience; We previously designed a new family of artificial proteins named aRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized aRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a ''filtration'' procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.7610 9 independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific aRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with K d values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between aRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that aRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties.

The pentameric ligand-gated ion channel (pLGIC) family plays an important role in the chemical-to-electrical transduction of neuronal signaling. Although human pLGICs are endogenously activated by acetylcholine, serotonin, zinc, glycine, or gamma-aminobutyric acid, some of them notably glycine and gamma-aminobutyric acid receptors are modulated by protons. The Gloeobacter violaceus ligand-gated ion channel (GLIC) is a prokaryotic proton-gated ion channel of the family and was the first pLGIC to be structurally resolved in both an open and closed conformation offering a structural perspective to the mechanism of activation for the pLGIC family. Determining the proton modulatory site(s) of receptors, including acid-sensing and ATP-sensitive K+ channels, may be a complicated endeavor. Although residues have been identified in these receptors as key proton-sensitive sites the underlying molecular mechanism of modulation is not always clear. GLIC has been shown to be a robust model of the pLGIC family, and understanding how a single or several proton(s) can stabilize the open conformation would shed light on the intricate mechanism involved in channel opening of human pLGICs. This comprehension might also pave the way to reveal proton modulatory mechanisms of other receptors. Here we represent a systematic functional evaluation of titratable residues in GLIC to pinpoint the key proton sensitive sites that play a role in the extracellular initiation of the activation mechanism. We have found two key inter-subunit interacting regions, distinct from the ligand-binding pocket, which play key roles in the opening of the channel.

International audience; GLIC is a prokaryotic orthologue of brain pentameric neurotransmitter receptors. Using whole-cell patch-clamp electrophysiology in a host cell line, we show that short-chain di-carboxylate compounds are positive modulators of pHo 5-evoked GLIC activity, with a rank order of action fumarate > succinate > malonate > glutarate. Potentiation by fumarate depends on intracellular pH, mainly as a result of a strong decrease of the pHo 5-evoked current when intracellular pH decreases. The modulating effect of fumarate also depends on extracellular pH, as fumarate is a weak inhibitor at pHo 6 and shows no agonist action at neutral pHo. A mutational analysis of residue-dependency for succinate and fumarate effects, based on two carboxylate-binding pockets previously identified by crystallography (Fourati et al. 2020), shows that positive modulation involves both the inter-subunit pocket, homologous to the neurotransmitter-binding orthotopic site, and the intra-subunit (also called vestibular) pocket. An almost similar pattern of mutational impact is observed for the effect of caffeate, a known negative modulator. We propose, for both di- carboxylate compounds and caffeate, a model where the inter-subunit pocket is the actual binding site, and the region corresponding to the vestibular pocket is required either for inter-subunit binding itself, or for binding-to-gating coupling during the allosteric transitions involved in pore gating modulation.