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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.

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

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.