Ludovic Sauguet

Structure and pharmacology of pentameric receptor channels: from bacteria to brain.

Orthologs of the pentameric receptor channels that mediate fast synaptic transmission in the central and peripheral nervous systems have been found in several bacterial species and in a single archaea genus. Recent X-ray structures of bacterial and invertebrate pentameric receptors point to a striking conservation of the structural features within the whole family, even between distant prokaryotic and eukaryotic members. These structural data reveal general principles of molecular organization that allow allosteric membrane proteins to mediate chemoelectric transduction. Notably, several conformations have been solved, including open and closed channels with distinct global tertiary and quaternary structure. The data reveal features of the ion channel architecture and of diverse categories of binding sites, such as those that bind orthosteric ligands, including neurotransmitters, and those that bind allosteric modulators, such as general anesthetics, ivermectin, or lipids. In this review, we summarize the most recent data, discuss insights into the mechanism of action in these systems, and elaborate on newly opened avenues for drug design.

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

Significance We describe the X-ray structures of the same pentameric ligand-gated ion channel (pLGIC) in both its liganded or ligand-free conformations. This provides the molecular basis for understanding the opening and closing (gating mechanism) of these key players in the fast transmission of chemical signals at synapses. As described with classical allosteric proteins, the tertiary changes of the subunits are linked together through the quaternary constraint by a marked reorganization of the interfaces between subunits and the associated binding pockets and cavities. The closed form displays a cavity that may allow a better understanding of the mechanism of action of pharmacological effectors of pentameric ligand-gated ion channels and the rational design of new modulators. 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.

Structural basis for potentiation by alcohols and anaesthetics in a ligand-gated ion channel.

Ethanol alters nerve signalling by interacting with proteins in the central nervous system, particularly pentameric ligand-gated ion channels. A recent series of mutagenesis experiments on Gloeobacter violaceus ligand-gated ion channel, a prokaryotic member of this family, identified a single-site variant that is potentiated by pharmacologically relevant concentrations of ethanol. Here we determine crystal structures of the ethanol-sensitized variant in the absence and presence of ethanol and related modulators, which bind in a transmembrane cavity between channel subunits and may stabilize the open form of the channel. Structural and mutagenesis studies defined overlapping mechanisms of potentiation by alcohols and anaesthetics via the inter-subunit cavity. Furthermore, homology modelling show this cavity to be conserved in human ethanol-sensitive glycine and GABA(A) receptors, and to involve residues previously shown to influence alcohol and anaesthetic action on these proteins. These results suggest a common structural basis for ethanol potentiation of an important class of targets for neurological actions of ethanol.

Structural basis for ion permeation mechanism in pentameric ligand‐gated ion channels

To understand the molecular mechanism of ion permeation in pentameric ligand‐gated ion channels (pLGIC), we solved the structure of an open form of GLIC, a prokaryotic pLGIC, at 2.4 Å. Anomalous diffraction data were used to place bound anions and cations. This reveals ordered water molecules at the level of two rings of hydroxylated residues (named Ser6′ and Thr2′) that contribute to the ion selectivity filter. Two water pentagons are observed, a self‐stabilized ice‐like water pentagon and a second wider water pentagon, with one sodium ion between them. Single‐channel electrophysiology shows that the side‐chain hydroxyl of Ser6′ is crucial for ion translocation. Simulations and electrostatics calculations complemented the description of hydration in the pore and suggest that the water pentagons observed in the crystal are important for the ion to cross hydrophobic constriction barriers. Simulations that pull a cation through the pore reveal that residue Ser6′ actively contributes to ion translocation by reorienting its side chain when the ion is going through the pore. Generalization of these findings to the pLGIC family is proposed.

Cyclodipeptide synthases, a family of class-I aminoacyl-tRNA synthetase-like enzymes involved in non-ribosomal peptide synthesis

Cyclodipeptide synthases (CDPSs) belong to a newly defined family of enzymes that use aminoacyl-tRNAs (aa-tRNAs) as substrates to synthesize the two peptide bonds of various cyclodipeptides, which are the precursors of many natural products with noteworthy biological activities. Here, we describe the crystal structure of AlbC, a CDPS from Streptomyces noursei. The AlbC structure consists of a monomer containing a Rossmann-fold domain. Strikingly, it is highly similar to the catalytic domain of class-I aminoacyl-tRNA synthetases (aaRSs), especially class-Ic TyrRSs and TrpRSs. AlbC contains a deep pocket, highly conserved among CDPSs. Site-directed mutagenesis studies indicate that this pocket accommodates the aminoacyl moiety of the aa-tRNA substrate in a way similar to that used by TyrRSs to recognize their tyrosine substrates. These studies also suggest that the tRNA moiety of the aa-tRNA interacts with AlbC via at least one patch of basic residues, which is conserved among CDPSs but not present in class-Ic aaRSs. AlbC catalyses its two-substrate reaction via a ping-pong mechanism with a covalent intermediate in which l-Phe is shown to be transferred from Phe-tRNAPhe to an active serine. These findings provide insight into the molecular bases of the interactions between CDPSs and their aa-tRNAs substrates, and the catalytic mechanism used by CDPSs to achieve the non-ribosomal synthesis of cyclodipeptides.

Allosteric and hyperekplexic mutant phenotypes investigated on an α1 glycine receptor transmembrane structure

Significance Pentameric ligand-gated ion channels (pLGICs) mediate neuronal communication in the central nervous system. Upon the neurotransmitter binding, these receptors undergo a rapid conformational change to open an integral ion channel. Mutations impairing the function of pLGICs are known to cause hyperekplexic, myasthenic, and epileptic syndromes. Here, we studied how the local perturbations caused by single mutations result in an alteration of the protein function. Using a chimeric protein assembled by the transmembrane domain of the human glycine receptors fused to the extracellular domain of the bacterial pLGIC GLIC, we performed functional experiments in parallel with X-ray crystallography. On this basis, we propose a molecular mechanism for channel opening that accounts for the phenotypes of several mutants causing hyperekplexia. 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 α1GlyR 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 α1GlyR 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 α1GlyR, 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 pore-opening mechanism for the α1GlyR, suggesting a structural explanation for the effect of some key hyperekplexic allosteric mutations. The first X-ray structure of the TMD of the α1GlyR solved here using GLIC as a scaffold paves the way for mechanistic investigation and design of allosteric modulators of a human receptor.

Crystallographic studies of pharmacological sites in pentameric ligand-gated ion channels

BACKGROUND Pentameric ligand-gated ion channels (pLGICs) mediate fast chemical transmission of nerve signals in the central and peripheral nervous system. On the functional side, these molecules respond to the binding of a neurotransmitter (glycine, GABA, acetylcholine or 5HT3) in the extracellular domain (ECD) by opening their ionotropic pore in the transmembrane domain (TMD). The response to the neurotransmitter binding can be modulated by several chemical compounds acting at topographically distinct sites, as documented by a large body of literature. Notably, these receptors are the target of several classes of world-wide prescribed drugs, including general anesthetics, smoking cessation aids, anxiolytics, anticonvulsants, muscle relaxants, hypnotics and anti-emetics. On the structural side recent progress has been made on the crystallization of pLGICs in its different allosteric states, especially pLGICs of bacterial origin. Therefore, structure-function relationships can now be discussed at the atomic level for pLGICs. SCOPE OF REVIEW This review focuses on the crystallographic structure of complexes of pLGICs with a number of ligands of pharmacological interest. First, we review structural data on two key functional aspects of these receptors: the agonist-induced activation and ion transport itself. The molecular understanding of both these functional aspects is important, as they are those that most pharmacological compounds target. Next, we describe modulation sites that have recently been documented by X-ray crystallography. Finally, we propose a simple geometric classification of all these pharmacological sites in pLGICs, based on icosahedrons. MAJOR CONCLUSIONS This review illustrates the wealth of structural insight gained by comparing all available structures of members of the pLGIC family to rationalize the pharmacology of structurally diverse drugs acting at topographically distinct sites. It will be highlighted how sites that had been described earlier using biochemical techniques can be rationalized using structural data. Surprisingly, the use of icosahedral symmetry allows to link together several modulation sites, in a way that was totally unanticipated. GENERAL SIGNIFICANCE Overall, understanding the interplay between the different modulation sites at the structural level should help the design of future drugs targeting pLGICs. This article is part of a Special Issue entitled structural biochemistry and biophysics of membrane proteins.

Sites of Anesthetic Inhibitory Action on a Cationic Ligand-Gated Ion Channel

Pentameric ligand-gated ion channels have been identified as the principal target of general anesthetics (GA), whose molecular mechanism of action remains poorly understood. Bacterial homologs, such as the Gloeobacter violaceus receptor (GLIC), have been shown to be valid functional models of GA action. The GA bromoform inhibits GLIC at submillimolar concentration. We characterize bromoform binding by crystallography and molecular dynamics (MD) simulations. GLICs open form structure identified three intra-subunit binding sites. We crystallized the locally closed form with an additional bromoform molecule in the channel pore. We systematically compare binding with the multiple potential sites of allosteric channel regulation in the open, locally closed, and resting forms. MD simulations reveal differential exchange pathways between sites from one form to the other. GAs predominantly access the receptor from the lipid bilayer in all cases. Differential binding affinity among the channel forms is observed; the pore site markedly stabilizes the inactive versus active state.

Genuine open form of the pentameric ligand-gated ion channel GLIC.

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.

Neurotoxic phospholipase A2 from rattlesnake as a new ligand and new regulator of prokaryotic receptor GLIC (proton-gated ion channel from G. violaceus).

Neurotoxic phospholipases A2 (sPLA2) from snake venoms interact with various protein targets with high specificity and potency. They regulate function of multiple receptors or channels essential to life processes including neuronal or neuromuscular chemoelectric signal transduction. These toxic sPLA2 exhibit high pharmacological potential and determination of PLA2-receptor binding sites represents challenging part in the receptor-channel biochemistry and pharmacology. To investigate the mechanism of interaction of neurotoxic PLA2 with its neuronal receptor at the molecular level, we used as a model crotoxin, a heterodimeric sPLA2 from rattlesnake venom and proton-gated ion channel GLIC, a bacterial homolog of pentameric ligand-gated ion channels. The three-dimensional structures of both partners, crotoxin and GLIC have been solved by X-ray crystallography and production of full-length pentameric GLIC (with ECD and TM domains) is well established. In the present study, for the first time, we demonstrated physical and functional interaction of full-length purified and solubilized GLIC with CB, (PLA2 subunit of crotoxin). We identified GLIC as a new protein target of CB and CB as a new ligand of GLIC, and showed that this non covalent interaction (PLA2-GLIC) involves the extracellular domain of GLIC. We also determined a novel function of CB as an inhibitor of proton-gated ion channel activity. In agreement with conformational changes observed upon formation of the complex, CB appears to be negative allosteric modulator (NAM) of GLIC. Finally, we proposed a possible stoichiometric model for CB - GLIC interaction based on analytical ultracentrifugation.