Samuel Murail

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.

Structural basis for alcohol modulation of a pentameric ligand-gated ion channel

Despite its long history of use and abuse in human culture, the molecular basis for alcohol action in the brain is poorly understood. The recent determination of the atomic-scale structure of GLIC, a prokaryotic member of the pentameric ligand-gated ion channel (pLGIC) family, provides a unique opportunity to characterize the structural basis for modulation of these channels, many of which are alcohol targets in brain. We observed that GLIC recapitulates bimodal modulation by n-alcohols, similar to some eukaryotic pLGICs: methanol and ethanol weakly potentiated proton-activated currents in GLIC, whereas n-alcohols larger than ethanol inhibited them. Mapping of residues important to alcohol modulation of ionotropic receptors for glycine, γ-aminobutyric acid, and acetylcholine onto GLIC revealed their proximity to transmembrane cavities that may accommodate one or more alcohol molecules. Site-directed mutations in the pore-lining M2 helix allowed the identification of four residues that influence alcohol potentiation, with the direction of their effects reflecting α-helical structure. At one of the potentiation-enhancing residues, decreased side chain volume converted GLIC into a highly ethanol-sensitive channel, comparable to its eukaryotic relatives. Covalent labeling of M2 positions with an alcohol analog, a methanethiosulfonate reagent, further implicated residues at the extracellular end of the helix in alcohol binding. Molecular dynamics simulations elucidated the structural consequences of a potentiation-enhancing mutation and suggested a structural mechanism for alcohol potentiation via interaction with a transmembrane cavity previously termed the “linking tunnel.” These results provide a unique structural model for independent potentiating and inhibitory interactions of n-alcohols with a pLGIC family member.

Class-B GPCR activation: is ligand helix-capping the key?

The class B family of G-protein-coupled receptors (GPCRs) regulates essential physiological functions such as exocrine and endocrine secretions, feeding behaviour, metabolism, growth, and neuro- and immuno-modulations. These receptors are activated by endogenous peptide hormones including secretin, glucagon, vasoactive intestinal peptide, corticotropin-releasing factor and parathyroid hormone. We have identified a common structural motif that is encoded in all class B GPCR-ligand N-terminal sequences. We propose that this local structure, a helix N-capping motif, is formed upon receptor binding and constitutes a key element underlying class B GPCR activation. The folded backbone conformation imposed by the capping structure could serve as a template for a rational design of drugs targeting class B GPCRs in several diseases.

Peptide agonist docking in the N-terminal ectodomain of a class II G protein-coupled receptor, the VPAC1 receptor. Photoaffinity, NMR, and molecular modeling.

The neuropeptide vasoactive intestinal peptide (VIP) strongly impacts on human pathophysiology and does so through interaction with class II G protein-coupled receptors named VIP pituitary adenylate cyclase-activating peptide (PACAP) receptors (VPACs). The molecular nature of VIP binding to receptors remains elusive. In this work, we have docked VIP in the human VPAC1 receptor by the following approach. (i) VIP probes containing photolabile residues in positions 6, 22, and 24 of VIP were used to photolabel the receptor. After receptor cleavage and Edman sequencing of labeled receptor fragments, it was shown that Phe6, Tyr22, and Asn24 of VIP are in contact with Asp107, Gly116, and Cys122 in the N-terminal ectodomain (N-ted) of the receptor, respectively. (ii) The structure of VIP was determined by NMR showing a central α helix, a disordered N-terminal His1-Phe6 segment and a 310 Ser25-Asn28 helix termination. (iii) A three-dimensional model of the N-ted of hVPAC1 was constructed by using the NMR structure of the N-ted of corticotropin-releasing factor receptor 2β as a template. As expected, the fold is identified as a short consensus repeat with two antiparallel β sheets and is stabilized by three disulfide bonds. (iv) Taking into account the constraints provided by photoaffinity, VIP was docked into the hVPAC1 receptor N-ted. The 6-28 fragment of VIP nicely lies in the N-ted C-terminal part, but the N terminus region of VIP is free for interacting with the receptor transmembrane region. The data provide a structural rationale to the proposed two-step activation mechanism of VPAC receptor and more generally of class II G protein-coupled receptors.

Microsecond simulations indicate that ethanol binds between subunits and could stabilize an open-state model of a glycine receptor.

Cys-loop receptors constitute a superfamily of ion channels gated by ligands such as acetylcholine, serotonin, glycine, and γ-aminobutyric acid. All of these receptors are thought to share structural characteristics, but due to high sequence variation and limited structure availability, our knowledge about allosteric binding sites is still limited. These sites are frequent targets of anesthetic and alcohol molecules, and are of high pharmacological importance. We used molecular simulations to study ethanol binding and equilibrium exchange for the homomeric α1 glycine receptor (GlyRα1), modeled on the structure of the Gloeobacter violaceus pentameric ligand-gated channel. Ethanol has a well-known potentiating effect and can be used in high concentrations. By performing two microsecond-scale simulations of GlyR with/without ethanol, we were able to observe spontaneous binding in cavities and equilibrium ligand exchange. Of interest, it appears that there are ethanol-binding sites both between and within the GlyR transmembrane subunits, with the intersubunit site having the highest occupancy and slowest exchange (∼200 ns). This model site involves several residues that were previously identified via mutations as being crucial for potentiation. Finally, ethanol appears to stabilize the GlyR model built on a presumably open form of the ligand-gated channel. This stabilization could help explain the effects of allosteric ligand binding in Cys-loop receptors.

Secondary and tertiary structures of the transmembrane domains of the translocator protein TSPO determined by NMR. Stabilization of the TSPO tertiary fold upon ligand binding

Numerous biological functions are attributed to the peripheral-type benzodiazepine receptor (PBR) recently renamed translocator protein (TSPO). The best characterized function is the translocation of cholesterol from the outer to inner mitochondrial membrane, which is a rate-determining step in steroid biosynthesis. TSPO drug ligands have been shown to stimulate pregnenolone formation by inducing TSPO-mediated translocation of cholesterol. Until recently, no direct structural data on this membrane protein was available. In a previous paper, we showed that a part of the mouse TSPO (mTSPO) C-terminal region adopts a helical conformation, the side-chain distribution of which provides a groove able to fit a cholesterol molecule. We report here on the overall structural properties of mTSPO. This study was first undertaken by dissecting the protein sequence and studying the conformation of five peptides encompassing the five putative transmembrane domains from (1)H-NMR data. The secondary structure of the recombinant protein in micelles was then studied using CD spectroscopy. In parallel, the stability of its tertiary fold was probed using (1)H-(15)N NMR. This study provides the first experimental evidence for a five-helix fold of mTSPO and shows that the helical conformation of each transmembrane domain is mainly formed through local short-range interactions. Our data show that, in micelles, mTSPO exhibits helix content close to what is expected but an unstable tertiary fold. They reveal that the binding of a drug ligand that stimulates cholesterol translocation is able to stabilize the mTSPO tertiary structure.

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.

Molecular mechanism for the dual alcohol modulation of cys loop receptors

Cys-loop receptors constitute a superfamily of pentameric ligand-gated ion channels (pLGICs), including receptors for acetylcholine, serotonin, glycine and γ-aminobutyric acid. Several bacterial homologues have been identified that are excellent models for understanding allosteric binding of alcohols and anesthetics in human Cys-loop receptors. Recently, we showed that a single point mutation on a prokaryotic homologue (GLIC) could transform it from a channel weakly potentiated by ethanol into a highly ethanol-sensitive channel. Here, we have employed molecular simulations to study ethanol binding to GLIC, and to elucidate the role of the ethanol-enhancing mutation in GLIC modulation. By performing 1-µs simulations with and without ethanol on wild-type and mutated GLIC, we observed spontaneous binding in both intra-subunit and inter-subunit transmembrane cavities. In contrast to the glycine receptor GlyR, in which we previously observed ethanol binding primarily in an inter-subunit cavity, ethanol primarily occupied an intra-subunit cavity in wild-type GLIC. However, the highly ethanol-sensitive GLIC mutation significantly enhanced ethanol binding in the inter-subunit cavity. These results demonstrate dramatic effects of the F(14′)A mutation on the distribution of ligands, and are consistent with a two-site model of pLGIC inhibition and potentiation.

Spatial proximity between the VPAC1 receptor and the amino terminus of agonist and antagonist peptides reveals distinct sites of interaction

Vasoactive intestinal peptide (VIP) plays a major role in pathophysiology. Our previous studies demonstrated that the VIP sequence 6‐28 interacts with the N‐terminal ectodomain (N‐ted) of its receptor, VPAC1. Probes for VIP and receptor antagonist PG97‐269 were synthesized with a photolabile residue/Bpa at various positions and used to explore spatial proximity with VPAC1. PG97‐269 probes with Bpa at position 0, 6, and 24 behaved as high‐affinity receptor antagonists (Ki=12, 9, and 7 nM, respectively). Photolabeling experiments revealed that the [Bpa0]‐VIP probe was in physical contact with VPAC1 Q135, while [Bpa0]‐PG97‐269 was covalently bound to G62 residue of N‐ted, indicating different binding sites. In contrast, photolabeling with [Bpa6]‐ and [Bpa24]‐PG97‐269 showed that the distal domains of PG97‐269 interacted with N‐ted, as we previously showed for VIP. Substitution with alanine of the K143, T144, and T147 residues located in the first transmembrane domain of VPAC1 induced a loss of receptor affinity (IC50=1035, 874, and 2070 nM, respectively), and pharmacological studies using VIP2‐28 indicated that these three residues play an important role in VPAC1 interaction with the first histidine residue of VIP. These data demonstrate that VIP and PG97‐269 bind to distinct domains of VPAC1.—Ceraudo, E., Hierso, R., Tan, Y.‐V., Murail, S., Rouyer‐Fessard C., Nicole, P., Robert, J.‐C., Jamin, N., Neumann, J.‐M., Robberecht, P., Laburthe, M., Couvineau, A. Spatial proximity between the VPAC1 receptor and the amino terminus of agonist and antagonist peptides reveals distinct sites of interaction. FASEB J. 26, 2060‐2071 (2012). www.fasebj.org

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.