4-BrC 4-Bromocinnamic acid CPZ Chlorpromazine PAM

pLGICs pLGICs Pentameric ligand-gated ion channels GLIC Gloeobacter violaceus pentameric ligand-gated ion channel ELIC Erwinia chrysanthemi pentameric ligand-gated ion channel sTeLIC symbiont of Tevnia jerichonana pentameric ligand-gated ion channel DeCLIC Desulfofustis pentameric ligand-gated ion channel nAChR Nicotinic acetylcholine

ACKNOWLEDGEMENT

I am really grateful for working at Pasteur with many excellent scientists during the past three years. It was extremely hard for me at beginning of my Ph.D. I would not complete this thesis without receiving help and support from my supervisor, my colleagues, my collaborators, my friends, my families.

Thanks to Marc, for your constant guidance and support throughout my Ph.D.

You spent lots of time with me, to discuss about my projects, to solve the problems I met, to correct my poor English. It is my pleasure to work with you. I learn a lot in the lab.

Thanks to Jérôme, Ludovic, Zaineb, for many great discussions and good time in the lab, for the great contribution to my work. You really make me feel warm in the lab.

SUMMARY

Ligand-gated pentameric ion channels (pLGIC) are one of the major families of transmembrane receptors. They allow rapid signal transduction in the central and peripheral nervous systems via neurotransmitters binding. PLGICs are also present in archaea and bacteria. Only two bacterial pLGICs have been biochemically and structurally characterized so far (GLIC and ELIC). They serve as working models for many scientists and have been extensively studied both at the functional and structural levels.

In the first part of my thesis, I purified, crystallized and solved the crystal structure of a new pLGIC from gamma-proteobacterial symbionts of Tevnia jerichonana (sTeLIC). Functional experiments show that sTeLIC is activated by alkaline pH, and is selective for monovalent cationic ions and inhibited by divalent cations. The crystal structure solved at pH 8.0 displays a widely open pore that is the first of this kind to be characterized in the pLGIC family. In addition, we identified a strongly positive modulator that binds to the "vestibule site" in the extracellular domain, and we solved the crystal structure of this complex. Functional experiments show that sTeLIC shares many features with ELIC. ELIC and sTeLIC are the archetypes of a new class of pLGICs, whose active form is characterized by a much more open pore than other pLGICs.

In the second part of my thesis, the proton sensor residues in GLIC have been mapped. All titratable GLIC residues were tested by site-directed mutagenesis to discover proton sensors involved in the triggering process. We have demonstrated that the residue E35 is a key residue, whose charged form stabilizes the resting state, and the protonated form the active state. We have also demonstrated that the proton response is dependent on two distinct networks at the ECD-TMD interface, which stabilize the open state of GLIC.

In the third part of my thesis, I cloned, purified, crystallized and determined the crystal structures of the open and closed forms of DeCLIC, a pLGIC of Desulfofustis proteobacterium. Each subunit contains a large N-terminal additional domain consisting of two subdomains (NTD1 and NTD2). This is the first structure of a pLGIC which contains a non-canonical additional extracellular domain.

Dans la troisième partie, j'ai cloné, purifié, cristallisé et déterminé les structures cristallines des formes ouvertes et fermées de DeCLIC, un pLGIC de la protéobactérie Desulfofustis. Chaque sous-unité contient un grand domaine additionnel N-terminal constitué de deux sous-domaines (NTD1 et NTD2). Il s'agit de la première structure d'un pLGIC qui contient un domaine supplémentaire extracellulaire non-canonique.

The cell membrane is comprised of two major components: lipids and proteins.

Phospholipids are the dominating membrane lipids that can self-assemble into a lipid bilayer. The hydrophobic fatty-acid tails interact with each other and thereby are isolated from the solvent environment, while the hydrophilic solvent-exposed heads face outward. Thus, the heads of the lipids border both the cytoplasm and the exterior of the cell. The two-layer structure, termed phospholipid bilayer, can be found in many cell organelles and control many fundamental biological processes as shown in Figure 1. Different kinds of lipids may also be found in the cell membrane and affect its function and some of them uniquely exist in animal cells, for instance, cholesterol.

Cholesterol is biosynthesized by all animal cells and is a key component of all animal cell membranes 3 . It functions as a special type of lipid molecule and controls the rigidity of the membrane as well as determines the membrane integrity and thereby cell viability 4 . Cholesterol can also interact with membrane proteins and thus affect the membrane protein structure and function 5 .

The membrane proteins can be roughly classified into three groups: peripheral proteins, lipid-anchored proteins and integral proteins. Peripheral proteins include some enzymes, polypeptide hormones and toxins. Lipid-anchored proteins are covalently attached to single lipid molecules or multiple lipid molecules. The lipid groups interact with the membrane and can directly modulate the protein function.

Finally, the integral proteins contain one or more membrane-spanning domains, which consist of α-helical or β-barrel motifs, such as water channel proteins (aquaporin 6 ), G protein-coupled receptors (rhodopsin), as well as all kinds of ion channels and ion transporters.

Ion channels and ion transporters.

Ion channels and ion transporters are two of the major integral membrane protein families. They control the flow of ions across the cell membrane, thereby establishing a resting membrane potential. They control electrical signals including the shape of action potentials, which are the change in electrical potential associated with the passage of an impulse along the membrane of a muscle cell or nerve cell. In humans, ion channels are involved in many cellular processes and many clinical drugs have been developed and used to target ion channels for the treatment of many diseases. Ion channels and ion transporters constitute the ionophoric proteins, which are present in the membranes of all cells. The major differences between ion channels and ion transporters can be summarized as:

i) The speed of ions transported through the channels.

ii) Requirement of metabolic energy for ion passage.

iii) Direction of ion transport with respect to its chemical gradient Ionophoric proteins are pore-forming membrane proteins that comprise the ion channels and ion transporters according to their different ion translocation mechanisms. Ion transporters need the energy provided by hydrolyzing ATP molecules to transport ions against their chemical gradient.

Voltage-gated ion channels respond to change of membrane potential. Ligand-gated ion channels respond to special ligands (agonists). also termed as P-type ion pumps, because the change of the transporters' conformation is coupled to their phosphorylation state via ATP interaction.

The diversity of ion channels.

There are many ways to classify ion channels: by the nature of their gating, the species of ions transported through the channels, the localization of the ion channels, the number of channel pores, as well as the subunit composition when the channels are formed by several homologous monomers 8 .

Ion channel classification by gating.

Voltage-gated ion channels (VGICs).

The activity of VGICs is regulated by the potential change of membrane. According to the species of ions conducted, VGICs can be further classified as voltage-gated calcium channels 9 , voltage-gated sodium ion channels 10 , voltage-gated potassium ion channels 11 , voltage-gated chloride ion channels 12 , as well as voltage-gated proton channels 13 . Cloning of bacterial voltage-gated ion channels has enabled purification of the channels that resulted in high-resolution diffraction of subsequent crystals. For example, the crystallographic structural resolution of a bacterial voltage-gated sodium ion channel has been reported (Fig 4). They provide structural models at the atomic level for understanding the structural basis of the voltage-dependent gating mechanism, ion preference, in addition to modulator binding properties. Generally, the overall architecture of each VGIC subunit comprises six transmembrane segments (S1-S6) with a voltage sensor domain (VSD) and a pore-forming domain, which is also termed the ion conducting pore module (PM). Recently, a high resolution structure of a eukaryotic voltage-gated sodium ion channel from an American cockroach has been resolved by Cryo-electron microscopy (Cryo-EM) at 3.8 angstrom resolution 14 . From prokaryote to eukaryote, VGICs share the same core architecture, namely the voltage sensor domain and the pore module. However, in eukaryotes, VGICs contain a larger auxiliary domain that may modulate channel properties. Additionally, the VGICs from eukaryotes are subjected to many posttranslational modifications, for instance, glycosylation, phosphorylation, and palmitoylation. These multiple posttranslational modifications also modulate the function of VGICs 15 . The bacterial voltage-gated sodium channels are formed by four monomers organized along the four-fold symmetry axis perpendicular to the cell membrane.

Each monomer contains two core domains, namely a voltage sensing domain and a pore-forming domain. These two core domains are highly conserved between receptors from prokaryotes and eukaryotes.

Ligand-gated ion channels.

Ligand-gated ion channels, also termed as ionotropic receptors, commonly contain two domains: a ligand-binding/extracellular domain (ECD) and a poreforming/transmembrane domain (TMD). In response to specific ligand molecules binding to the ECD, the receptors will undergo a global conformational change, and this conformational change will trigger the channel gate's opening. As a consequence, ions will permeate through the channel down their electrochemical gradient, which changes the plasma membrane potential 16 . Ligand-gated ion channels can be further classified by the oligomeric state, such as trimeric acid-sensing ion channels (ASICs) 17 , tetrameric glutamate-gated ion channels including NMDA receptor, kainate receptor, AMPA receptor (Figure 5), and pentameric ligand-gated ion channel (pLGICs).

Ion channels gated by other mechanisms.

Light-gated ion channels: Receptors in this family are open by photons, such as channel-rhodopsins 18 .

Mechanosensitive ion channels: Receptors in this family are open in response to pressure change and/or displacement 19 .

Temperature-gated ion channels: Receptors in this family are sensitive to the change in environmental temperature. Either the cold or the heat will trigger a conformational change of the ion channel pore. The representative receptors in this family are transient receptor potential channels (TRPCs) 20 .

My research will focus on the receptors from pLGIC family.

Pentameric ligand-gated ion channels (pLGICs).

The concept of a 'receptive substance', which localizes to the synapse, can be traced back to the beginning of 20 th century and the work of John Newport Langley on the observation that nicotine stimulates sympathetic nerve cells by a direct action upon them and curare behaves as the blocker that directly blocks this procedure.

Pentameric ligand-gated ion channels (pLGICs), a major family of transmembrane proteins, are expressed on the plasma membrane and mediate fast signal transduction in the central and peripheral nervous system. They were identified as the targets of the 'receptive substance'. The first isolated membrane protein belonging to the pLGIC family was the nicotinic acetylcholine receptor (nAChR) 21 . It was first used for single channel recording by expressing it in the membrane of denervated frog muscle fibres 22 . Subsequent studies revealed that pLGICs are receptors which respond to neurotransmitters including glycine, gamma-aminobutyric acid (GABA), serotonin, glutamine, histamine, as well as zinc ions 23 . Receptors from this family were first referred to as acetylcholine receptor type ligand-gated ion channels (ART-LGICs), then as 'Cys-loop' receptors because of a pair of conserved cysteine residues (only strictly conserved in eukaryotic pLGICs) located at the interface of the ECD and TMD, then as pentameric ligand-gated ion channels (pLGICs) in order to make a distinction among glutamate-gated tetrameric receptors, ATP-gated trimeric receptors, and acid-sensing trimeric ion channels. Finally, with the classification of prokaryotic pLGICs which lack the strictly conserved cysteine residues found in their eukaryotic counterparts, the term Pro-loop receptors, because of the widely conserved proline at the tip of 'Cysloop', has replaced 'Cys-loop' when referring to both prokaryotic and eukaryotic receptors 24 .

pLGICs widely exist from prokaryotes to eukaryotes.

From invertebrates to vertebrates, pLGICs widely exist in metazoans. They have been extensively studied both at the functional and structural levels 25,26 . In 2005, Tasneem et al found that pLGICs also exist in bacteria as well as archaea (Fig 6).

Following this work, the bacterial receptors provided the first high-resolution crystal structures of intact full-length ligand-gated ion channels. In the following part, I will present and discuss the remarkable progress on pLGICs, mainly based on the structural information made available during the past decade.

Allosteric states of pLGICs

pLGICs exist in multiple allosteric states. However, three major states can be classified as: resting/closed/basal state, active/open state, or desensitized state 25 .

Each of these states has its unique affinity for the agonist and unique structural conformations. For instance, the resting state has the lowest affinity for agonists; whereas the desensitized state, for which the receptor adopts a closed ion channel pore, has a high affinity for agonists. In addition to these three major states, several research teams have reported several short-lived intermediate states that exist during the activation of nAChRs and glycine receptors (GlyRs) [27][28][29] . Finally, fast desensitized and slow desensitized states have also been described (Figure 7) 30 . These works provide the general architecture of pLGICs. Five identical or homologous monomers are arranged symmetrically along a five-fold symmetry axis.

The ECD of each subunit is organized by two sets of β-sheets that pack into a βsandwich. In eukaryotic receptors, the interfaces of the ECDs harbor the agonist/neurotransmitter binding sites. The TMD of each subunit is made of four membrane-spanning helical segments (M1-M4). The five M2 helices, which shape the channel pore, are shielded by the M1 and M3 helices. The M4 helices are peripheral and face the lipid environment. The intracellular domain (ICD) of each subunit, which is absent in prokaryotic pLGICs, is made of a flexible helical segment that connects the M3 and M4 helices. However, because of limited resolution, the structural details of side-chain conformations were unclear. High-resolution structural determination of receptor conformations with different channel architectures is needed to address the central mechanistic issues of: ion permeation, gating, ligand binding, as well as modulator binding.

Beyond metazoans, pLGICs also exist in the genomes of bacteria and Archaea 24,35 . In 2006, Bocquet et al cloned and purified one of these pLGICs, a pLGIC from Gloeobacter violaceus. This homomeric receptor, referred to as GLIC, is open by increasing the extracellular proton concentration. GLIC shows selectivity for cations. This receptor can be well expressed in E. coli as a pentamer when its N-terminal is fused with a maltose binding protein (MBP) 36 . Compared to pLGICs from the eukaryotic system, GLIC has no intracellular domain. Instead, a short loop connects the M3 and M4 helix. These results provide good evidence that GLIC is a suitable candidate for structural studies by means of crystallography and that it can serve as a model system to understand the structures of eukaryotic receptors.

Following this work, two different labs reported the crystal structures of GLIC with 2.9 Å and 3.1 Å resolutions, simultaneously 37,38 . Around the same time, the crystal structure of another pLGIC from proteobacteria Erwinia chrysantemi, termed as ELIC, was also reported at 3.3 Å resolution 39 . They have been extensively used as models to study the pLGIC family, including: potential gating mechanisms, ligand binding properties, and ion selectivity. In the following part, I will document the structural and functional details of these two bacterial pLGICs.

1.5 Structural information of pLGICs from bacteria.

Erwinia chrysantemi ligand-gated ion channel (ELIC)

ELIC shares 18% and 16% sequence identity with GLIC and nAChRα7, respectively. The first crystal structure of ELIC was determined using single isomorphous replacement with anomalous scattering (SIRAS), taking advantage of the signal coming from seleno-methionine. This receptor is a cationic channel and shows no discrimination among monovalent cations Na + , K + and Cs + . The overall architecture of ELIC adopts the same conformation as the nAChR from Torpedo marmorata constructed according to EM images. The most interesting feature of this receptor is the conformation of the ion channel pore. Near the extracellular part, the channel is interrupted by bulky side chains of Phe 246 (F16') and Leu 239 (L9'), the hydrophobic side chain of these two residues occlude a hydrophobic cavity and prevent the diffusion of ions 39 (Fig 9). In the nAChR, it has been demonstrated that hydrophobic residues in the center of the pore play an important role in the channel closing [40][41][42] . the sites in the ECD have no functional impact of ethanol inhibition. Thus, it seems that the pore binding site (6' position) is the dominant blocking site in EtOH inhibition of ELIC 54 . Isoflurane, one of the general anesthetics, inhibits ELIC by binding to the channel pore (6' and 13' positions). Both EtOH and isoflurane inhibition of ELIC may share the same mechanism by occupying the channel pore position and stabilizing ELIC in a closed channel conformation. This is further demonstrated by the crystal structure of ELIC with memantine, an anti-Alzheimer's disease drug. Memantine binds to the channel pore at the F16' position level to inhibit ELIC 55 .

Gloeobacter violaceus ligand-gated ion channel (GLIC)

Four different conformations of GLIC GLIC, a proton-gated pentameric ion channel, sharing 20% sequence identity with the human nAChR subtype α7, can be well expressed in the E. coli system when its N-terminus is fused with MBP tag. The 2.9 Å resolution crystal structure, which was determined in a pH 4 environment, represents the first crystal structure of the high resolution of the pLGIC family in apparently open conformation 37 (Figure 13). The overall architecture is the same as that of ELIC. As the crystals are grown at pH 4, the receptors are fully surrounded by the agonist, namely protons. This structure shows a funnel-shaped ion channel pore with a diameter ranging from 5 Å inside (cytoplasmic domain side) to 12 Å outside (extracellular domain side). The size of the ion channel pore of GLIC sharply contrasts with the closed conformation of ELIC structure, where the minimal pore diameter is 2 Å. Thus this GLIC conformation is suggestive of an open structure 37 . and locally-closed forms can coexist as discrete ones at pH 4 in same crystal form 59 . At pH 4, the ECD of GLIC is in an agonist-bound conformation; however, the TMD of GLIC can adopt both LC and open conformations (see Figure 15). Finally, a lipid-induced potential desensitized-state of GLIC was also reported by a complex structure of GLIC with docosahexaenoic acid (DHA), an omega-3 fatty acid that can enhance the desensitization state of GLIC in the same way as it affects human pLGICs 61 . The DHA binds to the periphery of the TMD region. The ion channel pore in the DHA-bound structure adopts a slightly different conformation with the intracellular-half constricted. In summary, different GLIC conformations have been captured with various either open or closed ion channel configurations. Attributing these different conformations to a special allosteric state is still debatable. For example, in the open state of GLIC, the minimum radius of the ion channel pore is around 2.5 Å at T2' position and this size doesn't allow a fully hydrated ion to pass through. The question should then be addressed whether GLIC can permeate partially hydrated ions. However, the molecular dynamic simulation predicts that this conformation is indeed a conductive form 62 . Further experimental studies should be carried out to answer this question.

Allosteric modulators of GLIC

GLIC has been captured in different conformations. Even though the nominal resolution of the resting form of GLIC is just around 4.35 Å, non-crystallographic symmetry (NCS) averaging over four copies in the asymmetric unit allowed for a better quality electron density map to be obtained. However, the high resolution of the open and LC forms, where the LC forms are believed to be intermediate states between the resting and the open states 63 , enable us to study the interaction of important therapeutic reagents in the same receptor with two allosteric states at the atomic scale.

Interestingly, GLIC is shown to be sensitive to clinical concentration of general anesthetics 64 , such as desflurane, halothane, isoflurane, sevoflurane, and propofol. These compounds show inhibition of the currents through GLIC at clinically used concentrations and their principle targets are indeed human pLGICs 65 . Structural information from propofol/GLIC and desflurane/GLIC complexes reveal a common general anesthetics binding site in the upper part of the TMD 66 . Deciphering the molecular mechanism of general anesthetics requires the structural description of the complexes with eukaryotic pLGICs. However, the structures of propofol/GLIC and desflurane/GLIC provide a prototype to understand the structural details of general anesthetic binding sites. Ethanol, one of the most widely used reagents in human life, can alter nerve signaling by targeting human pLGICs. Interestingly, ethanol weakly potentiates proton-induced currents in GLIC. This phenotype is similar to some eukaryotic pLGICs, which are also potentiated by ethanol. However, one of the GLIC mutants, F14'A, can convert GLIC into a highly ethanol-sensitive channel. This mutant provides a useful model system for structural characterization of ethanol binding sites in GLIC 67 . In contrast with the ethanol-binding sites in ELIC (pore inhibitory binding site at the 6' position), the ethanol-binding site in GLIC (open form) is located at the transmembrane inter-subunit cavity. This may suggest that the inter-subunit cavity is a potentiation ligand-binding site. Barbiturates can induce anesthesia by targeting and modulating human pLGICs 68 . GLIC has also been shown to be sensitive to barbiturates at clinical concentrations. Crystal structure information shows that barbiturates preferentially stabilize the closed state of GLIC and that the binding site is located in the ion channel pore [START_REF] Fourati | Barbiturates Bind in the GLIC Ion Channel Pore and Cause Inhibition by Stabilizing a Closed State[END_REF] . This may suggest that the ion channel pore site is an inhibitory ligand-binding site.

GLIC and ELIC are cationic receptors from bacteria and they just contain the ECD and TMD. This means that GLIC and ELIC mimic the minimum core structure required for the receptor to be functional. They provide models to study many aspects of pLGICs at an atomic level with high-resolution. In parallel, structural information of pLGICs from eukaryotic systems has also emerged recently. In the following part, I will document the available six eukaryotic receptors whose structures have been determined either by X-ray crystallography (cationic channels: a 5-HT3A receptor from mouse, a α4β2 nicotinic receptor from human; and anionic channels: αGluCl receptor from caenorhabditis elegans, GABAA receptor from human, α3 glycine receptor from human) or by Cryo-EM (anionic channel: an α1 glycine receptor from Zebrafish).

Structural information of pLGICs from eukaryotes at an atomic level.

Crystal structure of the 5-HT 3A receptor

The X-ray structure of the mouse serotonin 5-HT3A receptor was solved by Hassaine and Deluz et al at 3.5 Å resolution. This receptor was crystallized in the presence of crystallization chaperone nanobodies, a specific antibody to the 5-HT3A receptors. The structural model contains three domains (ECD, TMD, and part of the ICD) [START_REF] Hassaine | X-ray structure of the mouse serotonin 5-HT3 receptor[END_REF] . To date, five subunits of serotonin-gated 5HT3 receptor, termed A to E, have been identified in humans. The A and B subunits are expressed widely throughout the brain [START_REF] Lummis | 5-HT3 Receptors[END_REF] . The determined crystal structure of the 5-HT3A receptor contains a hydrophobic constriction at the level of (L9') with a radius around 2.3 Å, too narrow for a cation to pass with its full hydration shell. However, the backbone conformation and the pore radius at the backbone level look very similar to the open form of GLIC.

In addition, if we consider the slight fluctuations of the side chain conformation, for example L9', the 5-HT3A receptor conformation may allow for ion passage. The ionic selectivity of 5-HT3A is located at the bottom of the M2 helix and is composed of D-4' and E-1'. The ICD, which comprises 70 to 150 residues, is important for vertebrate receptor trafficking and clustering at the synapse [START_REF] Zuber | Structure and superorganization of acetylcholine receptorrapsyn complexes[END_REF] . The crystal structure of this 5-HT3A doesn't have an exit pathway for the ions at the ICD level. Local conformational changes, either along the channel axis or the lateral portals, have to be taken into account to allow for the ions to exit. Recently, a low-resolution (12 Å) Cryo-EM structure of 5-HT3A also shows the interactions between the densely packed receptors in lipids, and such 2D-organization were mainly due to interaction between the ICDs 73 .

Crystal structure of an α4β2 nicotinic receptor from human (α4β2-nAChR)

The structure of the human α4β2-nAChR resolved at 3.9 Å resolution in the presence of nicotine represents the first heteromeric receptor determined at highresolution by X-ray crystallography in the pLGIC family [START_REF] Morales-Perez | X-ray structure of the human α4β2 nicotinic receptor[END_REF] . Assigning the subunits into the model was facilitated by the co-crystal structure with 5-Iodo-A85380. 5-Iodo-A85380 is a potent agonist, specifically binding to the same α-β interface binding site as nicotine. This structure in complex shows that 5-Iodo-A85380 only exists in the assigned α-β interface as revealed by the anomalous signal coming from iodine. The overall architecture of α4β2-nAChR resembles that of GLIC and ELIC. However, there are details in the conformation that differ. The minimum diameter of the α4β2-nAChR channel pore is around 3.8 Å, which is too narrow for hydrated sodium ions with a single water molecule to pass through (diameter of a single sodium is 1.9 Å, diameter of a water molecule is 2.8 Å). Additionally, according to electrophysiology, in the presence of nicotine, the receptor shows fast desensitization. Thus, together with the structural analysis, this α4β2-nAChR conformation may represent a desensitized form.

Detailed structural analysis of this receptor reveals why only the α-β interface can accommodate the agonist nicotine, while the β-α interface and α-α interface [START_REF] Morales-Perez | X-ray structure of the human α4β2 nicotinic receptor[END_REF] cannot. This is due to the conserved aromatic residues in the α-β interface that can specifically recognize and accommodate for nicotine.

The crystal structures of GLIC, ELIC, 5-HT3A, and α4β2-nAChR paved the way to understand the cationic receptor sub-family of pGLICs. In the following part, I will document the available structural information of eukaryotic anionic pLGIC receptors.

Caenorhabditis elegans Glutamate-gated chloride ion channel (GluCl)

GluCl is an inhibitory anion-selective pLGIC isolated from Caenorhabditis elegans. The 3.3 Å X-ray crystal structure of the GluCl-Fab (Fab, an antibody of GluCl, used as a crystallization chaperon) complex was solved in the presence of an allosteric agonist ivermectin. Ivermectin is an antiparasitic agent that has been widely used to treat river blindness, a disease that is caused by infection with the worm Onchocerca open channel blocker, could bind to GluCl at the T2' position as revealed by the crystal-complex structure of GluCl with picrotoxin [START_REF] Hibbs | Principles of activation and permeation in an anionselective Cys-loop receptor[END_REF] . Later, two additional conformations of GluCl have been determined. One is an inactive state and the other is in complex with a potentiator 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). In an inactive state, the constriction of the channel is located at L9', with a diameter around 2.8 Å. Thus, this structure may represent a resting state with the shut gate located in the middle of the ion channel pore. This closed conformation precludes the dehydrated chloride ion from passing through. In the GluCl-POPC complex, the site occupied by POPC molecules overlaps with the ivermectin binding site found from the GluCl-ivermectin complex. The constriction part of the ion channel in GluCl-POPC complex is also located at the L9' with the diameter around 4.8 Å. Thus, the POPC bound state seems to be conductive for hydrated chloride ions. This is consistent with the functional experiments that show that POPC can compete with ivermectin and potentiates glutamate binding [START_REF] Hibbs | Principles of activation and permeation in an anionselective Cys-loop receptor[END_REF] . The three different conformations of GluCl allow to investigate the global conformational changes of the same receptor in the different allosteric states.

Crystal structure of a human GABA A receptor

The human β3-GABAA homopentameric receptor was determined at 3 Å resolution in the presence of the benzamidine, an agonist capable of inducing desensitization of the β3-GABAA receptor at micromolar concentration levels [START_REF] Miller | Crystal structure of a human GABAA receptor[END_REF] . The binding site of benzamidine is located at the conventional agonist-binding cavity beneath the Loop C. The narrowest region along the M2 helices in the channel is around 3.15 Å diameter as defined by the A-2' region. This diameter is too narrow to allow for chloride ion passage. This may suggest a nonconductive state and can be attributed to an agonist-bound desensitized state. Additionally, several N-linked glycosylation sites were also found in this structure. A noticeable site, N149 on the β7 strand is quite conserved among GABAAR, nAChR and 5HT3R. This glycosylation site is believed to be associated with modulation of the GABAA receptor [START_REF] Buhr | Functional characterization of the new human GABA(A) receptor mutation beta3(R192H)[END_REF] . However, until now, just one structural conformation of GABAA receptor is available.

Crystal structure of human Glycine receptor-α3 (α3-GlyR)

The structure of α3-GlyR was determined in a complex with strychnine at 3.0 Å resolution. Strychnine, a potent and selective antagonist of GlyRs can stabilize the resting form of GlyRs at nanomolar concentration [START_REF] Huang | Crystal structure of human glycine receptor-α3 bound to antagonist strychnine[END_REF] , binds GlyRs competitively with their agonist such as glycine. The co-crystal structure of α3-GlyR-strychnine reveals that the strychnine-binding site overlaps with the conventional agonist-binding cavity. Along the ion channel pore of this structure, the narrowest constriction ring is located at Leu261 (L9') in the middle point of the channel with a diameter around 2.8 Å.

Therefore, this conformation is consistent with a closed, non-conducting state. The non-conductive state of α3-GlyR is also consistent with the electrophysiology experiments showing that competitive antagonists can induce pLGICs in a resting/closed state. Later, the homopentameric structure of α3-GlyR receptor was determined in the presence of both agonist (glycine) and analgesic potentiator (AM-3607) [START_REF] Huang | Crystal structures of human glycine receptor α3 bound to a novel class of analgesic potentiators[END_REF] . In contrast to the strychnine-bound structure, the agonist/potentiator bound GlyR structure shows a constriction ring located at P-2' with a diameter of 2.8 Å. Thus, the agonist/potentiator bound state may represent a desensitized state. In this structure the glycine is bound to the conventional agonist-binding site, the intersubunit cavity beneath the Loop C. However, the AM-3607 is found to bind in a novel allosteric site located near to the top of ECD at the interface between the subunits. This novel allosteric binding site is observed for the first time in the pLGIC family [START_REF] Huang | Crystal structures of human glycine receptor α3 bound to a novel class of analgesic potentiators[END_REF] . Additionally, co-crystal structure of α3-GlyR with ivermectin, a positive modulator of several Pro-loop receptors including α3-GlyR, reveals that ivermectin binds to a common transmembrane intersubunit site among pLGIC family [START_REF] Huang | Crystal Structures of Human GlyRα3 Bound to Ivermectin[END_REF] .

Cryo-EM structures of glycine receptor from zebrafish (α1-GlyR)

The structures of glycine receptor from zebrafish in three different states were determined by the Cryo-EM method with resolution ranging from 3.8 Å-3.9 Å [START_REF] Du | Glycine receptor mechanism elucidated by electron cryo-microscopy[END_REF] However, at the resolution ranging from 3.8-3.9, it is risky to assign the conformation of side chains.

Summary and Aims of my thesis.

In summary, significant progress has been achieved in the field of the pLGIC family both at the structural and functional level during the past decade. It seems that in the resting/closed form, the constriction gate is located at the middle of the channel: I9' in the resting form of GLIC, I9' in the locally closed form of GLIC, L9' in the closed form of ELIC, L9' in the resting state of human α3-GlyR, L9' in the resting state of zebrafish α1-GlyR, L9' in the resting state of GluCl. In addition, in ELIC, the upper part of M2 helices near the extracellular domain harbors another constriction ring (F16').

Those resting states are either captured in the absence of agonist (GLIC-closed, ELIC and GluCl-closed) or in the presence of antagonist (human α3-GlyR-closed and zebrafish α1-GlyR-closed). The desensitized state of pLGICs is also a nonconductive state and it seems that the constriction gate of this state is located at the end of the channel pore: the desensitized state of human α4β2-nAChR (E-1'), the desensitized state of human β3-GABAA receptor (A-2'), the desensitized state of human α3-GlyR (P-2'). Those desensitized states are captured in the presence of agonists (human β3-GABAA-D and human α4β2-nAChR-D) or in the presence of agonists together with potentiators (human α3-GlyR-D and zebrafish α1-GlyR-D). Finally, in the open form of zebrafish α1-GlyR, the minimum channel diameter of this conformation is around 9 Å that is large enough to allow the chloride ion pass through with its hydration shell.

However, this raises questions about the open form of GLIC and GluCl. Their crystal structures are solved in the presence of agonists or together with potentiors. Both of the channel pores only allow the partially hydrated ions to pass through. Their Cα backbone conformations are most similar to that observed in the desensitized state of α4β2-nAChR and desensitized state of β3-GABAA (Fig 16).

It has been suggested that ELIC is a representative of a novel branch of the pLGIC superfamily with its unique properties [START_REF] Gonzalez-Gutierrez | The atypical cation-conduction and gating properties of ELIC underscore the marked functional versatility of the pentameric ligand-gated ion-channel fold[END_REF] . However, only a closed channel conformation of ELIC is currently available. Considering the sequence diversity of pLGICs in prokaryotes, more information of bacterial pLGICs should be explored both at functional and structural levels.

In chapter II, I have cloned, purified, crystallized and solved the crystal structure of a new pLGIC (referred to as sTeLIC) from a gamma-proteobacterium.

Functional experiments show that sTeLIC is activated by alkaline pH, is selective for monovalent cations and inactivated by divalent cations. The crystal structure, which was resolved at pH 8.0, displays a widely open pore that is the first of this kind to be characterized in this pLGICs family. Furthermore, we found a strong positive modulator that binds in the "vestibule site" in the extracellular domain, and we solved the crystal structure of this complex, which should prove useful for designing pharmacological allosteric modulators of human neurotransmitter receptors.

Strikingly, functional experiments show that sTeLIC shares many features with ELIC.

Together with ELIC, sTeLIC defines a new class of pLGICs and its widely open pore provides a model for the active form of this class.

In chapter III and chapter IV, we attempt to map proton sensor residue(s) in GLIC and explain how proton binding stabilizes the open state of GLIC. We performed mutational mapping of all titratable residues in GLIC to determine proton-sensors involved in the gating process. We found that one amino acid E35 is a bona fide proton sensor, whose charged form stabilizes the resting conformation, whereas protonation favors the active state. Then, taking advantage of open/active and closed/resting form of GLIC, we used the Poisson-Boltzmann/Debye-Hückle (FD/DH) method to predict the pH sensing residues. The results showed that E35, which was identified as a key proton-sensor residue, possesses a high deviation of predicted pKas in both forms.

Thirdly, we show that contribution of proton binding to channel gating depends on several key sites located the on Loop F motif, the pre-M1 region and the Pro-loop motif. And finally, we propose two distinct networks in the domain interface that stabilize the open form of GLIC.

Unlike animal pLGICs, the bacterial receptors display greater diversity in their domain architectures, while preserving the core modules consisting of the extracellular ligand binding domain and the transmembrane channel forming domain, as described by Tasneem et al 35

Summary

Pentameric ligand-gated ion channels (pLGICs) constitute a widespread class of ion channels, present in archaea, bacteria, and eukaryotes. Upon binding of their agonist(s) 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 non-active (closed) form, such as Erwinia chrysantemi pLGIC (ELIC), is still lacking. Here we provide the X-ray crystallographic structure at 2.3 Å in an active conformation of a new gamma-proteobacterial pLGIC from an endo-symbiont of Tevnia jerichonana (sTeLIC), whose sequence is 28% identical to ELIC. Electrophysiology shows that it is a cationic channel activated at alkaline pH that is inhibited by Ca++ ions but not by large quaternary amines such as tetramethylammonium, just like ELIC. Together with ELIC, sTeLIC defines a new class of pLGICs and its widely open pore provides a model for the active form of this class. sTeLIC is allosterically activated by derivatives of aromatic amino acids, such as 4-bromo-cinnamate, whose co-crystal structure points to a classical two-state allosteric mechanism and whose binding site is equivalent to a vestibular site already described for benzodiazepines in ELIC.

Significance

Pentameric ligand-gated ion channels (pLGICs) mediate fast signal transduction in animal nerve cells through neurotransmitters. Mutation of some of these receptors in the brain causes severe nervous-system diseases. The high sequence diversity of prokaryotic receptors makes them unique model systems to understand what was conserved throughout evolution to achieve the same gating function. We present here the 2.3 Å X-ray structure of a new pLGIC (sTeLIC) from a gamma-proteobacteria that is shown to be activated at alkaline pH. The structure solved at pH 8.0 displays an unusually open pore. The structure of the complex with a positive allosteric modulator is identical to the unbound receptor but with much lower Bfactors, indicating a typical two-state allosteric stabilization mechanism.

Introduction

Pentameric ligand-gated ion channels (pLGICs), also known as Cys-loop receptors 1,2 or Pro-loop receptors 3 , play an essential role in nerve impulse transmission in animals by mediating the transduction of a chemical signal (release of a neurotransmitter in the synaptic cleft) into an electrical signal (modification of the membrane potential) in the millisecond range. The receptors are divided into two different functional families, cationic channels namely nicotinic acetylcholine (nACh-R) and serotonergic type 3 receptors (5HT3A-R) and anionic channels including glycinergic (Gly-R) and GABA-ergic type A receptors (GABAA-R), yet they retain a very similar folding topology and molecular architecture 1 . Following the binding of agonist(s) in the extracellular domain (ECD), the receptors undergo an allosteric conformational change, whereby the transmembrane domain (TMD) switches from a closed to an open state, letting ions permeate through the pore down their electrochemical gradient.

Dysfunction of Cys-loop receptors in the brain causes severe neurological diseases and these receptors are the subject of many functional, structural, and biophysical studies 4 . Interestingly these receptors are the targets of numerous natural and synthetic compounds of pharmacological interest, including nicotine 5 , alcohols 6,7 , benzodiazepines 8 , barbiturates 9 , and general anesthetics 10 -12 . Therefore, understanding their molecular properties and the mechanisms of the gating-process during channel opening, at an atomic level, remains a central issue in structural neurobiology and pharmacology with potentially far-reaching consequences for human health.

There are also numerous members of the pLGIC family throughout the bacterial world and their physiological role is not known, although they are likely involved in bacterial cell-to-cell communication, such as chemotaxis and/or quorum sensing 13,14 . The first two full-length crystal structures of members of the pLGIC family were obtained with bacterial pLGICs, the Erwinia chrysanthemi and Gloeobacter violaceus pentameric ligand-gated ion channels (ELIC and GLIC) 15 -17 , followed by GluClα, an anionic channel from Caenorhabditis. elegans 18 . Since then, all major eukaryotic classes of pLGICs had at least one member whose structure was solved either by crystallography or cryo-electron microscopy, both anionic ones (a human homopentameric β3-GABAA-R 19 , a human homopentameric α3-Gly-R 20 , a zebrafish homopentameric α1-Gly-R 21 ) and catonic ones (a mouse homopentameric 5HT3A-R 22 , a human heteropentameric α4β2-nACh-R 5 ). Paradoxically, there has not been any new high-resolution structure of bacterial pLGICs from other species since 2009, even though they certainly display a wide range of pharmacologies and allosteric conformations. Thus, the wealth of information contained in the diversity and adaptability of bacterial pLGICs has not been fully explored. Also, to understand their evolutionary history, it is important to understand all the differences between eukaryotic and bacterial pLGICs at the molecular level 23 .

GLIC was the first pLGIC to be solved both in an apparently open form, at resolution of 2.4 Å 24 , and in the relaxed/closed form. The latter form reached only a resolution of 4.35 Å but contained four pentamers in the asymmetric unit, which allowed the unambiguous tracing of all the chains after non-crystallographic symmetry averaging 25 . In molecular dynamics simulations, the structure of GLIC obtained at low pH relaxes to a slightly more open form where some permeation events could be identified 24 . Another crystal form, called the Locally-Closed form (LC form) was identified through mutations introducing disulfide-bridges at key positions, or even single-point mutations in either the M2 α-helix TMD region 26 or the M2-M3 loop 27 . It is possible that this LC form represents a pre-activation state, on the reaction pathway between the closed to open forms, as suggested both by experimental 28 and computational studies 29 . It was also found in one crystal form as co-existing with the open form at pH 4 in the same asymmetric unit 25 . Recently, another crystal form possibly representing one of the desensitized forms was found 30 . In general, these structural studies match very well with other biochemical studies, such as EPR spectroscopy studies in solution 31 , for instance. Finally, GLIC proved an excellent model system to study allosteric modulation at the structural level by general anesthetics 6,7,11,32 , alcohols 7 , xenon 33 and barbiturates 9 . Structural studies correlate very well with chemical affinity labeling studies in solution 34 . It is not presently known how many crucial proton-binding sites there are and where they are located in GLIC structure, nor if all the results obtained on this receptor can be extrapolated to other pLGICs sensitive to agonists with canonical neurotransmitters binding sites.

The other bacterial receptor (ELIC) for which structural information is available, has also many entries (17) in the PDB, involving several mutants and some very informative complexes with divalent cations 35 , benzodiazepines 8 and general anesthetics 36 . Electronic-spin resonance studies have also been conducted on this channel 37 . However, its crystal structure has only been obtained in the closed form thus far. Indeed, all ELIC crystal structures are found in a closed-pore conformation, even in the presence of a bound agonist and for mutants known to favor the active form 38 , therefore the functional state represented by the crystal structure is still not completely understood. It was even suggested that this closed form is actually an uncoupled form, reminiscent of the uncoupled form of the acetylcholine receptor 39,40 . Also, due to its peculiar behavior in the presence of quaternary amines that are usually channel blockers, it was suggested that ELIC could be a representative member of a new sub-family of pLGICs, whose gating properties should be extrapolated only with great care to other pLGICs 41 . Analysis of the aromatic residues at the interface of M1-M3 and M4 α-helices also suggested that ELIC's TMD is dissimilar to GLIC's 40 .

For these reasons we decided to get more structural information from bacterial pLGICs closely related to ELIC, especially in the gamma proteobacteria genus, in the hope of crystallizing it in an open form, or both its closed and open forms. In the following we describe our results for the endosymbiont of Tevnia jerichonana pLGIC (referred to as sTeLIC in the rest of the paper), a receptor from a gammaproteobacteria found in giant tube-worms that live close to hydrothermal vents 42 , with 28% sequence identity with ELIC.

Results

sTeLIC is activated by alkaline pH and its crystal structure at pH 8.0

Initial electrophysiological characterization of sTeLIC showed it is potentiated by MES buffer and inhibited by calcium ions. Therefore, HEPES and Tris buffers were used for all subsequent evaluations, after no effect was found for either buffer.

Calcium was also removed from the recording solution, keeping only Mg ++ as a divalent cation, whereas 1 mM CaCl2 was maintained in the wash solution only for experiments where long washes were needed. Under these conditions, sTeLIC was found to be open at basic/neutral pHs and closed at acidic pHs (Figure 1A). Using a perfusion buffer at pH 5 which keeps sTeLIC closed, pH jumps to more basic conditions elicit robust currents characterized by a fast activation. Small currents start to be seen at pH 7.5 and maximal currents occur at pH 9.5. Fitting all pH activation data according to the Hill model yielded a pH50 of 8.6 ± 0.4 (Figure 1A, inset).

Initial crystallization screens yielded crystals of sTeLIC diffracting only up to 6 Å. Extensive optimization, including seeding and dehydration protocols, gave crystals diffracting to 3.5 Å. After further optimization with various additives, we found that adding 6.5 mM nonyl-β-D-glucose (NDG) gave the best crystals, at pH 8.0, diffracting up to 2.3 Å resolution. The structure of sTeLIC was solved by molecular replacement using the open structure of GLIC (PDB: 4HFI) 24 as the search model. The final model of sTeLIC, which lacks only 6 residues at the N-terminus and 4 residues at the Cterminus, has excellent refinement statistics (Suppl. Table 1) and electron density maps of high quality (Suppl. Fig. S1). The crystal packing arrangement shows both parallel and anti-parallel, head-to-tail, molecule arrangements (Suppl. Fig. S2A-B), consisting of essentially two types, ECD-ECD (Suppl. Fig. S2C) or TMD-TMD, of pentamer interactions (Suppl. Fig. S2D), a phenomenon already seen in the GLIC 16 (PDB ID: 3EAM) and β3-GABAA-R 19 (PDB ID: 4COF) structures.

The overall architecture of the sTeLIC is the same as for other receptors from the pLGIC family. The five monomers are arranged along a five-fold symmetry axis perpendicular to the cell membrane with small deviations from C5 exact symmetry evaluated for pairs (1,2), (1,3), (1,4) and (1,5), respectively, as 0.17 Å, 0.09 Å, 0.14 Å and 0.14 Å. The dimensions of the receptor are given in Figure 1B. The TMD of one monomer is composed of a bundle of four trans-membrane α-helices (M1-M4) with M2 facing the ion channel pore (Figure 1C), flanked by M1 and M3 α-helices of the same subunit and the M1 α-helix from a neighboring (complementary) subunit. The ECD of the monomer contains ten β-strands (β1-β10), which form a compact curled βsandwich, where β4, β7, β9, and β10 compose the inner β-sandwich [in the principal (+) interface], and the rest form the outer β-sandwich [in the complementary (-) interface]. An amphiphilic α-helix α1 is inserted between the β3 and β4 strands, at the top ECD vestibule, with its hydrophobic part interacting with the top section of the βsandwich and its hydrophilic part facing the vestibule (Figure 1C). Other important regions, such as the β1-β2 loop, the M2-M3 loop, and the Pro-loop 3 (β6-β7 loop) at the TMD-ECD interface are also indicated in Figure 1C, as well as loops that are known to be important to bind the orthosteric-site agonist, such as Loop C (β9-β10 loop) and Loop B (β7-β8 loop) 43 . In addition, we define here Loop Ω (β4-β5 loop), that forms an almost continuous ring in the lumen due to C5 symmetry (Figure 1C and1D).

The structure shows a widely open ion channel pore

The columnar-shaped ion channel pore is constituted by the five M2 α-helices, whose pore-facing residues shape the ion translocation pathway (Figure 2A-B). The diameter of the pore ranges from 11 Å to 15 Å (Figure 2C) and the residues bordering the ion channel pore from the beginning to the end of M2 α-helix are D-4', D2', G6', L9', I12', A13', F16', T17', S20'. The two negatively charged residues, D-4' and D2', located in the cytoplasmic end of the pore, strongly suggest a cationic channel. The distribution of the pore lining residues is in accordance with those of other members of the Cys-loop receptor family (Figure 2E), with the exception of G6' (instead of S or T), D2' (generally polar but not charged) and K-1', which is found to interact through a salt bridge with D2' of the neighboring subunit (see below). In Figure 2C we show a selected set of pore-radius profiles for known pLGICs structures. Compared to other members of the pLGIC family, the ion channel pore of sTeLIC has the widest diameter, with a minimum value of 11 Å at the level of D2'. The closest comparable one is the open state structure of the α1-Gly-R, whose structure was determined by cryo-EM at 3.9 Å resolution 21 . Superimposition of sTeLIC with the open state of α1-Gly-R reveals that the top of the M3 α-helix and the M2-M3 loop move further outwards and the pre-M1 region moves closer to the M2 and M3 α-helices to form a compact helical bundle.

The widely open configuration of the pore is stabilized by two salt bridges, as illustrated in Figure 2D, one between D293 in M4 and R225 in M2, and one between K224 (-1') and D227 (2'). We assign this conformation to an open/active state as it would be capable of permeating sodium and potassium ions even with their hydration shells 44 . Finding an open conformation in the crystal structure resolved at pH 8 is compatible with electrophysiology experiments, which show that pH 8 corresponds approximately to an EC20 in functional tests (Figure 1A).

In the electron density map of the pore itself, we found five strong residual densities in the Fo-Fc map on the surface of the ion channel pore during the model building (Suppl. Fig. S3A-B), which were modeled by five NDG detergent molecules. NDG (Suppl. Fig. S3C) was used as an additive to get the high-resolution diffracting crystals; its curled aliphatic tail appears to be inserted into the crevice formed by two adjacent subunits while its hydrophilic sugar head faces the channel pore (Suppl. Fig. S3B). To exclude the influence of the NDG for the widely open channel pore conformation, we also collected data from crystals grown without NDG and we found that the conformation of the ion channel pore is unchanged (Suppl. Fig. S3E).

Structural characterization of the ECD-TMD interface

In Fig. 1D we have highlighted all strictly conserved residues resulting from a multiple-alignment of 11 bacterial pLGICs, including sTeLIC, ELIC and GLIC. It is apparent that many of these conserved positions are located at the level of the ECD-TMD interface.

As with other members of the pLGIC family, the Pro-loop, which lacks the disulfide bond conserved in the eukaryotic Cys-loop of pLGICs, interacts with the M2-M3 loop through the stacking interactions of F122 and P123 (Y119 and P120 in GLIC, and F119 and P120 in ELIC) and L247 (L246 in GLIC, and L255 in ELIC). The crucial salt bridge between R193 and D125 is the equivalent of R192-D122 in GLIC (R198-D122 in ELIC), whereas the third partner (D32) of this salt bridge in GLIC is replaced by Q25 (T28 in ELIC) in the β1-β2 loop (Suppl. Fig. S4B). The stacking interaction of W162 on the hydrophobic part of R193 side-chain is also conserved (W160 and R192 in GLIC, see Suppl. Fig. S4D), as well as the primordial role of Y198 side-chain (Y197 in GLIC). Interestingly, E161 of sTeLIC is also making a salt bridge with R193, opposite from the side of D125. E161 is well conserved in bacterial pLGICs but is absent in GLIC, where its role is fulfilled by D32 from β1-β2 loop. Strikingly, there is a strict covariation of the presence/absence of a negatively charged residue at positions 25 and 161 in sTeLIC (32 and 159 of GLIC). In this respect the sequence of Loop F in GLIC is an outlier within the cationic pLGIC family (Suppl. Fig. S4E), where the conserved short sequence motif (D/E)EW in bacteria (GEW in eukaryotes) is changed to TGW. This sequence motif (XEW) is otherwise well conserved in cationic ion channels, including sTeLIC and nAChR, but is absent in the anionic ones Gly-R, GABAA-R, and GluClα, where it is replaced by the conserved P(Q/S)F motif (Suppl. Fig. S4E).

At the level of the ECD-TMD interface, the fundamental role of the strictly conserved proline, P205, at the end of the second turn of M1 α-helix is confirmed in that it induces a characteristic kink that is further stabilized by an interaction of the carbonyl atom of M200 with the side-chain of N240 from (+) subunit (Suppl. Fig. S5A).

In addition, we noted the presence of two salt bridges that are however not conserved in GLIC, namely R249-D246, that connects two adjacent M2-M3 loops, and R201-D257, that links the pre-M1 region and the top of M3 α-helix from (+) subunit (Suppl. Fig. S5A-B).

Ion binding sites and ion flow in the lumen and in the pore

In order to experimentally investigate the interaction of sTeLIC with monovalent cations or anions, we performed crystal soaking experiments with Cs + or Br -that have both a substantial anomalous signal at wavelengths attainable in synchrotron beamlines. Crystals soaked at a final concentration of 150 mM CsCl gave anomalous difference Fourier maps that revealed two Cs + binding sites per monomer in the vestibule of the ECD (Figure 3B). There is one Cs + binding site around E106, N91, and Q90, as well as another one close to Loop F (with E28, E159, and E160).

Additionally, one strong Cs + anomalous peak is located at the bottom of the central ion channel pore on the C5 axis in between D-4' and D2'. These residues were mutated into an alanine and the D2'A (D227A) mutant was found to be non-functional while D-4' (D221A) has a marked loss of function phenotype (Figure 3C), namely a significantly higher pH50. On the other hand, soaking the crystals with 150 mM KBr gave no signal in the anomalous map, indicating no stable binding site for bromide anions.

Based on the structure, the electrostatic potential was calculated and mapped onto the protein surface (see Material and Methods, Figure 3B). Clearly there is an intermediate chamber in the lumen of the ECD with a strong negative potential, mainly from residues E69, E70, E106, D88, and E27, explaining the binding sites of the bound cations in the vestibule (Figure 3B). Altogether, these structural data strongly suggest that sTeLIC is a cationic ion channel. This prediction is consistent with the conclusion of the electrophysiological analysis presented below (see Figure 6). Surprisingly, the surface representation reveals that there are two constriction rings in the ECD, one at the level of K66 in the α1 amphipathic helix, and another at the level of D88-R86 in Loop Ω (β4-β5 loop) (Figure 3A). The carboxylate group of D88 interacts with atom Nδ of R86, which indicates a tautomeric form of the guanidinium group where the positive charge is carried by this atom. The distance of terminal Nε amino groups of symmetry-related mates is only 2.8-3.2 Å, showing potential hydrogen bonds between them. If these side-chains were frozen in this position in solution, they would prevent ion flow through the vestibule.

The functional significance of this feature was tested by site-directed mutagenesis and electrophysiological experiments in oocytes, which showed that suppression of the charged side-chains by mutation to alanine of R86 or K66, or both, did not significantly affect the pH50 (Figure 3C). R86 is not conserved in either GLIC or ELIC, whereas D88 (as D86) is present in both (Suppl. Fig. S12). Strikingly, D86, along with a neighboring D88, in GLIC bind soft divalent ions such as Ni ++ (PDB ID: 4NPP) 25 , while hard divalent ions such as Ba ++ bind to D86 in ELIC (PDB ID: 2YN6, Suppl. Fig. S6) 35 .

Interestingly, similar constriction rings in the lumen at the level of Loop Ω were also observed in other members of the pLGIC family. Indeed, this has been observed in the crystal structures of two eukaryotic receptors: i) in the 5HT3A-R structure (PDB ID: 4PIR) at the level of the five side-chains of K108 22 , that collectively bind a central sulfate ion and ii) in the GluClα structure (PDB ID: 4TNV) at the level of the five side chains of Y99 that bind to a central citrate ion Suppl. Fig. S6) 45 . Both constriction points would block ion flow if present in solution.

Characterisation of allosteric potentiator(s) of sTeLIC

To identify small molecules acting on sTeLIC, typical pLGIC agonists (nicotine, acetylcholine, 5HT3, GABA, glycine, glutamate, and histamine) and other compounds were tested for the potentiation and/or inhibition of the pH 7.5-elicited currents, with or without potentiator (Suppl. Table 2). Most compounds were inactive, but some amino acids were found to have a small potentiating effect (L-Met, L-His, L-Leu), with the aromatic amino acids L-Phe and L-Trp having the most promising potentiating effect (L-Tyr could not be tested beyond 1 mM due to its low solubility). The effect of aromatic amino acids and several of their derivatives was systematically studied. It was found that they robustly potentiate the pH 7.5-elicited currents, and their doseresponse curve at pH 7.5 was established (Suppl. Table 3). The most potent compound of the series is 4-BrC with an EC50 of 21 ± 8 µM (Figure 4A-C). All compounds, including the non-titratable Nα-acetyl-L-tryptophan ethyl-ester, do not elicit currents at pH 5, suggesting that they cannot activate the receptor by themselves and that they rather act as allosteric potentiators of the pH-elicited currents (Suppl. Fig. S7D). From the study presented in Suppl. Table 3, it appears that compounds with an aromatic ring are active at lower concentrations, which may indicate a better affinity for the site involved in potentiation. Substitution by bromine at the "para" position enhances the apparent affinity of the compound. The most potent potentiator, 4-BrC was therefore used in conjunction with pH activation for many experiments evaluating the channel characteristics of sTeLIC.

Derivatives of the hydrophobic amino acid initially identified as having a weak effect (L-Leu and L-Met), such as N-formyl-L-methionine, methyl-ester-L-methionine, and methyl-ester-L-leucine, were found to inhibit sTeLIC currents at high millimolar concentrations (Suppl. Fig. S7A-C). However, methyl-ester-L-leucine has a potentiating effect at low millimolar concentrations.

In order to gain structural information for molecular recognition between sTeLIC and 4-BrC, we solved the co-crystal structural complex of sTeLIC with 4-BrC at 3.0 Å resolution (Figure 5A). Both the Fourier Fo-Fc map (contoured at 3 σ) and the anomalous map (contoured at 5 σ) using data collected at the absorption edge of bromine allow unambiguous construction of 4-BrC within the density (Figure 5C).

The binding site of 4-BrC is deeply buried in the central vestibular-facing intra-subunit cavity of the β-sandwich, where the hydrophobic head interacts with the hydrophobic core of the β-sandwich via van der Waals interactions (Figure 5B). Its carboxylate tail forms a salt bridge with R92 from strand β5 and is exposed to the lumen. molecules can be assigned into the pentamer of sTeLIC, with one 4-BrC per monomer (Figure 5A), whereas just one molecule of FZM could be found in one of the intrasubunit sites for the whole pentamer of ELIC 46 . The 4-BrC binding site is also very close to the Br-Acetate binding site in GLIC 47 (Suppl. Fig. S8B).

It should be mentioned that the difference maps of sTeLIC crystals that were devoid of 4-BrC also revealed a large blob of unexplained electron density in both 2mFo-DFc and mFo-DFc maps at the level of the intra-subunit 4-BrC binding site but closer to ECD lumen (Suppl. Fig. S8D andS8H). We have tentatively assigned this density to a PEG 200 molecule (Suppl. Fig. S8E), the precipitant that we used during crystallization. This density is localized similarly to the homologous Br-Acetate site in GLIC (Suppl. Fig. S8F) and benzodiazepine FZM in ELIC (Suppl. Fig. S8G). This extra density may explain why sTeLIC is maintained in the open state at this pH but it must be emphasized that its assignment to a PEG 200 molecule is speculative at this stage.

Attempts to identify the bound molecule by mass spectrometry were unsuccessful.

Strikingly, this density goes away when 4-BrC is bound.

Electrophysiology confirms that sTeLIC is cationic

Ion selectivity of sTeLIC was examined using a change in pH from 5 to 8 (in the absence of potentiators, Figure 6B-C), as well as using potentiators at a Full substitution of the 6-carbon anion gluconate for Cl -produced a right shift of sTeLIC current reversal potential (from +10 ± 1 mV to +21 ± 4 mV), and full substitution of choline for Na + produced a left shift from +10 ± 1 to -41 ± 3 mV (Figure 6A). These results indicate a preferentially cationic permeability. Concurrently, simultaneous reduction of Na + and Cl -concentrations (kept equal and referred to as the NaCl concentration) produced a left shift of the reversal potential (Figure 6B), and the shift varied with the ratio of extracellular NaCl concentrations almost exactly as predicted by the Goldman, Hodgkin, and Katz voltage equation assuming PCl/PNa = 0 (Figure 6C). This analysis, which makes no hypothesis on intracellular ion concentrations, except that they are supposed to be constant, shows that PCl/PNa is near zero for sTeLIC and confirms it is a cationic channel.

Inhibition by Ca ++ , Ba ++ , and Zn ++ ions but not by quaternary amines

The inhibitory effect of several divalent cations was evaluated with pH 8 activation, and the most potent one was found to be Zn ++ with an IC50 of 2 ± 2 µM (Figure 7A). Calcium and barium ions have similar potencies at 180 ± 50 µM and 170 ± 90 µM respectively. A current trace showing the effect of increasing concentrations of Ca ++ ions is shown in Figure 7B and the corresponding dose-response curves for all ions in Figure 7C.

Typical pLGIC channel blockers were also tested on sTeLIC, and neither tetramethyl ammonium (TMA), tetra-ethyl ammonium (TEA), nor tetra-propyl ammonium blocked the channel (Suppl. Fig. S9A). It is interesting to note that similar results were obtained for ELIC with large channel blockers 41 . We have modeled the TMA molecule in the channel pore of sTeLIC based on its known complex with GLIC 48 and it is apparent that the pore is too wide to provide any side-chain interactions and stabilization for a TMA molecule at this level of the pore (Suppl. Fig. S9B).

Furthermore, it was possible to directly locate the binding site of Ba ++ in the sTeLIC structure by crystallography due to the anomalous signal of Ba ++ . Only two binding sites were found in the anomalous map using data collected at a longer wavelength and both were located in the pore, at the level of D2' and G6', respectively (Figure 7D). This data suggests that the molecular mechanism of Ba ++ and Ca ++ inhibition can be described by directly blocking the open channel. This is in contrast with three different binding sites found for Ba ++ in (the closed form) of ELIC: one on top of the ion channel entrance, one around Loop F and one close to Loop Ω in the lumen of the ECD 35 . We were unable to collect diffraction data in the presence of Zn ++ as the crystals did not stand any soaking treatment with this ion.

Discussion

sTeLIC shares many features with ELIC

Functionnally speaking, sTeLIC shares some striking similarities with ELIC: it is a cationic channel from the same gamma-proteobacteria family and it is also blocked by Ca ++ and Zn ++ ions 35 , but not by quaternary ammonium cations such as TEA or TMA 41 . It is also positively modulated by the same ECD intrasubunit vestibule site 46 .

In this way, it could be said that ELIC and sTeLIC are representative members of a new subfamily of pLGICs, with specific characteristics of the pore, a possibility already envisaged for ELIC by Grosman and colleagues 41 . However, sTeLIC is activated at alkaline pH, whereas ELIC is activated by amine containing compounds such as GABA or cysteamine 49 . Here we show that sTeLIC is indeed a second member of this sub-family and its high-resolution structure in the open form provides a model for the first time of their open channel conformation. It is striking to see that, if one models TMA in the pore of sTeLIC at the level of its position observed in GLIC 48 , one sees that the pore is too wide to provide any direct interaction stabilizing this position for the TMA molecule, thereby explaining the absence of an inhibitory effect for this kind of quaternary amines (Suppl. Fig. S9B).

sTeLIC shows canonical Loop C, Loop B and Loop F

The analysis of the known important loops for activation in pLGICs shows that Loop B conformation is "canonical" in sTeLIC, as compared to GLIC and other eukaryotic receptors, but confirms that Loop B is peculiar (an outlier) in GLIC (Suppl. The analysis of Loop F, emphasizing the role of E161 and its interaction with R192-D122 salt bridge, confirms the importance of this Loop in ELIC family, as seen recently with the identification of the binding site of chlorpromazine 51 . However, this region appears as an outlier in GLIC, both in sequence and in structure, compared to other cationic pLGICs. Sequence information also indicates that this region is different in anionic channels.

It is possible that Loop F also plays a role in the Ca ++ inhibition of sTeLIC by stabilizing its closed form. Indeed, one Ca ++ binding site in the closed form can be assumed to be similar to the one seen in ELIC 35 , possibly recruiting the side-chains of residues E159-E160 in Loop F. In this way, it would also block the alternative lateral cation pathway (see below).

Comparison with other open pores

The widely open pore of sTeLIC matches very well the one of the α1-Gly-R (Suppl. where the role of the "bubble" would be played by the ice ring seen at the level of the 6' position in the high resolution structure of GLIC (PDB: 4HFI).

In any case, on the structural point of view, sTeLIC's open form definitely appears as an extreme open form, which may correspond to functional properties specific of this family of channels, that may include the transport of large cations, as suggested for ELIC 41 .

Quaternary structural comparision

Overall, structures of the ECDs of sTeLIC and GLIC superimpose well after aligning the TMDs, with very little twist (tangential movement) and little bloom (radial movement), as seen in Suppl. Fig. S10B andS10E. The amphipathic α1-helix of sTeLIC and ELIC is absent in GLIC. The comparison of sTeLIC and α1-Gly-R ECDs give results similar to the comparison of sTeLIC and GLIC (Suppl. Fig. S10C andS10F). However, superimposing sTeLIC and ELIC in the same way reveals both significant twist and bloom movement in the ECD (Suppl. Fig. S10A andS10D), while the α1-helix appears to be shifted by one helical turn along its axis. In conclusion, the sTeLIC conformation in the ECD is more similar to GLIC's and α1-Gly-R's than to ELIC's, and this is at odds with one of the proposals for ELIC's crystal structure, namely that it might represent a "decoupled state" with an open ECD and a "closed" TMD.

Specificities of the ion permeation pathway(s)

The cation pathway in the pore is well illustrated by crystallographic studies in the presence of Cs + . However, this study also suggests there might be an additional entry pathway for monovalent cations through a lateral door located at the interface between the ECD and the TMD, as observed in GABAA-R 19 as well as 5HT3A-R, based on molecular dynamics studies 54 (Figure 3B). Changing the rotamers of E160 (Loop F) and E28 (Loop2) is enough to reveal a lateral tunnel, as shown in Suppl. Fig. S11.

In addition, the presence of two constriction rings in the lumen of the ECD (especially at the level of Loop Ω, the β4-β5 loop) is intriguing, but these constriction rings in the ECD are not uncommon among other members of the family (GluClα, 5HT3A-R). It is possible that they may play a role as an additional valve, forming a temporarily isolated chamber to regulate the flow of ions inside the lumen of the ECD.

To investigate a possible dynamic role of this region, we calculated lowfrequency all-atoms Normal Modes using the Elastic Network Model (http://lorentz.dynstr.pasteur.fr/) and looked for specific modes that could potentially open and close this valve. We found several modes that indeed display a coordinated and anti-correlated movement of this restriction ring (Loop Ω) and the pore itself. Interestingly, this dynamical behavior of the receptor mimics exactly the functioning of a (linear) "peristaltic pump". Whether or not this actually takes place in the cell will require further studies involving single-channel recording experiments of the receptor with the R86A mutation and measuring its conductance.

Allosteric activation of sTeLIC

sTeLIC data in the presence of 4-BrC indicates a positive allosteric regulation by the vestibular intra-subunit site in the ECD, as described previously for a benzodiazepine in ELIC. Attempts to explain the allosteric mechanism at the molecular level are limited by our lack of information on whether the same PAM molecule binds to the closed form of sTeLIC. Modeling of the closed form of sTeLIC from the ELIC structure is risky because it relies on the belief that the ELIC closed form is indeed the relaxed form of the channel, which is at the present time not known for certain 40 .

However, if one directly compares the structures with and without 4-BrC, it is puzzling to see that there is almost no difference (rmsd=0.22 Å). Comparison of the B-factors of the two molecules, crystallized in exactly the same conditions, shows a striking difference, with the bound-form more ordered than the unbound-form (Figure 5D). This is typical of the classical two-state allosteric model, where the binding of the PAM molecule can effectively redistribute the electrostatic energy without disturbing significantly the structure. This could result in a better anchoring of the β-sheets to the top of the ECD, a more stable ECD-TMD interface, and an overall stabilization of the open form.

Finally, we note that the results on methyl-ester-L-Leucine reveal opposite modulatory effects for this compound at different concentrations (Suppl. Fig. S7C), an inhibitory one above 10 mM and a potentiating one at 3 mM. This is similar to benzodiazepines that have two allosteric sites in ELIC 46 . The sites involved are i) the vestibular intra-subunit site and ii) the site located just below the orthosteric site. This dual modulation seems to be also valid for general anesthetics, both for ELIC and for GLIC, this time for the TMD binding sites 36,55 Further studies will be needed to identify and characterize all allosteric binding sites of sTeLIC both structurally and functionally.

In conclusion sTeLIC is a new bacterial ion channel that is relatively easy to work with and whose full functional and structural characterization reveals striking and unanticipated similarities with ELIC. It helps define a new class of pLGICs whose resting form remains to be characterized but whose active form is definitely widely open. Strikingly, the functional effect of a PAM molecule binding in the vestibule is best explained at the structural level by the classical two-state allosteric model. Here, the rotamers of just two residues around Loop F have been changed. A Lateral view. B Top view.

The two subunits in front were colored in green and red. Loop F and b1-b2 are highlighted. ClustalW2 and ESPript were used to perform and display the alignment with the following proteins sTeLIC, 5HT3A_mouse, AChRα4_Human, AChRβ2_Human, GluClα_C. elegans, GlyRα1-Zebrafish, GABA A β3_Human. The secondary structural elements of sTeLIC are indicated above the sequences. Strictly identitical residues are shown in red boxes. Well-conserved residues (with a similarity score greater than 70%) are boxed in blue and colored in red.

Supplementary

Materials and Methods

Cloning, protein expression and purification

The full-length gene of sTeLIC was codon optimized for expression in E. coli and chemically synthesized. The N-terminus of sTeLIC was fused to an MBP tag with a thrombin protease cleavage site. The recombinant gene was subcloned into the pET20b vector. The primers used to amplify the full-length sTeLIC are:

Forward primer: 5-CGACGTGCGGCCGCGATGGCAAGCCTGGCAGCAG-3

Reverse primer: 5-CTCGAGCTAATAGCTACGCCAAAAAAACAGCC-3

The plasmid containing the recombinant gene was transformed into E. coli C43 competent cells. The medium used to express the recombinant protein was 2YT. 1 mM ampicillin was added during cell culture and protein expression. The temperature was decreased to 20°C when the OD reached 0.6-0.8, with the addition of 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for overnight induction. The overnight culture was harvested and the pellet was suspended using buffer A (20mM Tris pH 8.0, 300 mM NaCl) supplemented with EDTA-free protease inhibitor (ThermoFisher) and Benzonase nuclease. Cells were disrupted by sonication and subsequently spun at 40,000 rpm for 1 h. The pellet was collected and dissolved using buffer A containing 4% n-dodecyl-β-D-maltoside (DDM) at 4°C for 3 h. The dissolved membranes were then spun at 28,000 rpm for 1 h. The supernatant was applied to an amylose resin (New England BioLabs) which was equilibrated with buffer A. Incubation of the supernatant with amylose resin was limited to around half an hour in order to avoid introduction of a contaminant, maltoporin. The amylose resin was extensively washed with buffer A containing 0.1% DDM followed by buffer B (buffer A containing 0.02% DDM).

Recombinant protein was then eluted by buffer B containing 50 mM D-(+)-maltose monohydrate (Sigma), subsequently concentrated, and applied to a Superose TM6 Increase 10/300 GL (GE Healthcare) gel filtration column, which was pre-equilibrated with buffer B. The fractions of the peak corresponding to a molecular weight of five times MBP-sTeLIC were pooled and concentrated. The MBP tag was removed by incubating with thrombin protease overnight. MBP and thrombin were then removed from the solution by another round of gel filtration. The fractions of the peak corresponding to the sTeLIC pentamer were pooled and concentrated to 12 mg/ml. All the purification steps were handled at 4°C. Protein was frozen and stored at -80°C for further use.

Crystallization and data collection

Crystallization experiments were set up at 18°C. The purified protein was mixed with an equal volume of the reservoir buffer containing 100 mM Tris pH 8.0, 150 mM MgCl2, 3% DMSO, and 32% PEG 200 at 1μl: 1μl volume ratio and the drops were equilibrated against a 1 ml reservoir solution using the hanging drop method.

The pH of the drop, around pH 8.0, was checked using pH strips. Crystals appeared after two days and grew to full size after around two weeks. For reproducibility, and in order to get larger crystals, the purified protein was mixed with 65 mM NDG at 4:1 volume ratio and incubated on ice for 20 min prior to crystallization set-up. Microseeding procedure was additionally applied to increase the reproducibility of the crystals.

Co-crystallization of sTeLIC with 4-BrC was performed by mixing the protein with 4-BrC at a final concentration of 3 mM (solubilized in 100% DMSO) and incubating at 4°C overnight. Crystals of the sTeLIC with 4-BrC appeared within two weeks and grew to full size within a month using the same method and the same mother liquor as described above.

Soaking experiments of sTeLIC crystals with Br -, Cs + , and Ba ++ ions entailed incubation with mother liquor supplemented with 100 mM KBr, 150 mM CsCl, and 150 mM BaCl2, for 5-30 min respectively.

Crystals were flash frozen in liquid nitrogen. Diffraction data sets were collected at ESRF (beamline ID23-2 and ID29) or Soleil (Proxima1 and Proxima2) and processed using XDS 56 and CCP4 57 software. The crystals belong to C2 space group with one pentamer in the asymmetric unit.

Structure determination and model refinement

The structure was solved by molecular replacement using GLIC (PDB ID: 4HFI) The best data set of sTeLIC with 4-BrC was collected at 0.873 Å wavelength.

The crystal was isomorphous to the native one. Taking advantage of the anomalous signal coming from the bromine atom, we could build the 4-BrC molecule into the model without ambiguity.

Sequence alignment was done using ClustalW2 62 and represented using ESPript 63 . Structural analysis and comparison were done by Pymol 64 and Chimera 65 .

The surface electrostatic potential map was generated by APBS server 66 s and read by Chimera 67 . HOLE software 68 was used to analyze the channel pore dimensions.

Pictures were prepared by Pymol and Chimera.

Two-electrode Voltage Clamp Electrophysiology

Xenopus laevis oocytes were obtained from the Centre de Ressources NaOH was used to obtain desired pHs of other HEPES solutions unless otherwise specified.

Evaluation of compound effects (Suppl Table 2) without and with a potentiator was performed using the specified test concentration in either the standard-solution or CaCl2-free standard-solution, and the standard-solution containing 30 mM MES or CaCl2-free standard-solution containing 10 µM 4-BrC, respectively.

Evaluation of the potentiation of currents by compounds was performed using CaCl2-free standard-solution superfused for 30 sec before and after applications of the compound at the specified concentration also in CaCl2-free standard-solution for 15 sec-1 min depending on compound. The wash time, using the standard-solution, was 15 min in-between sweeps. An initial test pulse to ensure receptor expression was performed using the maximum concentration followed by a 15 min wash before the recording of sweeps. A total of 6 sweeps per curve, in either ascending or descending concentration, was conducted on an individual oocyte. MES was applied for 1-3 min, after 30 sec in CaCl2-free standard-solution, and was then washed directly using standard-solution for varying times.

Evaluation of currents at different pHs was tested in either 10 mM HEPES or 10 mM Tris CaCl2-free solutions (100mM NaCl, 3mM KCl, 1mM MgCl 2 ). The stock solution was raised to pH 10.5 using NaOH and subsequently lower pH's were obtained using HCl with possibly the addition of the pH 10.5 solution to obtain the desired pH, thereby maintaining an equivalent Na concentration in all solutions. All pH testing was performed in the absence of CaCl2 throughout the entire experiment.

Experiments for selectivity analysis were performed using HCl and N-methyl-D-glucamine (NMDG) to obtain the desired pH of 10 mM HEPES solutions (100 mM NaCl and 1 mM MgCl 2 ). Sorbitol was used to maintain the desired osmolarity, where solutions containing 0 mM NaCl contained 200 mM sorbitol. Neither of these compounds were found to have an effect upon the receptor (data not shown).

Ascending ramps (0.1 V/s) from -100 to +100 mV were applied every 5s for a minimum of five times, with a 100ms hold at -100mV before ramping and a return to -100mV after the ascending ramp. A holding potential of -60mV was maintained between ramps.

Inhibition of pH8 currents by divalent cations was tested using CaCl2-free standard-solutions at pH 5. 1 min applications of pH 8 before and after a 1 min perfusion of the specified concentration of the cation at pH8 were subsequently followed by 2.5 min washes at pH 5.

For amino-acid derivatives, CaCl2-free standard-solution containing 10 µM 4-BrC was used. 1min applications of test-compound free solution before and after a 1 min perfusion at the specified concentration were followed by 15 min washes of the standard-solution in-between sweeps.

Evaluation of recordings

For concentration-dependent responses, the mean ± standard deviation is reported using the mean of the EC50 and Hill-slope obtained for each individually recorded oocyte. A sigmoidal dose-response fit to the peaks calculated, was used to obtain the EC50 and Hill-slope for the set of concentrations tested on an individual oocyte. Maximum and minimum refer to the absolute inward current (negativedirection) response.

For potentiation tests: peaks are calculated using the maximal response to CaCl2-free standard-solution subtracted from the overall maximum per sweep.

For pH tests: the holding current (at pH 5) before application of the desired pH is subtracted from the overall maximum per sweep.

For inhibition tests: the 'peak' is calculated as the minimum during cation/compound application divided by the maximum obtained before cation/compound application, where the leak current measured at the beginning of the sweep is removed from both measurements. Fits were constrained to have a minimal response of 0.

The graphs in the tables show the mean ± standard deviation of all normalized recorded peak currents for the specified concentrations, which are normalized to their recorded oocyte's maximal peak current, with one non-linear regression fit to all the data. These fits are not constrained. All calculations were made using GraphPad Prism 4 (GraphPad Software Inc.).

Whole-cell patch-clamp recording

Voltage-clamp currents were recorded using an RK-400 patch-clamp amplifier In this paper, I contributed to molecular biology and did all the crystallography part (14 mutants structures), analyzed the data together with Akos and the rest authors, wrote part of the paper. Through a systematic analysis by mutagenesis of every titratable residue, combining electrophysiology, and by X-ray crystallography, we find a novel unique position, Glu35, in the extracellular domain, nearby the transmembrane domain (TMD) interface that is the key proton sensor. We also pinpoint a few other key positions as proton sensor candidates, most being located nearby the TMD. Additionally, we demonstrate that a central His235 residue in the middle of the TMD, previously claimed to be the sole proton sensor, is not necessary for proton sensitivity since H235Q mutant is fully functional. Overall, protons are found to act simultaneously at several loci that are below the classical orthosteric sites of neurotransmitters, revealing a novel and original mode of activation within this important family of channel receptors. Beside these mechanistic insights, the combination of electrophysiology and crystallography implemented here allowed us to challenge previously accepted ideas that are currently used in the literature on ion channels, notably that amide derivatives of Asp and Glu (Asn and Gln) are good mimics of their protonated uncharged form.

Introduction

Pentameric ligand-gated ion channels (pLGICs) are key players of neuronal communication. They promote either cell depolarization or hyperpolarization with the passive permeation of ions through an intrinsic channel, whose opening is stabilized by the binding of specific neurotransmitters. pLGICs are ubiquitously expressed in virtually all neurons, contribute to central nervous system functions, including sensory and motor processing, central autonomous control, memory and attention, sleep and wakefulness, reward, pain, anxiety, emotions, and cognition [1].

As such they are important drug targets. At least one member of each major subfamily of vertebrate pLGICs has been structurally resolved in the recent years: a serotonergic receptor (5-HT3A, [2]) and a nicotinic acetylcholine receptor (α4β2-nAChR, [3]) from the cationic receptors, and a GABAergic receptor (β3-GABAA, [4]) and two glycinergic receptors (α1-and α3-GlyRs, [5,6]) from the anionic receptors. However, relating these 3D structures to the physiologically-relevant allosteric states that mediate pLGIC activation and desensitization remains an open and debated question [1].

At present, the best structurally-characterized pLGIC is the Gloeobacter violaceus ligand-gated ion channel (GLIC), of prokaryotic origin. Its structure shows a highly conserved fold with the subsequently resolved structures in the pLGIC family.

Each subunit consists of an extracellular domain (ECD), predominantly in a βsandwich fold, and a transmembrane domain (TMD) composed of four helices, labeled M1-M4. The available structures of pLGICs support that the GLIC globally shares a common gating mechanism with its eukaryotic cousins, although molecular details differ [1].

Due to the relative ease for over-expression in E. coli, as well as its biochemical robustness toward detergent-solubilization and mutations, the GLIC has been resolved in four distinct conformations. It is the first receptor to be resolved in an apparently open as well as three various closed channel conformations [7-9]. In addition the GLIC has been solved as a complex with a variety of allosteric modulators such as barbiturates, bromoform, lidocaine, propofol, and xenon, and barbiturates [10-14]. Membrane-inserted GLIC proteins have been studied by electron paramagnetic resonance spectroscopy and fluorescence quenching experiments following site-directed labeling, relating some local conformational changes to the activation and desensitization transitions monitored by electrophysiology [8,15-18].

The GLIC has also been studied by computational methods including molecular dynamic simulations [19-23]. This combined set of data support that, upon activation, the subunits' ECD regions move closer together, which precedes a concerted tilt of the pore-lining M2-α-helices to open the pore gate and activate the receptor.

Chimeras between the ECD of the GLIC and the TMD of the α1-GlyR were additionally shown to fold properly and to be functional, revealing compatibility between prokaryotic and eukaryotic domains [24,25]. However, the GLIC is a pHgated channel, activated by lowering the pH, with a maximal activation at pH 4 and a pH50 around 5 [26]. This sharply contrasts with most eukaryotic pLGICs, which are activated by a neurotransmitter binding to a well-described cavity in an inter-subunit interface of the ECD. Mammalian pLGICs are not directly activated upon pH changes, but the agonist-elicited responses are modulated by pH, notably for the α3β4-, α3β2-, and α4β2-nAChRs [27], α1-GlyR [28,29], and various GABAA receptors [30,31]. So far, a handful of invertebrate pLGICs were found to be directly activated by pH, a nAChR from C. elegans by low pH [32], and two insect GABAA receptors by high pH [33,34].

Fully understanding the molecular mechanism of GLIC signal-transduction thus requires identifying the locus where protons act to activate the channel and which titratable groups are crucial in this process. The observation that a chimera composed of the ECD of the GLIC fused to the TMD of the α1-GlyR (GLICECD-GlyRTMD known as

Lily [25]) or the Erwinia chrysanthemi ligand-gated ion channel (GLICECD-ELICTMD [35,36]) is activated by protons indicates that the major proton-sensing motifs are likely situated in the ECD. However, an inverse chimera, the ELICECD-GLICTMD, yields pH-gated currents when the gain-of-function mutation of I9'A is incorporated, suggesting either proton sensors too weak to activate in the ELIC ECD or their presence in the GLIC TMD [35,37]. Mutation of a His residue located in the middle of the TMD (His235) abolishes GLIC function, raising the possibility that it might be involved in pH sensing [38,39], but combination of this mutation with a gain of function mutation restores at least part of the pH-gated function [8,37]. Altogether, the location of the proton activation site(s) remain(s) essentially unknown thus far.

The present study provides an exhaustive mutational analysis of GLIC titratable residues, with pKa's in the pH 6 to pH 4 range (namely Asp, Glu, and His), given the pH50 of GLIC activation, to search for the proton-sensing site(s). Each GLIC subunit contains 19 Asp, 16 Glu, and 3 His that were individually and/or collectively mutated.

Mutation of an Asp/Glu/His residue may be expected to affect the GLIC function in two ways: (i) by altering direct proton sensing, when the protonated form of this residue stabilizes the active state, as compared to the non-protonated form, or (ii) by altering the gating equilibrium independently of side chain protonation. To try discriminating between these two possibilities, Asp/Glu residues were systematically mutated to Asn/Gln, replacing the carboxylate moiety by a non-titratable amide group tentatively mimicking a permanently protonated form, as well as to Ala, removing the titratable moiety. Selected mutants were also solved by X-ray crystallography to characterize the effect of the mutations on the local protein structure.

To identify the determinants of proton-modulation/gating, two-electrode voltage clamp electrophysiology, using expression in Xenopus laevis oocytes, was employed; activation was elicited by dropping the pH from neutral (pH 7.3-8) to lower values (min pH 3.7). All recordings showed a slow onset of the response, with no apparent desensitization during a 30-90s pH application. Distinguishing the kinetics of activation and desensitization as compared to the wild-type GLIC (Wt), proved difficult for the vast majority of mutants, therefore mutants were characterized on the basis of their pH50, defined as the pH eliciting half of the maximal current.

To minimize the influence of the intrinsic variability between oocyte batches, which show some variation in the Wt response to pH changes, mutants were also characterized by a ΔpH50, which corresponds to the variation of pH50 between each cell expressing a mutant, and the Wt cell(s) in the same batch of oocytes. Significance of the results in this report was determined as values larger than 0.5 pH units for the mean ΔpH50, and mean pH50, as compared to Wt, based on the standard deviation of 83 measurements of the Wt (with a standard deviation of 0.4 pH units). All values which fulfilled this criterion of significance also had a p value <0.01 in the Student's Ttest against the Wt. A pH50 could not be established for some mutants, often with minimal currents.

Apical ECD mutations weakly affect proton sensitivity/modulation

In the apical region, the residues either face the solvent (D13, E14, D55, E67, E69, D97), are located at the subunit interface (D49, E75, D136), or are located at the middle of the ECD β-sandwich (E104). Their mutation produces weak effects. D13N-E14Q, D49N-D55N, E67Q-E69Q, and E67Q-E75Q produced no significant change in pH50, and neither did the corresponding single mutants of D55N, E67Q, E67A, E69Q, and E75Q. The mutants D49N, E75A, D97N, D136N, and D136A display small increases in pH50, but do not meet the criterion for a significant effect. The effect of D49 replacement is corroborated in a recent study, whereas the same study showed D136A to have a decreased pH50 with an increased Hill-slope [36].

Finally, E104, which makes internal intra-subunit contacts showed no significant change when mutated to Q. Mutation to A meanwhile produced a significant ΔpH50 and a noticeable, yet non-significant, decrease in pH50 (Fig 2).

A complex network in Loop C modulates proton sensitivity

The β9-β10-loop (Loop C) was investigated in conjunction with R133 (Loop B), both of which are located on the principal (+) inter-subunit interface, a region that contributes to neurotransmitter binding in eukaryotic pLGICs. The mutation D185N at the base of Loop C did not have a significant effect (Fig 2). This position was also recently reported with Wt-like properties as both Asn and Ala mutations [36].

Removing all remaining titratable moieties in the sextuple mutant R133A-E177Q-D178N-R179Q-E181Q-K183Q produced a marked increase in pH50. In contrast, mutating only the three acidic residues (E177Q-D178N-E181Q) produced a marked decrease in pH50, whereas the individual Asp/Glu residue mutations show Wt-like responses with E181Q having a tendency to decrease the pH50. The mutations E177A and E181A were previously reported in a cinnamic acid study with pH50 values of 5.4 ± 0.1 and 5.6 ± 0.1 respectively, as compared to a Wt value of 5.2 ± 0.1 [40]. D178 was also recently reported as displaying a Wt phenotype for both the Asn and Ala mutations [36]. Evaluation of the basic residues shows R179 mutation increases the pH50, with R179Q producing a marked increase, whereas R179A only has a tendency to increase the pH50, showing that the removal of the side-chain guanidinium is not solely responsible for the phenotype at this position. Meanwhile, replacing the remaining basic residue of Loop C, K183Q, has no effect. The single mutation R133A on Loop B also has no effect, which was also previously shown in [40]. Despite containing the triple mutation that results in a decrease in pH50, the mutant which removes all titratable residues from Loop C along with R133A has a similar but stronger change in pH50 as compared to the single R179Q mutant, yet their ΔpH50s vary by 0.5 units. This suggests that the main player in this region is R179, along with a complex network of other side-chain interactions modulating GLIC activation at this level. All residues were also tested individually, with most mutations having no significant effect, although a large majority tend to decrease the pH50 (notably D86N/A, D88N/A, and D91N). Although D91A was found to insignificantly increase the pH50 as compared to Wt, D91N and D91A were recently reported to have a slight decrease in pH50 values [36]. In contrast to the non-significant individual mutations, E26, which is found interacting with the bottom of the β4-β5 loop of the principal (+) face, shows a robust decrease of the pH50 when mutated to both Q and A.

A combination of E26Q with mutations of vestibular facing residues that tend to decrease the pH50 produces an obvious decrease in pH50. The E26Q-D86N-D88N-D91N mutant exhibits strongly reduced maximal currents, which prevented reliable evaluation of the pH50 in most of the cells (Fig

A key mutation at the principal (+) face of the ECD increases proton sensitivity

The basal principal (+) face, below Loop C, contains E82 and H127, along with a cluster of Asp/Glu residues found close to the TMD: D31, D32, and E35 from Loop 2, as well as D115 and D122 from Loop 7.

Consistent with some of the previous studies of H127 [38] The effects of D32 and D122, both of which are engaged in salt-bridges with R192, have been previously studied in other pLGICs [42] as well as in GLIC [22,43].

D32A and D122A were shown to be non-functional, yet weakly expressed [43],

whereas D32N shows marked decrease in pH50 and in maximal currents ( [22]). D122N

showed no function, and subsequent expression studies showed no expression (Fig Finally, mutation of E35 produced the strongest effect; therefore a more extensive study of this position was performed. The mutations E35Q, E35A, and E35M

4B
(the GlyR-position equivalent) display a significant increase in pH50, whereas E35K

shows a weaker non-significant effect, and E35H appears to show the inverse phenotype with a marked decrease in pH50. Interestingly, fitting E35H data with a single sigmoidal curve yielded poor fits preventing reliable calculation of the pH50. Of 1). The poor fit of E35H indicates a more complex mechanism at play, which could possibly be elucidated with a more in-depth study of mutation of this position. Altogether these data show that the loss of charge at the E35 position is important to the gain-of-function effect found.

Three hydroxyl-containing residues that are situated near E35 and E82 on the complementary face (-) of the ECD were subsequently mutated: Y28, S29, and T158.

Individually removing each hydroxyl group shows a tendency to increase the pH50, with Y28F, near E82, having a significant increase. The mutation of T158A did not

Crystal Structures of ECD mutants show no significant change with the exception of E35A and E82Q.

A structural analysis was performed on E26Q, E26A, E35Q, E35A, E67A, E75A, E82Q, E82A, D86A, D88N, D88A, H127N, E181A, and H277Q mutants, by solving their cascading rotamer/conformational change in Y28 and F156, the latter of which flips out towards the aqueous environment. Interestingly E35A also produces the greatest structural deviations around Y28 along with neighboring residues. Of the few conformational changes seen it is interesting to note that Y28F had the greatest functional effect of the non-titratable mutations tested.

Mutations at the TMD decrease proton sensitivity

It has been proposed that H235 is a key residue mediating proton sensing [38,39], but recent data show that the GLIC can still gate when H235 is mutated to non-titratable residues in combination with a strong gain-of-function mutation [8,37].

It has also previously been shown that GLIC expression in bacteria is highly sensitive to mutation at H235 [7]. Both of these findings are confirmed, firstly with the mutation H235A, which abolishes expression in oocytes ( 1). H235Q does not allow for a charge or a change in protonation state at this position, and therefore one would expect a completely non-functional receptor if this residue were solely responsible for the proton modulated gating of the GLIC. Rather this finding confirms the structural importance of H235 that has been previously reported, along with the importance of a hydrogen-bonding network, between the M2 α-helix and the neighboring M3 α-helix, with the backbone carbonyl of I262 [7,38].

Among the other titratable residues of the TMD, neither a mutation of E272Q nor E282Q produced an overall significant effect, albeit a significant negative ΔpH50 was observed for E272Q which was previously reported to be non-functional when mutated to A [44]. In contrast, the mutation of either E222 or E243 as Q or A, as well as H277Q, all produced significant loss-of-function effects, pointing to the key role of these residues in activation. E222 (E-2'), at the beginning of the M2 α-helix, has been extensively studied as the key component of the selectivity filter, a charged constriction point in the lower part of the pore, for cationic pLGICs [45,46]. The E222A mutation has previously been resolved crystallographically and has no change in structure as compared to Wt at pH4 [10]. Therefore the strong loss-of-function mutation does not appear to affect conformation. E243 flanks the apical section of the TMD pore, making a pentameric ring at the end of the M2 α-helix. A mutation of E243C was shown to have a similar pH50 as Wt [18], whereas the mutation E243P is reported as non-functional and found in a locally-closed conformation [7]. Mutation of H277, which lies in the adjacent M3 α-helix, nearby E222, with possible electrostatic of interpretation with red and darker red as significant decreases and a decrease of greater significance, respectively.

Discussion and Conclusion

All of the titratable Asp/Glu/His residues of the GLIC were mutated and evaluated for their impact on the receptor sensitivity to protons. This approach allows for the identification of key regions controlling gating.

Several mutations within Loop C markedly alter the pH50. The decrease in proton sensitivity following neutralization of the E177/D178/E181 cluster, which is completely negated and reversed with the neutralization of the basic R133, K183, and R179 residues, as well as the importance of R179 mutations alone, suggests a role in gating rather than in proton sensing. This idea is in line with the observation that replacement of the entire Loop C with other pLGIC Loop C sequences or a poly-glycine segment is still compatible with a pH-gated channel [47]. Interestingly, this region was suggested to be the binding site for organic acids that act as negative allosteric modulators, including caffeic [40] and crotonic [48] acids. These data thus further document the key allosteric role of this region, which is homologous to the neurotransmitter binding site of eukaryotic receptors.

Mutants of E35 are unique in strongly increasing the proton sensitivity.

Neutralization of the E35 side chain by mutation to A, Q, or M produce a similar increase in proton sensitivity, while mutation of E35K exhibits the same behavior with half the increase. The E35H mutation, however, shows the inverse phenotype. This suggests that a charge at this level impairs activation, and a hydrophobic/polar sidechain is preferred for stabilization of the active-state. Interestingly, the structures of the Wt GLIC, E35Q, and E35A, at pH 4, show that the side chain of residue 35 is located in a hydrophobic environment bordered by the side chains of P113, L114, F116, F156, T158, and P247. In the GLIC structure at pH 7, which is representative of the resting state, the carboxyl moiety of E35 is more solvent exposed toward the inner vestibule, since the side chain of P247 is moved away following the key revolving motion of the

M2-M3 loop (Supp Fig 2)

. Therefore, the resting state would be predicted to better accommodate a charged residue, consistent with the functional data. It is noteworthy that E35K, while charged, does not decrease the proton sensitivity. However, the length of the lysine side chain likely places the charged ammonium away from the E35 carboxylate locus. Altogether, the data support that E35 is a bona fide proton sensor, whose charged form stabilizes the resting conformation, whereupon protonation favors the active state. The functional proton-gating of E35Q and E35A mutants additionally suggests that if E35 is a bona fide proton sensor, it can't be the only one in the GLIC.

In the lower part of the ECD, the principal (+) face also carries two acidic residues E82 and D115. E82Q and D115N display significantly higher proton sensitivity than E82A and D115A, respectively. Assuming that Gln and Asn faithfully mimic protonated side chains of Glu and Asp, this would suggest that residues E82 and D115 are also proton sensors. In this case, their protonated side-chain would elicit stabilizing interactions specifically in the active state. To investigate this idea, the structures of E82Q and E82A were solved at pH 4. The E82A structure is similar to that of the Wt pH 4 structure, whereas that of E82Q shows a marked local reorganization of the Q82 moiety. Assuming E82 to be in a protonated state at pH4, Gln is not a good mimic of a protonated Glu residue, since Q82 is engaged in a completely different set of interactions with neighboring residues. Therefore the E82Q mutant phenotype is likely due to local side-chain reorganizations of Y28 and F156 rather than being a proton-sensor itself.

Finally, D32 mutations significantly decreased the pH50. However, this residue is shown in the pH 4 structure to interact through a salt bridge with the already saltbridged pair of R192 and D122. This interaction inter-locking Loop 2, Loop 7, and the pre-M1 loop is often conserved throughout the pLGIC family [23], with notable exception of the ELIC and the α-glutamate-gated chloride channel from Caenorhabditis elegans. Thus, it is unlikely that it undergoes side-chain protonation at pH 4, and likely contributes in gating rather than in proton sensing [22].

Mutation of Asp/Glu residues at the complementary (-) face, in general, decreased the proton sensitivity of the GLIC, with additive effects in many cases, especially for D88 and D86. However, the pattern of phenotypes does not allow discriminating whether those residues are involved in proton-sensing or gating, since mutations to Asn/Gln also produced decreased proton sensitivity. The key mutants at E26, D86, and D88 were solved at pH4, each showing a structure similar to that of the

Wt (Supp Fig 2). E26 clearly is an important residue in GLIC function, but other

techniques are required to elucidate its mechanistic role.

Mutations at both entrances of the ion channel, E243 (outer ring) and E222/H277 (inner rings), as well as at the middle of the TMD within each subunit four-helix bundle (H235), produced significant decreases in proton sensitivity, but again here the pattern of phenotypes does not allow discriminating between protonsensing and gating. H235 was previously proposed to be the major proton-sensing residue for GLIC activation, but the mutation H235Q, which is no longer titratable, still produces a functional proton-sensitive receptor, questioning its role as the sole proton sensor. Clearly H235 is a very important residue for functionality, although its protonated state would be hardly accommodated by the hydrophobic environment of the TMD in both known apparently open and closed forms.

Molecular dynamics simulations using a string method optimization made predictions of the change in protonated state and sensitivity of titratable residues throughout the gating-mechanism of the GLIC [23]. The report identified E26 and E177 to have loss-of-function effects when mutated. E26 indeed results in a loss-offunction effect, whereas E177 seems to have no effect. Additionally, the prediction identified E35, E75, and E243 as gain-of-function locations if mutated, where E75 and E243, when mutated, have either no effect or the opposite effect.

Global robustness of GLIC gating toward Asp/Glu/His mutations

Despite combined mutation attempts, channels displaying constitutive activity were not observed, nor was there a complete abatement of function. This shows that proton sensing is not mediated by a single Asp/Glu/His residue, but rather by several residues located on different parts of the protein. Individual residues in many places may contribute partially to proton-sensitive channel gating, unraveling a mode of proton-controlled activation quite different from that of classical agonists of pLGIC family receptors, which act at a well characterized orthosteric site.

The results indicated that E35 is an important proton-sensor, as well as the existence of a number of other proton sensing candidates, notably E26 and the D86/D88 pair in the ECD, E243 at the top of the channel, and E222/H277 at the bottom of the channel. E35, E26, and E243 are all located nearby the ECD-TMD interface, suggesting that at least part, if not all, of the pH-elicited activation of GLIC bypasses the orthosteric site. E222/H277 might also contribute to pH sensing, implying in this case the diffusion of protons from the extracellular compartment, possibly through the ion channel itself. The E222/H277 pair most likely confers the intracellular proton concentration sensitivity of the GLIC, which was reported from inside-out patch clamp experiments [49].

Complementary approaches that would be too cumbersome to perform on all the mutants evaluated in this study, such as reporting on the open probability, channel kinetics, and linking the efficiency of gating to the apparent affinities identified are necessary to elucidate the specific effects of the key residues in the complex gatingmechanism of the GLIC.

The recent studies employing chimeras between ELIC domains and GLIC domains validate the possibility of multiple proton sensing sites. Interestingly the pH50 of Lily, and the GLICECD-ELICTMD mimicking Lily (GELIC), and E35Q/A mutants all closely hover around pH 6.5 [25,36]. Altering the TMD residues surrounding E35 could have the same effect as charge neutralization. However, another GLICECD-ELICTMD mutant (GE) which lacks the mutation Y119F and the C-terminal conversion of RGITLELIC to LFFGFGLIC has a reported pH50 of 3.63 [35]. It is important to note that neither the study of Lily nor of GELIC tested below a pH of 5, and both found significantly reduced currents; whereas, GE displayed robust currents at pH's below 5.

These discrepancies point to the intricate interactions between the ECD and TMD, which influence gating and possibly proton sensitivity. These studies combined with the current results also clearly show that the principal component of proton activation lies within the ECD and not the TMD as previously assumed. The ELICECD-GLICTMD chimera is not susceptible to proton activation until further mutation by I9'A, which has previously been shown to destabilize the recovery time for the receptor to return to the resting-state [26].

Residues other than Asp/Glu/His may also contribute to proton sensing, notably Arg/Lys side-chains, that can display pKa's in the 4-6 range when located in very hydrophobic environments [50], or aromatic residues through cation-pi interaction with hydronium ions [51].

Cases studies of other pH-sensing ion channels.

Among pH-gated ion channels, two were studied in molecular details. First, the bacterial potassium channel KcsA was found to be inhibited by a local network of ionic/H-bond interactions between two Glu, two Arg, and a single His residue. A disruption of this network upon protonation allows channel opening [52]. In this case, E to A mutation of the two key residues increased the sensitivity of the channel to protons. Such a "suppression of charge to activate the channel" mechanism on GLIC is observed for the single E35 residue.

The pattern of phenotypes observed here is reminiscent of acid-sensing ion channels (ASICs). Indeed, ASICs have been extensively studied, but efforts to map the sites for proton binding have so far yielded inconclusive results, since mutation of multiple individual Asp and Glu residues independently produce changes in proton sensitivity [53]. Simple mutation of basic Arg/His/Lys residues cannot mimic a protonated state either, therefore further limiting this approach to study ASICs.

Proton sensitive positions and their involvement, or lack of, in gating

The combined mutagenesis data support that, for several pH sensitive Asp/Glu positions, notably E26Q, D32N, E82Q, D86N-D88N, D122N, E222Q, and E243Q the change of charge does not contribute to activation. In these cases the side-chain carboxylic acid may engage in active-state stabilizing interactions with neighboring residues or water molecules. Using the site-directed mutagenesis approach may only conclusively evaluate the perturbation of residue titratability, as Asn and Gln residues may in fact be poor mimics of protonated Asp and Glu residues. Either this is the case or the aforementioned residues must maintain their de-protonated state at pH4 and provide stability to the active-state as charged moieties, contradicting pKa predictions.

Additionally, it is expected that a good mimic would maintain the same conformation as the protonated version of an Asp/Glu residue, and that this should be witnessed in a crystallographic structure. Yet the E82Q structure differs from Wt GLIC at pH4 where E82 would be presumably protonated. As previously mentioned, replacement of His residues may also only evaluate the removal of their titratability, whereas a simple mutagenesis cannot mimic the His protonated-state. The functional results indicate either a sensitivity to protons or a structural importance for both H235 and H277, neither of which are crucial for proton elicited gating of the GLIC.

Overall, the data suggest a complex network of H-bonds and polar interactions, with important positions below the orthosteric site, in the pH-sensitive channel opening mechanism of the GLIC. A specific importance of the ECD-TMD interface was identified with the position E35 acting as a key sensor next to the D32-R192-D122 triad involved in the signal transduction between domains [43].

current with the holding current subtracted. The average of the two recorded peak values for a given pH was plotted in GraphPad Prism 4 (GraphPad Software, Inc, La Jolla, CA) against the pH and fitted with a non-linear sigmoidal dose-response fit to obtain one (n-unit) value of pH50. The given number of injections and number of recorded oocytes per construct are listed in Suppl. Table 1. .with generally 2-4 oocytes recorded per injection. Fits with an R 2 <0.9, a Hill-slope <0.6 or >4, or an absolute Imax <0.9µA were excluded from inclusion into mean-values, and therefore not counted in the n either. The Imax cut-off was chosen due to endogenous current at pH4 or lower that appears on occasion. To be sure that this current does not greatly influence the pH50 calculation, a cut-off greater than three-fold was chosen. The arbitrary cut-offs for Hill-slope and R 2 were chosen to remain consistent in the removal of data that could not be properly fitted as a result of any circumstance. In order to minimize the influence of the intrinsic variability between oocyte batches, which show some variation in the Wt response to pH changes, mutants were also characterized by a ΔpH50, which corresponds to the variation of pH50 between each cell expressing a mutant, and the Wt cell(s) in the same batch of oocytes, measured with the same solutions of pH. The pH50 values and ΔpH50 are reported as mean ± standard deviation.

Protein Production and Purification

GLIC variants were purified as previously reported [54]. PET20b vectors carrying the GLIC constructs fused with an N-terminal maltose binding protein (MBP) tag were used to transform E. coli C43 cells, cultured at 37°C in the 2YT medium containing 100mg/ml ampicillin. At an optical density (OD) of 0.8, the cultures were cooled to 20°C and 0.1mM IPTG was added for an overnight induction. All of the purification steps were carried out at 4°C. Proteins were extracted from the cell membrane with a Tris-buffered saline solution (TBS, 300mM NaCl, 20mM Tris pH 7.6) containing 2% n-dodecyl-β-D-maltoside (DDM). Solubilized proteins were subsequently isolated by ultracentrifugation, loaded onto an amylose resin, and incubated for ~1 hr. The resin was extensively washed using a TBS containing 0.1% DDM and subsequently with a TBS containing 0.02% DDM. Thrombin enzyme was added into the MBP-GLIC-bound resin and incubated overnight. The GLIC protein was eluted using a TBS containing 0.02% DDM and 20mM maltose. A further purification step was carried out by size exclusion chromatography on a Superose 6 10/300 column (GE Healthcare), which was equilibrated with a TBS containing 0.02% DDM.

The fractions of the peak corresponding to the molecular weight of the GLIC pentamer were collected and concentrated to around 10mg/ml for crystallization.

Crystallography

The concentrated protein was mixed at 1:1 volume ratio with a mother liquor solution containing 12-14% PEG 4000, 400mM NaSCN, 15% glycerol, 3% DMSO and 0.1M NaAcetate pH4. The crystallization procedure was performed at 20°C using the hanging drop method. Micro-seeding was performed after an initial crystallization setup. Crystals appeared overnight and grew to full size in two weeks. The crystals were flash frozen using liquid nitrogen. The diffraction data sets were collected either on the beam-lines Proxima-1 of the Soleil Synchrotron or the European Synchrotron Radiation Facility (ESRF) ID29 and ID23A. The data sets were processed with xdsme [55] and further processed by CCP4 programs [56]. The structures were solved by molecular replacement in PHASER [57] using the GLIC (PDB ID: 4HFI) as the initial model. Further refinement was carried out using BUSTER refinement [58]. The quality of the structural models was checked by Molprobity webserver [59]. Statistics for all crystal structures are listed in (Supp Table 2).

Supplementary information

Supp. Table1: Electrophysiological Values

Introduction

Pentameric ligand-gated ion channels (pLGICs), also known as Cys-Loop receptors in animals 1 , or, more recently, Pro-loop receptors 2 , mediate rapid signal transduction in the central and peripheral nervous systems 1 . Activation of the receptors is triggered by binding of agonist(s) at the extracellular domain (ECD) to lower the transition energy between closed and open states 3 . This leads to a global allosteric conformational change and promotes the opening of the selective ion pore at the transmembrane domain (TMD). Dysfunction of pLGICs can cause severe nervous-system diseases and they are the targets of many therapeutic compounds 4 .

Available structural information of this family derives from a number of prokaryotic and eukaryotic channels [5][6][7][8][9][10][11][12][13] . Despite substantial diversity of sequences, structural information shows high conservation of tertiary and quaternary architectures between eukaryotic receptors and their bacterial homologs. Besides the covalent linking between ECD and TMD through the pre-M1 region, the interface of ECD and TMD is associated with highly conserved loop regions: β1-β2 loop, loop F (β8-β9 loop), Pro-loop (β6-β7 loop) and M2-M3 loop, see Figure 1A 1,14 . Of all pLGICs, by far the best two structurally characterized receptors are the prokaryotic ELIC from Erwinia chrysanthemi 12 and GLIC from Gloeobacter violaceus 13,15,16 . Notably, GLIC has been captured in four different states. Following the first apparently open/active state of GLIC that crystallized at pH 4 13,15 , several different 'locally closed' states (LC) have been solved [17][18][19] . Those LC forms are believed to be pre-active states as suggested by fluorescence quenching experiments 20 . Recently, the closed/resting state of GLIC was solved at neutral pH 16 . Finally, a desensitized state of GLIC has also been reported 21 . GLIC has been extensively used as a model to characterize the binding properties of important pharmacological reagents, such as propofol 22 , bromoform 23,24 , ethanol 23,25 and barbiturates 26 , while ELIC has also been used as a model system for the binding of general anesthetics and benzodiazepine molecules [27][28][29] . In addition, a number of biophysical methods have also been

Results

Using Poisson-Boltzmann electrostatics and the FD/DH method to predict potential pH-sensing residues in GLIC

Activation of GLIC is triggered by lowering the pH from neutral (around pH 7.0) to acidic (around pH 4.0) with a pH50 value of 5.1 ± 0.2, at which half the maximal current is reached (Table 1). We are looking for those residues whose protonation will most profoundly affect the conformational transition between the closed and the open forms of the channel. These are likely to be either Asp, or Glu or His residues. There are 35 carboxylate residues in each monomer, along with 3 Histidine residues (see below). It is expected that the pKas of residues that do respond to proton activation should be significantly shifted from model pKas (Asp = 3.8-4.0; Glu=4.2-4.4; His 6.5). However, this is a necessary but not sufficient condition to predict/identify the proton-sensing residues. Indeed, following Sazanavets and Warwicker 37 The output of FD-DH gives a list of putative salt bridges in the structure, along with their calculated energy. The strongest ion pairs (with an interaction energy around 9 kT in the open form, but 6 kT in the closed form) involve the triplet R192, D122 and D32, a well-known and strongly conserved feature of all pLGICs. However, at the same level (8.5-9 kT) in the list comes the less wellknown network made of Y197-Y119 and K248 of the next subunit. We will come back to these two "triplets" below, which are actually linked together by a 5 kT interaction between Y197 and R192, in the open form (but not in the closed form). The list of the strongest electrostatic interactions given by FD-DH will be supplied in Supp. Information. between the open and the closed forms (ΔpKa ≥ 1) (Figure 1C), indicating that the local environment of these residues notably changes between the two states, but in a zone quite distant from pH50, therefore there are more likely to be pHcoupled residues. As noted above, D32, D122 together with R192 form a salt bridge, which is stronger (9 kT instead of 6 kT) in the open state compared to the resting state. However, D122 and R192 (and, to a lesser extent, D32) are conserved in almost all pLGIC, even in receptors not triggered by a pH change.

Mutations at these positions often impair expression of the receptors on the surface of oocytes or display a total loss-of-function phenotypes (Table 1).

Therefore, they are not expected to play a role in proton-sensing per se but rather to be essential for maintaining the structural integrity of the receptor.

E181 is located in Loop C (β9-β10 loop), which experiences a considerable conformational change between the two states and would be a good candidate for proton-sensing. However, its predicted pKa is not in the zone of pH50 +/-1.

Furthermore, mutations of each titratable residue in loop C show no or small effects on the proton activation in the pH 7.0-4.0 range, as shown by several studies 38,39 . Indeed, deleting the entire loop C or replacing it by 10 glycines has no influence on pH activation 18 .

To further characterize the potential candidates for pH-sensing, sitedirected mutagenesis and FT-IR spectroscopy were employed in order to experimentally determine their pKa values (E26, E35, E75, E104, E181). Other residues (D86, D88, E67, D97) served as experimental controls (see Material and Methods section). The phenotype of these mutants was recently tested by electrophysiology elsewhere, especially in a systematic approach (Nemecz et al., submitted) and the results are summarized in Table 1. Here we focus on the experimental determination of their pKa and their structural characterization.

Experimental measurement of the pKa values of potential pH-sensing residues using FT-IR

Figure 2A and 2B show pH-induced FTIR spectra for GLIC wild type and for the GLIC E35A mutant, respectively, reconstituted in POPE/POPG lipid bilayer. The experimental device is shown in Suppl. Figure S1A and typical spectra and control experiments are shown in Suppl. Figures S1B-E. Reference spectra were measured at pH = 7.0, while sample spectra were calculated by measuring the difference at various lower pH's down to pH ~ 2.0. Negative peaks represent the structural features that appear at pH 7.0, while positive peaks represent structural changes that occur at lower pH. Peak positions of the bands in both the observed spectra of wild-type and E35A mutant are almost identical and their differences are smaller than 2 cm -1 . The structural effect of the point mutation could not be identified from the direct analysis of the observed spectra. On the other hand, the relative intensities of the observed bands are slightly different among different measurements. These are mainly due to slight differences in lipid/protein ratio among the different sample preparation. All observed bands can be assigned to either the protein (1718~22, 1655, 1630, 1573, 1540, 1520, 1400 cm -1 ), the lipids (1738, 1466, 1456 cm -1 ) or the buffer (1364, 1321, 1218 cm -1 ) (For a detailed band assignment, see Suppl. Note S1).

Among the protein bands, bands at ~1720 cm -1 (ν(C=O)), ~1573 cm -1 (νas(COO -)), and 1400 cm -1 (νs(COO -)) are assigned to the carboxylic acid/carboxylate group of Asp or Glu residues. The increase of the intensity of the ν(C=O) band due to the carboxylic acid (1720 cm -1 ) at lower pH is due its protonation along with the decrease of the intensity of νas(COO -) and νs(COO -) bands. Since intensities of these bands indicate the extent of protonation state of Asp and Glu residues in the protein, a plot of the intensity of these peaks as a function of pH provides a titration curve for the carboxyl groups. However, a definite determination of peak positions and absolute intensities of ν(C=O) and νas(COO -) are difficult to obtain as these bands closely overlap with lipid ester band at 1738 cm -1 and amide II band at 1540 cm -1 , respectively. Therefore, we employ νs(COO -) band at 1400 cm -1 as a marker for the intensity analysis of carboxylic group, because it is sufficiently isolated from other bands.

Figure 2C shows the normalized peak heights at 1400 cm -1 for wild-type (black cross) and E35A mutant of GLIC (red dots). The intensities are normalized to be zero at pH = 7.0 and -1 at pH 2.0 for wild-type. In Asp or Glu mutants that are replaced by residues that do not contain any carboxyl groups such as alanine, the normalized factor of -0.97 at pH 2 was employed because the total number of the carboxyl groups is one less than that of wild-type, hence a normalized factor of 34/35 (For the definition of the normalized factor, see the Suppl. Note S2).

These plots provide a titration curve of all Glu and Asp residues in GLIC. Since each GLIC monomer contains 16 Glu and 19 Asp residues, the overall titration curve display a broad sigmoidal shape in a wide pH range due to the overlap of the individual titration curves from each residue. Notably, the pH titration curves from E35A mutant shows significant deviations from that of the wild type receptor in a pH range starting around 7. Open and filled dots represent data sets from different experiments with different sample preparations. Note that individual features of these titration curves are highly reproducible except for E181A (orange), which shows a deviation at pH range between 6 and 4 but converges to the same trace as the wild-type at lower pH region. (Suppl. Figure S2).

Probing residues around E35 by site-directed mutagenesis

Because E35 is not located in the expected agonist binding site of a member of the pLGIC family, namely close to Loop C and Loop B, we performed a systematic mutagenesis study centered on E35 to probe its immediate environment.

a) T158 and the role of Loop F

Examination of the structure of GLIC indicates that E35 establishes polar interactions with T158 from Loop F (Figure 4C). To determine whether this interaction is involved in signal transduction, i.e. linking proton binding to channel gating, we probed by site-directed mutagenesis this residue as well as those immediately adjacent to loop F (G159, W160). The currents of mutants were measured by two-electrode voltage-clamp electrophysiology in Xenopus oocytes. We decided to make a mutation into a cysteine so as to later perform chemical labeling of this cysteine. First we checked that the so-called Cys-less mutant of GLIC (C27S) has the same properties as the wild type of GLIC and is unaffected by treatment of MMTS (S-Methyl methanethiosulfonate), a reagent that converts the side chain of cysteine into -S-S-CH3 group, or DTT (Dithiothreitol), that reduces S-S bonds. We found that replacement of T158 by a cysteine doesn't affect the function of the receptor. However, when GLIC T158C mutant is labeled with MMTS, the current is decreased by 50%. This phenotype can be reversed by reducing and removing the MMTS labeling (Figure 4A).

Cysteine replacement of G159 totally abolishes the pH-induced currents and generates a nonfunctional receptor as well as the replacement of W160 by phenylalanine (Table 2), indicating that these residues located in loop F are important for proton-induced activation.

b) A network of water molecules (not seen by the FD-DH method) at the ECD-TMD interface near Loop F stabilizes the open form structure

Conserved loops at the interfacial region between the extracellular agonist-binding domain and transmembrane ionotropic domains are strong candidates to play the role of connecting the binding of an agonist to channel gating 19,40,41 . The 2.4 Å resolution crystal structure of the open form of GLIC allows to study in detail the network of bound water molecules in this region 31 .

Indeed, a closer analysis of this high-resolution model uncovers the existence of an elaborate hydrogen bonds network at the ECD-TMD interface. The interfacial residue Q193 interacts with the backbone amide nitrogen atom of G159 from loop F, as well as with the carbonyl oxygen atom of K248, which is located at M2-M3 loop from the adjacent subunit through hydrogen bonds mediated by water molecules. This hydrogen bonds network at ECD-TMD interface extends to residue T158 of Loop F, which is in turn interacting with E35, as discussed in the previous paragraph. This network has also been confirmed in a new wild-type open-form crystal structure, which was determined at a higher resolution of 2.22

Å and has even better refinement statistics than the 2.4 Å structure (Figure 4B, 4C and Table 3). In the vicinity of Q193, there is a critical arginine (R192)

surrounded by two negatively charged residues: D122 (located in the Pro-loop) and D32 (located in β1-β2 loop). This salt bridge is highly conserved among pLGIC Pro-loop receptors and has been shown to be important for channel activation 19,42 . In addition, the strictly conserved P120 (from Pro-loop) interacts with Y119 and Y197 (Pre-M1) through hydrophobic interactions (Figure 4D).

Additionally, the hydroxyl group of Y197 interacts with the side chain of R192 by forming a hydrogen bond, as picked up by the FD-DH program and presented earlier. These two tyrosine residues will be studied in more details below.

interaction between Y197 (pre-M1) and R192 is highlighted by indicating their average distance calculated from five equivalent positions.

c) The interfacial hydrophilic crevice occupied by Q193 is involved in coupling proton binding to channel gating

In the higher resolution open form of GLIC structure, Q193 side-chain penetrates deeply into a crevice, which is formed by loop F and M2-M3 loop from neighboring subunits. Three water molecules can be modeled into this crevice.

Distance measurements confirm the existence of a hydrogen bonds network in this region. Sequence alignment reveals that other pLGICs possess at this position either a hydrophilic or charged residue. Here, the role of residue Q193

in GLIC has been tested by electrophysiology and crystallography. First, Q193

has been replaced by the hydrophobic and nonpolar residues methionine and leucine, separately. Electrophysiology experiments show that both Q193M and Q193L yield functional receptors; however, both mutants are less conductive and exhibit a loss-of-function phenotype with pH50 = 4.53 ± 0.02 and pH50 = 4.48 ± 0.05 compared to that of wild-type pH50 = 5.10 ± 0.20 (Figure 5C and Table 2).

These results indicate that disrupting the hydrogen bond network in the crevice hinders the signal transition from ECD to TMD.

In order to document at the structural level the effect of these mutations, we solved the crystal structures of Q193M and Q193L. Both mutants express readily in E. coli cells. We crystallized Q193M and Q193L using conditions similar to those used to obtain the open form of GLIC (pH 4.0) and solved the structures at 2.95 Å and 3.39 Å resolution, respectively (Table 3). Unexpectedly, both mutants adopt the conformations previously described as "locally closed" 17 or "fully-liganded closed-channel" form 18 . Compared with the open form of GLIC, M2-M3 loop extends and shows the conformation previously described in "LC1" form (Figure 5A and Suppl. Figure S4). When superimposed to GLIC H11'F (PDB ID: 3TLC)) 17 , the Q193M and Q193L mutants show a root mean square deviation (RMSD) with the LC1 form of 0.61 Å and 0.59 Å, respectively. On the other hand, all five trans-membrane M2 α-helices are kinked at the level of the I9' position, the upper portion of M2 helices tilt and rotate clockwise around the five-fold symmetry axis along the ion channel pore, and consequently narrow the ion permeation pathway, generating a nonconductive state of the ion channel (Figure 5B). Compared to the wild-type open-form structure, the side chains of M193/L193 rotate by 90° and do not protrude into the interfacial crevice any more (Figure 5D and5E). Thus, on the structural level, breaking the hydrogen bond network by replacing Q193 with hydrophobic residues hinders the signal transmission that bridges proton binding to channel gating and destabilizes the open form compared to the locally closed form.

d) Further characterization of the proton-coupled Q193 using a cysteine mutant and chemical labeling

To probe further the role of Q193, we replaced Q193 with cysteine.

Compared to glutamine, the side chain of cysteine is shorter and less polar. The phenotype of Q193C was almost identical to that of the wild-type as was shown by electrophysiology (Table 2). Moreover, we grew crystals of Q193C at pH 4.0 and determined its structure at 2.58 Å resolution. Consistently, the structure of Q193C adopts the same conformation as the GLIC open state (RSMD = 0.31 Å)

(Figure 6A). The structure shows a water molecule in the interfacial crevice region, that connects the thiol group of C193 to the nitrogen atom of G159 (Loop F), and the nitrogen atom of P250 (M2-M3 loop) from neighboring subunits (Figure 6B). Therefore, we postulate that the putative new hydrogen bond network maintains the ion channel open. Since hydrogen bonds formed by thiol groups are rather rare and weak, additional experiments were performed in order to perturb this new hydrogen bond network. First, we blocked the polar side chain of cysteine with MMTS. The electrophysiology experiments show that after applying Q193C with 100 μl MMTS, the current was inhibited by more than 75%. After treatment with the reducing agent DTT for five minutes, the current recovered around 50% (Figure 6D). After treatment with DTT for nine minutes, the current recovered almost 100% (Suppl. Figure S5B). The same results were observed when applying Q193C with MTS-ET and MTS-ES (Suppl. Figure S5C and Suppl. Figure S5D). Thus, blocking the thiol group of residue Q193C destabilizes the open form of the channel.

Next, we solved the crystal structure of Q193C co-crystallized with MMTS.

To do so, we incubated Q193C protein with 10 mM MMTS overnight before setting-up crystallization trials. Several crystals of GLIC Q193C+MTS were tested and the best one diffracted to 3.5 Å. As expected, the structure of Q193C+MMTS

shows an LC1 conformation similar to that Q193M and Q193L (Figure 6C) and the 2mFo -DFc map shows density for the MMTS covalently linked molecule.

Histidine mutants show no effect on the global pH titration curve of carboxyl groups

We also tested by site-directed mutagenesis and FT-IR all the histidine residues (H127, H235, H277) -see Figure 7A for their localization in the structure, even if none of them had a significant value of predicted |Δ pKa| value (Figure 7B). with other vibrational modes from the background 44 . However, we studied the effect of all Histidine mutants by monitoring the effect of the pH titration at 1400 cm -1 . It appears that mutating all histidine residues of GLIC has no effect on the pH titration curve of their carboxyl group (Figures 7C and7D).

Interestingly, all three Histidine residues are closely interacting with a Tyrosine having a high |Δ pKa| value (Figure 7B): Y129 with H127, Y221 with H277 and Y263 with H235, respectively. In the wild-type open form of GLIC, the aromatic ring of Y197, together with Y119, P120, F121 (Pro-loop) and L246 (M2-M3 loop) form a hydrophobic stacking. We therefore replaced Y197 with alanine to not only disrupt the interaction between Y197 and R192, but also to reduce the hydrophobic stacking between both the Pro-loop and the M2-M3 loop (Figure 4C). The Y197A mutant totally abolishes the channel current, generating a non-active receptor (Table 2).

Therefore, our data indicate that the aromatic ring of Y197 play a crucial role in the coupling of proton binding to channel gating.

The Pro-loop points toward a cavity bordered by pre-M1 region, M2-M3 loop and the end of the M4 helix within the same subunit. Contacts between these regions are predominantly mediated through aliphatic or aromatic side chains. Several studied have pointed out that P120 in GLIC, the only cis isomer proline that is strictly conserved in the pLGIC family, is highly sensitive to mutagenesis 45 . Next to P120, Y119 is also predicted to have the largest ΔpKa (ΔpKa =2.7) between the open and closed forms (Figure 7B). Therefore, we generated the Y119F mutant and studied its structural and functional properties.

The crystal structure of Y119F displays a nearly identical conformation as the wild-type open form of GLIC and this is consistent with the fact that the replacement of Y119 side chain by a phenylalanine has the same behavior as the wild-type receptor in the oocyte system. However, even though the Y119A mutation generates a nonfunctional receptor 19 , the crystal structure that we determined at 2. Upon channel opening, the upper portion of the M2 helix tilts and anticlockwise rotates around the symmetry axis of the ion channel. This rearrangement causes the tightly packed bundle of five M2 helices to detach from each other and move closer to M3 helices. This also reshapes a cavity behind the M2 helix. Previous studies show that this intra-subunit cavity is essential for general anesthetics binding 22 . The mutation I201W should block the M2 helices movement by introducing a bulky amino acid in this cavity. Indeed, the mutant I201W at top M1 region generates a nonfunctional receptor as was revealed by a previous study 20 . In order to elucidate this functional phenomenon at the structural level, we grew crystals of this mutant and determined its structure at 2.9 Å resolution. Interestingly, this model shares the same conformation as the structure of E243G with the wild-type like conformation of M2-M3 loop and an unwound top turn of the M2 helix (Figure 9B). In the wildtype open-form of GLIC, the side chain of I201 contacts the hydrophobic residue F238 (F14'), L241 (L17') and V242 (V18') within the M2 helix of the same subunit (Figure 9D and9E). The new bulky residue in I201W occupies the cavity behind the M2 helix and hinders the movement of the upper part of the M2 helix.

It is noteworthy that the variant V242W also generates a nonfunctional receptor 22 , suggesting that the V242W substitution has a comparable role as I201W.

In summary, I201 is also a member of the hydrophobic network originating from the Y197-Y119 interactions that reach out to the M2-M3 loop (L246) of the same subunit (as well as E243, N239 and H235 of the same subunit). In this way, the two ramifications of the networks containing mutants stabilizing an agonist-binding but inactive form completely encircle the ECD-TMD interface of the pentamer (Figure 10A).

Discussion

There is a lot of interest in identifying the so-called "allosteric pathway(s)" in allosteric proteins in general and in pLGICs in particular 46 . This has recently been made possible by the availability of three-dimensional structures of couples of open and closed forms of the ion channel, especially in the case of GLIC 16 , GluCl 5 and GlyR 10 . Here we used GLIC as a model system to understand the gating transition at the molecular level, using a combined computational and experimental approach. Previous work has focused on the use of coarse-grained methods to predict transition pathways from the pair of structures and simplified models of the proteins based on an elastic network model -ENMalone 16 or in combination with experimental phi-values 47 . Molecular dynamics studies have also been used 48 and culminated recently with the generation of possible pathways for GLIC gating transition using massive MD simulations and the so-called strings method 49 . Other similar studies concerning ELIC 50 and GluCl 51 have also appeared. Obviously, for GLIC there is the additional difficulty to assign the protonation state of all Asp, Glu and His residues in the two end states. Presently, it is not known for certain which titratable residues get protonated concomitally with the conformational transition, because MD simulations do not allow the change of protonation state of titratable residues during a run.

It has been known for some time that several special zones at the interface of the ECD-TMD are crucial for the gating transition (β1-β2 Loop, the Pro-Loop, the M2-M3 Loop, see for instance the recent study by Bertozzi et al., 2016 19 ), but the exact role of all 36 titrable carboxylate residues in the drastic conformational change that occurs upon dropping the extracellular pH transition has up to now remained elusive. There is much interest in identifying the protonsensing residues responsible for the gating transition; recently, a systematic study was undertaken to identify this (or these) residue(s) through site-directed mutagenesis of all Asp or Glu residues and electrophysiology (Nemecz et al., submitted). However, these studies have an inherent limitation in that the apparent pH50 of the transition does not give a direct access to the dissociation constant between the two forms, unless one knows the pKas value of the pHsensing residue in both the inactive and the active forms 52 . There is also the problem of the existence of a third form, the so-called desensitized form, so that in theory it would be better to conduct these experiments on a mutant that does not desensitize at all. Also it is likely that there are several titrating residues participating in the driving the conformational change. According to Sazanevets and Warwicker 37 , a more sure sign of a pH-sensing residue is the amplitude of the transition when the charge of the pH-sensing residue is suppressed. Here we have used the FD/DH pKa predictions to reduce the list of the proton-sensing residues for further experimental investigation. In our study, we have performed a pH titration of a selected set of Asp/Glu residues of GLIC by means of sitedirected mutagenesis and FT-IR spectroscopy. We compared the titration curves of mutants selected by the FD-DH method against the wild-type receptor. From the deviation with the titration curve of wild-type, the pKa value of E35 was determined to be 5.9, which is significantly shifted from the model pKa value of Glutamic acid (pKa = 4.2-4.4). We could not determine directly the pKa values of H127, H235, H277 because the spectral change of histidine residues could not be clearly identified, but their role is clearly minor in the conformational transition associated with gating the channel. Considering the coincidence of pH range for both the conformational change (measured by electrophysiology, i.e. the opening of the pore) and the titration zone of some residues, one can say that the protonation of E35 has a clear relationship with the channel gating mechanisms.

The importance of Loop F and the bypass of the agonist binding site

E35 is located at the end of the β1-β2 loop in ECD of GLIC structure, and it is closely associated with Loop F of the next subunit through T158. We could show that labeling T158C with MMTS impairs channel opening.

Loop F has been shown to be responsible for the inhibition of some pLGICs by divalent ions, such as Ca ++ ions for nAChR, as well as by modulators such as chlorpromazine for ELIC 27,[53][54][55] . Thus, it is not surprising that it is involved in channel gating in GLIC. Also it is clear from the examination of the structure that any change to W160 in Loop F will affect the stability of the R192-D122-D32 salt bridge. Furthermore, compared with the resting state of GLIC, a marked backbone shift of Loop F is observed in the open state of GLIC, and the Cterminal half of Loop F moves closer to its neighboring subunit 16 .

In the eukaryotic pLGIC family, it has been shown that the gating equilibrium is governed by neurotransmitter binding under LoopC/Loop B region and this was also demonstrated by extensive mutational studies in loop C region 14 . The key proton sensor E35 found in this study is not close to Loop C and Loop B; it could thus be said that proton-gating in GLIC bypasses the "classical" pathway down from Loop C, Loop B -> Loop E, D and G to Loop F across the subunit interface. However, the backbone of Loop C shifts markedly between the open and closed states, meaning that the proton-induced channel activation simultaneously induces the rearrangement of the orthosteric site.

On the Locally-closed form

The crystal structures of both apparently open state and the so-called LC state of GLIC are captured at pH 4, an environment with a high concentration of protons. Previously it was shown that mutation of single residues either on the M2-M3 loop or in M2 helices converts the open form into an LC form [17][18][19] .

Recently, it was shown that mutations further down in M2 helix (N239 and H235) also induce this conformational change (Fourati et al., submitted). Here we go further by producing mutants in the pre-M1 region that also stabilize the LC form (Q193M, Q193L and Y197F). The two forms can even co-exist in the same crystal at pH 4 with C-terminal of the receptor tagged with 10 histidines 16 . This is due to the fact that the energy barrier of these two conformations is small and this allows reversible interconversion between them 16 . This unique property enables us to map the important residues located on the activation pathway that upon mutation breaks the balance of these two states. The LC forms have a closed channel pore but co-exist at pH 4, where proton binding is not adequate for the activation of the channel, or where the coupling between the ECD and TMD transition is shut down. For instance, in the LC2 conformation (E243G and E243C-I201W mutations in this study), the deformation of the end turn of M2 helices stabilizes the closed pore. In the LC1 conformation (Q193M/L, Q193C+MMTS and Y197F mutations in this study), the mutation in the pre-M1 region presumably impairs the coupling of ECD and TMD. In We see that H235 is not protonated at pH 4.5, but that H127 should be protonated and (maybe) H277 as well. There is very little variation of predicted pKas in both forms for all three Histidines.

Table&1:&Summaries!!of!electrophysiological&values&&

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! NA!represents!that!data!is!not!available.!NF!
Here are the results also for Tyrosines We see that Y129, Y197, Y221 and Y263 have a marked difference of predicted pKas in the two forms. On the structural level, Y129 is stacked on H127 and Y221 is stacked on H277 (in the the TMD) while Y263 interacts with H235. Y197 is important for the gating transition. 1400 cm -1 was chosen for the analysis of protonation state of the carboxyl groups in Glu or Asp groups. Peak height differences between 1400 cm -1 and 1417 cm -1 , where the former represents peak position and the latter represents the bottom edge of the peak as a baseline, were taken at every measured pHs from each data sets as representations of the relative contents of deprotonated carboxyl groups. The peak height differences without any normalization process are shown in the Figure S3 (A). Because of differences in the experimental conditions, the intensities were varied from -0.005 to -0.011 (~ 45 %) at pH = 2 for different samples. For normalization of these varied intensities, we introduced following assumptions. First, we assumed that all carboxyl groups in the proteins are protonated at pH = 2. Although the curves in Figure S3 (A) does not saturate at pH = 2, which is partially due to instability of the lipid layer against the acidic solution environment, we took this assumption that major carboxyl groups that we are focusing were all protonated, so that the normalized factor of wild-type at this pH was set to one. Then, the second assumption is that the normalized factor at pH = 2 for those mutants that replaced Glu or Asp to other non-carboxylic amino acid was set to 0.97. A wild-type GLIC monomer contains a total of 35 carboxyl groups contributing from 19 of Asp and 16 of Glu.

Res

Therefore, the carboxyl intensities of the mutants should be (35-1)/35 = 0.97 because their number of the carboxyl group are one less than those of wild-type.

This assumption is based on the assumption that there are not many differences among the absorption coefficients of each carboxyl groups. The plot of the normalized carboxyl band intensities vs pH is shown in Figure S3 (B).

Comparison of the pH titration curves for each mutant was made on the basis of these normalized curves.

Materials and method

pKa calculation

The original Fortran code from J. Warwicker 57 was adapted by one of us (PK), to allow it to handle larger protein assemblies such as GLIC full pentamer.

Default values of the parameters were adopted: the ionic strength is 0.15 M NaCl and the dielectric constant is taken as 4 inside the protein and 78 in the bulk solvent. No special treatment was used to model the membrane, as it was shown for two membrane proteins to have little effect on the results 37 . The reported pKa values are the mean values averaged over the five monomers.

Protein expression and purification.

Purification of GLIC mutants followed the same procedure as previously reported 13 . Plasmids containing N-terminal GLIC gene fused with MBP gene were transformed into E. coli C43 cell lines. The cells were cultured in 2YT medium in presence of 100 mg/ml Ampicillin and were incubated at 37°. 0.1-0.2 mM of IPTG was added into the medium when the OD reached to 0.8-1.0, followed by a decrease of the incubation temperature to 20°. GLIC was extracted from E. coli membrane with buffer A containing (300 mM NaCl, 20 mM Tris pH 7.6) with 2% DDM. Solubilized proteins were loaded onto amylose binding resin, which was pre-equilibrium with buffer A, and incubated around 30-40 minutes. Amylose binding resin was washed with buffer A containing 0.1% DDM and then washed with buffer A containing 0.02% DDM. The MBP tag was cut off by incubating recombinant MBP-GLIC bound resin with thrombin enzyme overnight. The elution containing GLIC was further purified by size exclusion chromatography (Superose 6 10/300) with buffer A containing 0.02% DDM. The fraction with the peak corresponding to GLIC pentamer was collected and concentrated to 8-10 mg/ml and the protein was flash frozen using liquid nitrogen and then stored at -80° for further use.

Reconstitution of GLIC in Lipid bilayer

0.2 mg of detergent solubilized GLIC was added in 100 µl buffer solution (150 mM NaCl, 20 mM Tris, 0.02 % DDM, pH=7.6) containing 0.5 mg POPE/POPG (5/2 weight ratio) mixture, and stored at 4°C for 3 hours. The molar ratio between GLIC:Lipid was around 1:120 in this concentration. 10 mg of biobeads were added to the solution and incubated overnight. Subsequently, biobeads were removed and the solution was centrifuged twice at 15000 rpm for 15 minutes. After the supernatant was removed, the sediment was resuspended in 100 µl of 20 mM Tris-HCl buffer with pH=7.6. The final concentration of the reconstituted GLIC was around 6 mg/ml. The prepared samples were immediately used for FTIR measurement in order to avoid degradation.

Protein crystallization and structure determination.

All of the crystals were grown with the hanging drop evaporation method at 18°. 1 μl protein was mixed with 1 μl buffer containing 12%-14% PEG 4000, 400 mM NaSCN, 15% glycerol, 3% DMSO and 0.1M NaAcetate pH 4 against 1 ml of mother liquor. In order to improve the quality and reproducibility of crystals, the micro-seeding procedure was performed after setting up crystallization. The crystals were flash frozen using liquid nitrogen directly. The diffraction images were collected either at ESRF (European Synchrotron Radiation Facility, beamlines ID30, ID29 and ID23) or Synchrotron-Soleil (beamlines PX1 and PX2).

The data sets were indexed and analyzed by software xdsme 58 and further processed by CCP4 programs. The phase problem was solved by molecular replacement using Molrep 59 . Further refinement of the models was performed using BUSTER refinement 60 alternated with manual building in COOT 61 . The quality of the final structure models was checked by Molprobity webserver 62 .

The structural pictures were prepared by Chimera 63 . HOLE software 64 was used to analyze the channel diameter.

CHAPTER V 5 Crystal structures of a pentameric ligand-gated ion channel decorated with a large N-terminal additional domain

This short report is about an exciting but not completely finished project. Some parts of the structural models of the additional domains are not completely built, whereas both the LBD and TMD domains are well defined. Here I will mainly focus on analyzing the structures of the LBD and TMD domains in the two forms (open and closed).

Summary

Pentameric Ligand-gated ion channels (pLGICs) mediate rapid chemoelectrical transduction between neurons through binding neurotransmitters.

pLGICs also exist in bacteria and archaea and display a great diversity in their domain architectures. We present here the crystal structures of DeCLIC, a pLGIC form proteobacteria Desulfofustis, in both its closed and open conformations.

Introduction

Pentameric ligand-gated ion channels play a central role in synaptic transmission in the central and peripheral nervous systems 1,2 . They are key targets for a great variety of therapeutic regents that are widely clinically used, and for allosteric modulators 3 . They have been therefore the subject of intensive functional and structural studies since the first member of nicotinic acetylcholine receptor (nAChR) was isolated 4 . Orthologs of the eukaryotic pLGICs have also been identified from bacterial and archaeal species 5,6 . Structural data derived from several invertebrate and bacterial pentameric channels show a remarkable conservation of the compact core modules embodying the extracellular ligand binding domain (LBD) and the transmembrane pore-forming domain (TMD) [7][8][9][10][11][12][13][14][15][16][17][18] .

Whereas most bacterial pLGICs lack the large flexible intracellular domain (ICD) that specifically exists in eukaryotic pLGICs, many of them possess an N-terminal extension domain (NTD) 5,6 . Strikingly, other distinct animal neuronal receptors also contain a large NTD domain, such as the ionotropic tetrameric glutamate receptors that are functionally similar to eukaryotic pLGICs 19 . Molecular understanding of pLGICs decorated with the additional N-terminal extension domain has been hampered by the lack of structural information.

Recently, the crystal structure of a pLGIC from an endo-symbiont of Tevnia jerichonana (sTeLIC) has provided molecular insight into this family of receptors that display widely open pore conformations in their active state at high resolution. Through sequence database searches, we found several prokaryotic homologs with their LBD and TMD sharing high similarity in length and sequence to the sTeLIC (above 45% sequence identity), fused directly with a large N-terminal additional domain. Their NTD domain encompasses about 300 residues that fold into two subdomains (referred to as NTD1 and NTD2) with predicted β-strand-rich conformations. To glean insights regarding their molecular architecture and gating mechanism at the atomic level, we determined the x-ray structure of one of them, DeCLIC, a pLGIC from the proteobacteria Desulfofustis, in its two distinct conformations.

M1 and M3 appear to shield and stabilize the pore. The M4 helix is peripheral.

The M3-M4 region, which displays additional intracellular domains in eukaryotic pLGICs, is replaced by a short loop that is made of three residues as is seen in other prokaryotic receptors (Figure 1).

Ion channel pore

At the transmembrane domain level in the closed state, three physical sites of constrictions are observed at 16'Phe, 9'Leu and 2'Glu positions from the extra-cellular side to the cytoplasmic side. 16'Phe and 9'Leu are highly conserved residues that reside on the M2 helices. Inspection of the ion channel pore radius reveals that the pore is most constricted at the 16'Phe level with the hydrophobic side chain of phenylalanine restricting the pore radius to ~1. Overall, LBD and TMD rotate and bloom in opposite direction. To better quantify the twist, we analyzed the cumulated rotations of Z slices between the two states.

As illustrated in Figure 4, the ECD rotates counterclockwise reaching a maximum angle around +10° and the TMD rotates clockwise reaching a maximum angle of -27°. On the other hand, we found a unique behavior of upper part of the M2 helices, which display a concerted rotation in the same direction as LBD and in opposite direction to the rest of TMD (Figure 4). In order to better inspect the overall blooming, we performed the measurement of the distances of several pairs of identical residues placed in two opposing subunits. Strikingly, the LBD Ca 2+ binding sites shown here have also been identified as the modulation sites for other regulatory divalent ions, such as Ca 2+ in ELIC 22,23 .

CHAPTER VI

6 General discussion and perspectives

sTeLIC and ELIC define a new family with an unusually widely open pore

Quaternary-ammonium cations, such as TMA + and TEA + , can block nAChR and GLIC at a low millimolar concentration 1 . This sharply contrasts with the fact that neither of TMA + nor TEA + blocks ELIC and sTeLIC. It has been shown that TMA + and TEA + can pass through the ELIC channel 1 . Modeling TMA + or TEA + in the pore of sTeLIC at the level of its position observed in GLIC, we found that the pore is too wide to provide any direct interaction for the TMA + or TEA + molecule, thereby explaining the absence of an inhibitory effect for this kind of quaternaryammonium cations. sTeLIC and ELIC share higher sequence similarity than other pLGICs and both of them are from the family of gamma-proteobacteria. sTeLIC Deprotonation of these positively charged residues may be responsible for the activation of sTeLIC. However, this hypothesis is far from being established. In GLIC Loop C is deletable without affect its gating equilibrium by proton. Actually, we did find a residue in β1-β2 loop that is responsible for GLIC activation by proton (See below). So one could expect that other titratable residues in other locations may also responsible for sTeLIC activation at alkaline pH. Various invertebrate pLGICs were demonstrated to be directly responsive to basic pH, such as two GABAA receptors from insects 6,7 . Even though finding the hydroxyl group sensor residues is challenging, uncovering the hydroxyl group acting site(s) will be interesting in allosteric proteins and these kinds of ion channels.

sTeLIC and symbiont of Tevnia jerichonana

What is the functional role of sTeLIC for symbionts of Tevnia jerichonana?

The symbionts are enclosed in specialized host cells in an organ termed and human health is drawing great interest and has been under extensively investigation in recent years 11 . Is it possible that these gut bacteria communicate with the host through pLGICs beyond other already known mechanisms? So, much more research should be done to identify pLGICs in gut bacteria and study their role in communicating with the host nervous system.

The apparently open form of GLIC: is it really open?

The open form of GLIC just allows partially hydrated sodium ions to pass

Water pentagon at S6' level.

Actually, the open form of GLIC is indeed a conductive state, as shown by molecular dynamic simulation 12 . This open state is solved at pH around 4.0, a high proton concentration environment that normally energetically favors a desensitized conformation of the receptor. One of the possibilities is that other factors lead to the stabilization of the open conformation in the crystal state. For instance, the self-stabilized water pentagon located at the level of S6' position in the pore that is observed in the 2.4 Å higher-resolution structure 12 . We also note that one water pentagon has also been seen in the desensitized form of a human α2β4-AChR at the level of the S6' position 13 .

6.7 Detergent molecules in the channel pore.

Attentively, the presence of six detergent molecules in the channel pore suggest how GLIC might adopt an open conformation, in what could be described as a foot-in-the-door mechanism (detergent-in-the-pore). A loss of channel function was observed in a eukaryotic nAChR solubilized in DDM detergent and this supports the idea that crystal structures of membrane protein obtained in the presence of detergents can be artificial 14 . Detergents can affect the membrane structural dynamics and this lead to ambiguity for the structural conformation 15 . Further experiments should be done to address the effect of DDM detergent on GLIC.

The major proton-sensing site in GLIC and the stabilization interfacial networks

Protons are ubiquitous modulators of proteins, and major classes of ion channels are sensitive to the extracellular or intracellular pH, notably acidsensing ion channels (ASICs) and potassium KcsA 17 receptor, for which protons are the major endogenous activator. However, our understanding of the mechanisms underlying proton sensing is limited for most ion channels.

Addressing such an issue is indeed challenging since protons potentially act on a subset of the numerous titratable residues present in proteins to elicit conformational changes. Each monomer of GLIC contains 19 Asp, 16 Glu and 3

His. We systematically mutated Asp/Glu residues into Asn/Gln, replacing the carboxylate moiety by a non-titratable amide group, tentatively mimicking a permanently protonated form, as well as by Ala, removing the titratable moiety.

We found that E35 is the main proton sensor. How does protonated E35 stabilizes the channel in its open form? Many studies have pinpointed that Loop F undergoes a dramatic conformational change during channel activation. In our study, we show that protonated E35 is stabilized by residue T158 in Loop F.

Then, the hydrogen bond network mediated by water molecules between the subunits at the ECD-TMD interface and the hydrophobic network within the subunits contribute together to maintain the open channel conformation. Most of the human pLGICs are activated by binding a neurotransmitter to a wellcharacterized cavity at the ECD-ECD domain interface. Thus, the question is whether the stabilization mechanism we proposed here can be generalized to the other pLGICs. These ECD-TMD loop regions contribute to both the lifetime of the open-state and the kinetics of desensitization 18 . The functional consequences of mutational perturbations in these loop regions have been extensively investigated 15,19,20 . However, investigations at the structural level are still lacking in human pLGICs. We believe that the mechanism involving the hydrophilic network mediated by the polar or charged residues at the pre-M1 region (equivalent position is Q193 in GLIC), and the hydrophobic network within the subunit underlying at the structural level then could be generalized to other pLGICs.

The shut gate in the LBD

We have determined the crystal structures of DeCLIC in two different

I

  still remember the time that Jérôme helped me to prepare the department presentation. I am really glad to work with you. Your positive spirit affects me a lot. Thanks to Sandrine, Irina, Pierre, Phillipo, for your company and selfless support, for the wonderful time in the lab. Thanks to members of Channel Receptors unit, Pierre-Jean, Ákos, Anaïs, Marc, Solène, for their significant contributions to my work, for the very open and friendly discussions. Thanks to Benoit, for your great help for my thesis writing. Always see the smile on your face. Thanks to the Chinese community at Pasteur … to Ailing, Shuai, Jia for the wonderful trip of Camino de Santiago. Thanks to Hualin, Kuiying, Bin, Xiaohan, Jun, Chuande, Longfei for the great time in Paris. Thanks to my parents and Miss haiyue, for their endless love and support. Thanks for the three years in Pasteur, in Paris.

Figure 2 .

 2 Figure 2. Family of ionophoric proteins.

Figure 4 .

 4 Figure 4. Structural organization of voltage-gated sodium channels (Picture adapted from ref. 11).

Figure 5 .

 5 Figure 5. Tetrameric ligand-gated ion channels including NMDAR, AMPAR and KAR (figure adapted from ref. 18). The general domain organization of receptors from this family is comprised of an amino-terminal domain (ATD), a ligand binding domain (LBD) and a transmembrane domain (TMD).

Figure 6 .

 6 Figure 6. A phylogenetic tree based on protein sequence alignment containing the putative pLGICs (Pictures are adapted from ref. 27). Bacterial branches and animal branches are colored blue and magenta, respectively. Notably, putative pLGICs from bacteria, besides the core ligand-binding domain and transmembrane domain, are either directly decorated with an additional N-terminal domain or share the same operon with some periplasmic proteins.

Figure 8 .

 8 Figure 8. Ribbon diagrams of the first whole heteropentamer nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata constructed from the electron microscopy images (EM) and refined at 4 Å resolution. Picture adapted from32.

Figure 15 .

 15 Figure 15. GLIC open form and LC form coexist at pH 4 (Picture adapted from ref. 59). Energy landscapes inferred from the pH 7 crystal form and the pH 4 form is described in C.

  volvulus. By targeting the GluCl, ivermectin can activate GluClα at nanomolar concentrations. The binding site of ivermectin in GluCl is located at an intersubunit site in the TMD. Additionally, GluCl-Fab has been co-crystallized with L-glutamate in the presence of ivermectin. The GluCl is activated by glutamate only after activation by ivermectin. This suggests that binding to ivermectin may cause a global conformational change of GluCl that will enlarge the conventional agonist-binding cavity (beneath the Loop C) that can then accommodate glutamate. The open channel pore conformation of GluCl resembles the open form of GLIC with a minimum diameter around 4.6 Å and the constriction part at P-2' position. This suggests an open conformation that allows dehydrated chloride ions to pass through. Picrotoxin, an

  . All of these three states are solved in the presence of ligands. Strychnine blocks the receptor in a non-conductive state representing the antagonist-bound closed state with the constriction gate located at L9' (the same position as we observed in the co-crystal structure of α3GlyR-strychnine). The glycine-bound receptor adopts a largely open pore conformation with the minimum diameter at the P-2' position that is large enough for a hydrated chloride ion to pass through. This conformation may indicate an agonist-bound open state. Finally, the glycine and ivermectin together block the receptor in a partially open state with a minimum diameter at the P-2 position around 5 Å, which is smaller than the size of a fully hydrated chloride ion. However, this partially open conformation can allow the partially dehydrated chloride ions to pass through. Importantly, the middle gate of the channel found in the antagonist-bound state is open in the agonist-bound state, whereas the lower gate (P-2') is further narrowed in the agonist/potentiator (glycine/ivermectin) bound partially open state.

  . To date, little is known about the molecular architecture of pLGICs decorated with auxiliary domains. In chapter V, I have cloned, purified, crystallized and determined the crystal structures of DeCLIC, a pLGIC from proteobacterium Desulfofustis, both at closed and open conformations. Each subunit contains a large N-terminal domain constituted of two subdomains (NTD1 and NTD2). The closed state obtained in 200mM concentration of Ca 2+ delimits a closed pore with a 1.0 Å radius hydrophobic constriction ring that occlude the ion conductive pathway. On the contrary, the open state possesses a conductive aqueous pore with no obstruction at the membrane domain level. The NTD1 and NTD2 form mobile regulatory domains by establishing extensive interactions with the LBD domain. Despite the absence of electrophysiology characterization, this pairs of DeCLIC structures, with N-terminal extension domain, provides the first structural framework for understanding the gating mechanism of pLGICs modulated by auxiliary domains. In chapter VI, I will discuss the findings and implications of my work. A better understanding of pLGICs based on the studies of different pLGICs from different bacterial systems further allow us to elucidate the gating mechanism of pLGIC superfamily. This will provide novel insight to design novel modulators that could be used either against pathogenic bacteria or human diseases. pH defines a new family with an unusually widely open pore This chapter is in submission in the Journal of P.N.A.S. by Haidai Hu, Ákos Nemecz, Zaineb Fourati, Catherine Van Renterghem, Ludovic Sauguet, Pierre-Jean Corringer and Marc Delarue In this chapter, I did the molecular biology experiments. Purified and crystallized the protein. Optimized the crystals and solved the structures. I analyzed the structures and wrote the initial manuscript together with Marc. The electrophysiology experiments were done in Pierre-Jean Corringer's Lab mainly by Ákos Nemecz, Catherine Van Renterghem.

Figure 4 .C

 4 Figure 4. Modulation of sTeLIC response to pH by 4-BrC.

Figure 6 .

 6 Figure 6.Cationic selectivity of sTeLIC. A-C. Ion selectivity was evaluated measuring the reversal potential of the sTeLIC current using voltage ramps, with an oocyte two-electrode voltage-clamp setup. A. Current-to-voltage plot of typical 100 µM 4-BrC elicited currents in 10 mM HEPES/NMDG, pH7.5, with 1mM MgCl2 solutions containing either 100 mM NaCl, 100 mM NaGluconate, or 100 mM CholineCl. For each solution, the current recorded in the absence of compound was subtracted from the current recorded in the presence of 4-BrC on the same oocyte. The measured reversal potentials (mean from three separate oocytes ± standard deviation), indicated by the dotted lines, were: 10±1 mV (NaCl), 21±4 mV (NaGluconate), and -41±3 mV (CholineCl). B. Current-to-voltage plot of typical pH 8, from pH 5, elicited current, in 10mM HEPES/NMDG, with 1mM MgCl2 solution, simultaneously and equally diminishing the extracellular concentrations of Na + and Cl -(denoted [NaCl]) counterbalanced with sorbitol. For each [NaCl] value, currents recorded with the solution at pH 5 were subtracted from currents recorded at pH 8 on the same oocyte. C. Plot of the shift in reversal potential (Erev-Erevref), measured in experiments as shown in B, as a function of the ratio [NaCl]/[NaCl]ref. Erevref denotes the reversal potential measured with the reference concentration, 100mM NaCl ([NaCl]ref). Data, given as mean ± standard deviation (error bar is smaller than the symbol), are from three oocytes. The theoretical values of the shift in Erev for anionic and cationic channels, assuming perfect selectivity for Cl -over Na + (PNa/PCl=0), or for Na + over Cl -(PCl/PNa=0) are also shown. Experiments shown in A-C indicate that sTeLIC is cationic.

Fig. S4B )

 S4B Fig. S4B). Loop C in sTeLIC displays no cavity and is in close contact with the complementary subunit, as expected in an active form. It is still unknown what is the effect of pH at the level of the agonist site, although the presence of several histidines around Loop C may be an indication of a possible mechanism for pH-sensing (Suppl.

Fig. S4A )

 S4A Fig. S4A), perhaps through a redistribution of electrostatic energies and/or dielectric relaxation 50 .

  Fig. S10L), while it is more open than in GLIC structure at acidic pH. It has indeed been suggested from EPR experiments 52 that this GLIC structure may be only semi-open, in which case one may wonder what is the place of this conformation in the kinetic pathway 53 . The interpretation suggested in this view is that GLIC's crystal open form may represent an intermediate along the reaction path from closed to open forms, trapped because of crystallization conditions. Considering the updated and refined kinetic pathway proposed by Auerbach and colleagues 53 , it would be tempting to assign GLIC's open state to the one just before the final active state (wet channel),

Figure

  Figure S6 Common ECD constriction rings at the level of Loop Ω in various pLGICs.

Figure S11 .

 S11 Figure S11. Possible lateral pathways for cation penetration in the lumen.

Figure S12 .

 S12 Figure S12. Sequence alignment of sTeLIC with a representative set of prokaryotic pLGICs.

Figure S13 .

 S13 Figure S13. Sequence alignment of sTeLIC with eukaryotic pLGICs of known structures.

  as the search model by Phaser-MR in Phenix58 . Before molecular replacement, the PDB file of GLIC was modified by the program Chainsaw in CCP4, keeping the side chains atoms if the residues are the same, otherwise truncating the side-chain of the model to the C-beta atom. The result of the molecular replacement gave Log-Likelihood-Gain and top translation-function Z-score values of 282 and 10.3 respectively. The refinement was performed using Buster 59 , alternating cycles of refinement and manual building in Coot60 . During the refinement, auto non-crystallographic symmetry (NCS) restraints were applied. We also used the fivefold averaged NCS map to trace the chains during model building. There are 25 methionines in the sTeLIC pentamer (five in each monomer, omitting the first one), therefore we collected one diffraction data set at the sulfur edge (1.7 Å). Taking advantage of the anomalous signal coming from sulfur, we confirmed the correct assignment of the protein sequence in the model. The best data set of sTeLIC was crystallized with NDG and processed at 2.3 Å resolution. Clear electron-density coming from NDG can be seen on the surface of the ion channel pore in the TMD. One NDG molecule was assigned for each monomer with the sugar head facing the ion channel pore and its curly alkyl tail inserted into the hydrophobic cleft formed by two adjacent subunits. The final model contains residues ranging from Glu7 to Phe316, missing only six N-terminal and four C-terminal residues. 662 water molecules were built into the model. Model quality was assessed in Coot60 and by Molprobity 61 . 97.66% residues fall into the preferred regions of the Ramachandran plot and the rest (2.34%) fall into the allowed regions.

  Biologiques-Rennes, France. Defolliculated oocytes were maintained at 4°C in a modified Barth's saline solution (88 mM NaCl, 1 mM KCl, 0.8 mM MgSO4, 2.4 mM NaHCO3, 0.3 mM NaNO3, 15 mM HEPES/Na pH 8) with 0.7 mM CaCl2. After intranucleus injection of ~30 nL cDNA (80 ng/µl specified clone cDNA with 20 ng/µl of GFP cDNA), using a compressed air microinjection system, the oocytes were transferred to 18°C. 1-2d later they were evaluated for GFP expression, and subsequently maintained at 15°C. Recordings were made 1-5d after injection using low-resistance (0.2-2 MΩ) electrodes filled with 3 M KCl, and clamping the voltage to -40mV unless otherwise specified. The standard solution superfusing the oocyte during recording at room temperature was 100 mM NaCl, 3 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES pH 7.5 using NaOH. Any subsequent reference to standard-solution uses this formulation.

( 3

 3 Bio-Logic, France) with an HK-R-410/08 dual resistor headstage, connected to a computer using a Digidata 1550 digitizer interface and the program pClamp 10 (Axon Instrument). Currents were low-pass filtered at 1 kHz, and digitized at a sampling frequency of 10 kHz. Digital filtering by 40 to 1 points averaging was further applied to display traces in the Figures. Pipettes were pulled from thick wall (0.37 mm) borosilicate glass (1.5 mm o.d., 0.75 mm i.d.), and fire-polished to resistances of 2-4 MΩ in the solutions used. The pipette solution (intracellular face of the membrane) was composed of: 155 mM NaCl, 1 mM MgCl2, 10 mM BAPTA and 10 mM HEPES adjusted to pH7.5 with NaOH The solution superfused (extracellular face) was composed of: 170 mM NaCl, 1 mM MgCl2, and 10 mM HEPES, adjusted to pH 8 withNaOH, followed by HCl (2M) to reach other pH values. The culture dish was rinsed and filled with this solution supplemented with 1 mM CaCl2 (pH 7.5). Before any sTeLIC current recording, a 20-s pretreatment at pHo 5, was applied, ending 10-s before sTeLIC stimulation, in order to reverse desensitization/inactivation. Solutions were applied locally over the cell, using a gravity driven, multiway perfusion system converging to a single tip (50-100 µL.min-1). A 3 M KCl, 5 g.L-1 agar bridge was used to isolate the reference electrode from changes in the extracellular solution.For all voltage-clamp experiments, electric current flowing inward through the cell membrane is counted negative and represented downwards.CHAPTER III Full mutational mapping of titratable residues helps to identify protonsensors involved in the control of channel gating in the Gloeobacter violaceus pentameric ligand-gated ion channel This chapter is in submission in the Journal of Plos Biology by Ákos Nemecz, Haidai Hu, Zaineb Fourati, Catherine Van Renterghem, Marc Delarue, Pierre-Jean Corringer.

Fig 3 3 . 3 . 3

 3333 Fig 3: Loop C residues modulate activation through a complex network of interactions. Stick representation of the labeled Arg, Asp, Glu, and Lys residues evaluated, color coded based upon effect, with Asp/Glu pink residues producing a strong decrease in pH50 when mutated in combination, and yellow residues a non-significant/weak effect. R179 is shown in teal. The main subunit is shown in black with ribbon and cartoon representation whereas the complementary neighboring subunit is represented in white ribbon, with important motifs/βsheets labeled. Distances of intrasubunit contacts are shown between D136 and R179 as well as R133 and E181. Table of the pH50 and ΔpH50 are shown below with mean values ± standard deviation for evaluated mutants. Light red and green zones with dashed lines represent the respective ½ log decreases and increases from Wt.Values outside this region are color coded for ease of interpretation with red as decreases and green and dark green as varying degrees of increases.

  , H127N and Q both produce Wt-like currents (Fig 4). A structure was resolved for H127N, which resulted in no appreciable difference from the open form of GLIC (4HFI/3EAM) (Suppl. Fig 1).

, Fig 5 ).

 5 D31, which points towards the vestibule, has no effect when mutated to D31N in this study, whereas a slight loss of function, insignificant with the criterion and to the Wt value of this study, was observed with both Ala and Asn mutations by Alqazzaz et. al. [36]. Mutants of the solvent exposed D115, further away from this triad, on Loop 7, exhibit Wt-like properties, yet the D115N and D115A mutations show opposite phenotypes, with Asn having a tendency to increase the pH50 and Ala to decrease it. Of the mutants which had both Asn/Gln and Ala mutations tested only the pair of E82Q and E82A follows the same pattern (Fig 4B).

  the 13 recorded oocytes (over four injections), only two recordings could be fit properly (Fig 1B&C, Fig 4B, Supp Table

structure at pH 4 .

 4 All were in the apparently open conformation. Each residue's RMSD and Cα RMSD was evaluated in relation to the intrinsic variability between subunits and across the two Wt structures (3EAM and 4HFI) at pH 4.6 and pH 4 respectively, as a means to control for crystal variability. There are no significant differences, measured as five-fold over Wt, seen in the backbone Cα residues of any of the resolved structures, with the exception of E35A and E82Q (Fig 6, Supp Fig 1). The E82Q sidechain takes on a different rotamer causing a local change in the backbone, as well as a

Fig 5 )

 5 , indicating a structural importance of the residue, and secondly, with the mutation H235Q, which does actually produce functional receptors, albeit with a strong decrease in pH50 and reduced maximal currents (Fig 5, Supp Table

Fig 7

 7 Fig 7: TMD residues decrease sensitivity. Stick representation of the labeled Glu and His residues evaluated. Glu residues are color coded based upon effect, with residues in red producing a significant decrease in pH50, and in yellow, an insignificant/weak effect, whereas the dark purple and magenta for His residues are synonymous to the red of Glu residues. The main subunit is shown in black with ribbon and cartoon representation with the αhelices labeled, whereas neighboring subunits are represented in white ribbon. The M2 α-helix of neighboring subunit (labeled M2') is also highlighted in cyan and in cartoon representation. A table of the pH50 and ΔpH50 is shown below with mean values ± standard deviation for evaluated mutants. Light red and green zones with dashed lines represent the respective ½log decrease and increase from Wt. Values outside this region are color coded for ease

  one can divide residues whose pKas are shifted from model pKas values in two classes: pH-sensors and pH-coupled. The pH-sensors are expected to change ionization during the conformational change between the two forms, namely around pH50 +/-1 37 . GLIC x-ray structures have been solved in both its open/active form (pH 4.0, PDB ID: 3EAM, 4HFI) and in its closed/resting form (pH 7.0, PDB ID: 4PQN). In principle, Poisson-Boltzmann (PB) electrostatic calculations allow to predict individual pKas of a protein with known structure. Finite Difference Poisson-Boltzmann/Debye-Hückel (FD/DH) calculation is a refined Poisson-Boltzmann method, that has been shown to be significantly better at predicting pH-sensing residues compared to normal FD-PB methods. It takes into account the flexibility of charged residues at the surface and uses the Debye-Huckel theory for those charged side-chains exposed to the solvent and screening by salt 37 .

Figure

  Figure 1A shows the localization in the three-dimensional structure of those Asp and Glu residues predicted by FD-DH to have significantly different pKas in GLIC both open and closed states, as shown in Figure 1B, while Figure 1C emphasizes their differences. The residues lying on the diagonal have the same predicted pKas in both states, suggesting that they are not involved in pH sensing. E26, E35, E75, E104 are predicted to change protonation state around or above the pH50 determined by electrophysiology and also have a predicted pKa that differs in the two forms (Figure 1B); they represent potential pHsensing residues. Also, the pKas of D32, D122 and E181 differ substantially

Figure 2 .

 2 Figure 2. The pH-induced different FTIR spectra of GLIC reconstituted in POPE/POPG bilayer for (A) Wild-type and (B) E35A mutant. Reference spectra were taken at pH = 7.0 for both. The solution pH was then successively lowered up to below pH ~2.0. Negative peaks represent the structural components that were reduced from pH = 7, while positive peaks represent the structural components that were gained by lowering the pH. (C) The pH titration curves derived from the normalized intensities of carboxylate band at 1400 cm-1. Wild-type (+), E35A (red). Open and filled markers represent data sets from different experiments.

Figure 3 .

 3 Figure 3. The trace deviation of the pH titration curve of mutants from wild-type GLIC. The color codes are used to represent•: E26A, •: E35A, •: E75A, ○: E104A, •: E181A The cross marker (+) represents trace deviation of wild-type between two different experiments, which sets the extent of the reproducibility error. The solid curves represent results of fitting the data points by the Henderson-Hasselbalch equation for E35A, E75A, E181A.

  Mutants H => A were difficult/impossible to express and the only successful ones were H => Q or N. The structures of these mutants have been probed elsewhere: H127Q is open, H235Q adopts the LC-form and H277Q is open. Their phenotypes measured by electrophysiology showed a slight loss-of-function phenotype for H127Q, a severe loss-of-function for H235Q and a wild-type phenotype for H277Q 43 (Fourati et al., submitted and Nemecz et al., submitted). Here we also studied them by FT-IR. Detection of specific FT-IR bands from His during pH titration remains challenging because of their relatively low extinction coefficient compared to the bands of carboxyl groups, and this extinction coefficient overlaps

Figure 7 .

 7 Figure 7. FD-DH predicted pKas for all His and Tyr residues. (A) Location in the threedimensional structure of His and Tyr residues predicted to have strong differences in their pKas in both forms. (B) Plots of the DpKa as a function of residue number. (C) FT-IR titration at 1400 cm-1 of all three His mutants. (D) Difference spectra for all three His mutants.

  8 Å adopts an open conformation (Figure 8E left panel).Surprisingly, unexplained electron density appeared in the Fourier mFo-DFc difference map (average peak height around 7.5σ) and it can be well modeled by one detergent molecule (DDM) in each of the subunit. DDM, a detergent that we used during the purification and crystallization of the receptors, is positioned in such a way that its sugar moiety is exposed to the ECD lumen and its hydrophobic tail inserts into the cavity vacated by the removal of phenylalanine side chain (Figure8Eright panel). Interestingly, this cavity largely overlaps with the cavity that has been shown to contribute to lipid-solubilized propofol and desflurane binding22 . Therefore, we propose that in this case the hydrophobic tail of DDM fulfills the role of the aromatic ring of Y119 to artificially maintain the channel open.Finally, for the sake of completeness, we generated the Y263F mutant because it is the tyrosine position predicted to have one of the largest ΔpKa (ΔpKa=-3.0), as shown in Figure 7B. It turns out to have a wild-type pH50 and an open conformation in the crystal, implying that it has no role in the gating transition (Figure 8F).

4. 3 . 6

 36 Other mutations that block the channel in a locally-closed conformation a) Connection to the entrance of channel pore (E243) and link with the N239-H235 cavity implicated in General Anesthetics (GA) bindingOne of the main predictions of the FD-DH calculation is the existence of a triplet of strong electrostatic interactions involving Y119-Y197 and K248(+) that extends the R192-D122-D32 well known triplet. Y197 can actually interact with two different M2-M3 loops, one from the adjacent subunit (through L246 and N245) via the Pro-Loop and one from the same subunit (through K248 and E243). E243 marks the entrance of the channel at the end turn of M2 helix.Strikingly, its side chain can adopt two different conformations in the open form of GLIC31 . Previous studies showed that replacement of E243 with proline abolished the proton-elicited currents17 . The structure E234P was solved and showed a locally-closed (LC2) conformation. This structure shares the same M2-M3 loop conformation as the wild-type open-form, except that the top turn of the M2 helix is unwound and that the upper part of the M2 helix is detached from the M1 and M3 helices bundle. It thus looked as if this position is important for gating. However, electrophysiology recordings show that E243C has almost same pH50 value as the wild type20 . In order to gain structural insight on the importance of this position, diffracting quality crystals of E243C were grown in the same conditions as the wild-type GLIC at pH 4 and their diffraction was collected. Consistent with electrophysiology results, there is no distinguishable difference between the conformation of E243C and the open form of GLIC (RSMD = 0.215 Å) (Figure9A). To further probe the site of E243, we substituted E243 by a glycine, a more flexible amino acid with no restriction on the main-chain atoms imposed by the side-chain. Again this substitution results in a nonfunctional channel same as E243P (Figure9C). The E243G structure was solved at 3.25 Å resolution and shows the same "LC2" conformation as E243P with the top turn M2 helix unwound and the RSMD value 0.31 Å. Collectively, our data suggest that E243 is probably not a key residue for proton binding; rather, it is crucial for maintaining the stability of the upper part of M2 helix during the signal transduction. Strikingly, if one goes down the M2 helix starting at E243 by steps of 4 residues, one finds N239 and H235 that all have been implicated in GA binding and whose mutation ends up in the LC form in the crystal(Fourati et al., submitted). In conclusion, the connection Y197-K248-E243-N239-H235 further extends the network that originates from E35 and Q193, deeply into the TMD from one subunit as well as to the TMD of the adjacent subunit. b) Extending the hydrophobic network to I201 and to the TMD intra-subunit cavity previously shown to bind general anesthetics (GAs)

Figure 9 .

 9 Figure 9. X-ray structure of GLIG mutants at positions E243 and I201. (A)-(C) Ribbon representation TMD of E243C, I210W-E243C and E243G mutant structures. M2 helices and M2-M3 loops are colored in green, magenta, blue, respectively. (D)-(E) Superimposition of the structures of E243C (open form), E243G and I210W-E243C (closed form) showing M2 helix, M2-M3 loop and M3 helix as a side-view (D) or viewed from the top (E). The side chain of W201 is shown as sticks (magenta) in the structure of I201W-E243C and the side chains of F238, L241, V242 are shown as sticks (green) in the structure of E243C.

4 . 4 . 3 A 4 . 4 . 4 A

 443444 Figure 11A, we summarize a plausible model for the energy landscape of the three forms, open, LC and closed, at both pH 7 and pH 4. Clearly, mutations that are crucial for gating will affect the equilibrium between LC and open forms, and divert the transition from Closed -> Open to Closed to -> LC. We now describe in more molecular details a plausible scenario for the full gating transition, namely how does the protonation event of E35 propagate to stabilize an open form of the pore. Hydrophilic network across subunit helps open the gateThe interfacial hydrogen bond network mediated by water molecules identified in the ECD-TMD crevice at the level of Q193 can be found in all the equivalent five positions in the pentameric receptor and provides a plausible link from the proton sensing residue E35 to the ECD-TMD interface. In the open state, the pentagon-shaped hydrogen bond network is formed by the side chain of Q193, three water molecules, oxygen atom of carboxyl group of K248 (K24' in M2-M3 Loop) and Nitrogen atom of G159 from loop F. In the LC1 conformation, because of the M2-M3 helix moves towards the ion channel pore, the pyrrolidine ring of P250 occupies the position of the carboxyl group oxygen atom of K248 (K24'). This movement is further stabilized by a hydrogen bond between the oxygen atom of the carboxyl group of P250 and the nitrogen atom of F195 (pre-M1) of the neighboring subunit. The interfacial crevice in the LC1 form is smaller and much more hydrophobic and we observed no water molecule density in this crevice in the high resolution of a LC1 structure. Hence, we point out that the hydrophilic environment in this crevice is a determinant factor linking the proton binding to channel opening (Figure10Aleft panel). In addition, there is a clear electrostatic network of interactions, uncovered by FD-DH calculations, that extends further the well-known D122-R192-D32 network that was studied recently by mutagenesis in both ELIC and GLIC19 , namely the network of Y197 (pre-M1)-Y119 (Pro Loop)-K248 (M2-M3 Loop). Hydrophobic cluster within the same subunit also mediates gating How does the Pro-loop contribute for the channel activation? In the open state of GLIC, the interfacial hydrogen bond network connects together the loop F (T158, G159), pre-M1 region (Q193) and the neighboring M2-M3 loop (K248), as described above. Within one subunit, the conserved salt bridge D32-R192 is sandwiched by W160 and packed against the conserved F121-P120-Y119, thus linking loop F, pre-M1 region and the Pro-loop. In addition, L246 (L22') side chain of M2-M3 loop establishes a hydrophobic association with the aromatic ring of Y119 in the open state of GLIC. Replacing L22' with alanine generates a non-functional receptor16 . This inter-subunit hydrophobic association is almost parallel to the plasma membrane. Together with our data, this supports the idea that the coupling among the pre-M1 region, Pro-loop and M2-M3 loop is mediated by a hydrophobic cluster within each subunit that stabilizes the open form of the channel (Figure10Aright panel). Interestingly, the Y197F mutant adopts LC form in the crystal but shows a similar pH50 than the wild-type in electrophysiology. This contradiction may be explained by the different membrane environment (detergent vs membrane) of the two experimental measurements56 . The slight differences might be enough to explain how the

Figure 11 .Figure S2 .

 11S2 Figure 11. A model for the coupling of proton(s)-binding to channel gating during activation of GLIC. (A) Energy landscape inferred from crystal structures of resting GLIC, LCform, GLIC Open. (B-D) Sequential model for GLIC activation. The arrows represent the major changes in the structure during activation. The key proton-sensor residue (E35) is indicated in deep red color in LC form and open form. Residues shown to be experimentally important for channel activation are indicated. The two stabilization networks are shown in panel D with the hydrophilic network represented in green and the hydrophobic one in orange.

Figure S6 .

 S6 Figure S6. (A) and (B) shows the electron density quality of I201W-E234C. (A) 2mFo-DFc electron density map (blue) is calculated at 2.8 Å and controlled at the level of 1 σ and is shown superimposed on one of the subunits (magenta). (B) Electron density quality of the E234C-I201W mutant pre-M1 region. Electron density map was prepared the same as in E and residue of 201W was labeled.

4. 7 . 4

 74 pH titration ATR-FTIR measurement 10 µl of the lipid reconstituted GLIC solution was added on the surface of Diamond IRE. The buffer solvent of the sample solution was evaporated under an argon stream. Subsequently, the sample was re-hydrated with 8 ml of a buffer solution (150 mM NaCl, 20 mM HEPES, 20 mM MES, pH=8.3). Most of the rehydrated sample remains at the vicinity of the IRE surface. Followed by the rehydration reaches to an equilibrium (> 30 minutes), a reference spectrum was measured at pH = 8.3 with 512 spectral co-addition. For the investigation of the 'channel opening' process, 2 to 20 µl of 1 M HCl solution was added to the sample solution and stirred by pipetting. One addition of HCl solution typically dropped 0.2 to 0.4 pH unit and final pH was checked by pH meter directly mounted to the ATR optical cell (Figure 1). When the solution becomes stable, a sample spectrum was measured with 512 spectral co-addition. A set of sample spectra was measured with various pH values by repeating the former procedure until the pH value reached 2.0.

  Each subunit contains a large N-terminal domain that is constituted of two subdomains (NTD1 and NTD2). The closed conformation obtained at 200mM concentration of Ca 2+ shows a closed pore with a 1.0 Å radius hydrophobic constriction ring that occludes the ion conduction pathway. Conversely, the open conformation possesses a conduction aqueous pore with no obstruction. The NTD1 and NTD2 form mobile regulatory domains that establish tight interactions with the LBD. Unexpectedly, in the open state, the LBD harbors a shut gate, indicating that that local movement of the loop regions in the lumen can control the ions access to the pore. Despite the absence of electrophysiology characterization, this pair of DeCLIC structures, with the N-terminal additional domain, defines a structural framework for understanding gating mechanism of the pLGICs modulated by an auxiliary domain.

  0 Å, followed by a ~1.4 Å radius constriction ring at the 9'Leu level. Both of the hydrophobic constriction rings are too narrow for the conduction of a fully dehydrated sodium or potassium ion, thereby fully closing the channel. This indicates that they serve as the hydrophobic physical gates at the upper part of the M2 helices and prevent the diffusion of ions. Below the hydrophobic constriction rings, the third gate, contributed by five 2'Glu with their side chains extenting to the center of the pore axis, define a pore radius of ~1.8 Å.Intriguingly, the mFo-DFc and 2mFo-DFc maps show strong electron density peaks at the 2'Glu level and we speculatively modeled there a Na + ion, the most preponderant monovalent ion in the crystallization buffer. This sodium ion is coordinated by the five negatively charged glutamates located at the end of first turn of M2 helix, in agreement with what is seen in GLIC, where a Na + ion could be located at the same level20 . Thereby, this hydrophilic constriction ring near the cytoplasmic side could serve as another gate. In the open state of DeCLIC, because of the outward movement and twist of TMD, all of the three constriction gates observed in the closed state disappear: 16'Phe and 9'Leu rotate away from the five-fold axis, yielding a pore radius of ~5 Å at the upper part of the pore. At 2'Glu region, the ion channel pore expands to a radius around 6 Å. Therefore this open channel allows the permeation of fully hydrated sodium or potassium ions with an ionic radius estimated 4 Å and 3 Å, respectively (Figure2). The overall open channel architecture is similar to that observed in sTeLIC and the closed channel architecture is similar to that observed in ELIC10 , however, the details

Figure 2 :

 2 Figure 2: DeCLIC pores. A, Side view of the water-accessible region of the pore of the closed (left) and open forms (right). The two front subunits are removed for clarity. B, Transmembrane parts in DeCLIC two states viewed from the extracellular side. C, Side view of DeCLIC M2 helices. In the side view, only two opposing M2 helices are shown. Helix backbones and side chains facing the pore are depicted.

Figure 3 : 5 . 3 . 4

 3534 Figure 3: Tertiary changes at the LBD between the open and closed states. Superimposition of a single LBD subunit of DeCLIC open (violet red) and DeCLIC closed (Cyan) subunits. The LBD regions are shown in cartoon representation. The left structures are viewed from the outside of the pentamer and the right structures from the inside.

5 . 3 . 5

 535 (i) In the LBD. In the closed form, the distances between the two Cα atoms at the positions of Ala385 on α1 helices and the two Cα atoms at positions of Trp407 in Loop Ω are around 27.9 Å and 23.6 Å. In the open state, these distances shrink to 15.9 Å and 18.6 Å, respectively. The α1 helices that are parallel to the pore axis in the closed state have undergone counterclockwise rotation by changing their angle of orientation by an estimated 40° in the open state. (ii) At the cytoplasmic side of TMD, the distances of two pairs of Cα atoms of residues Asp541 and Gly604 in M1-M2 loop and M3-M4 loop are 23.5 Å and 35.5 Å in the closed form, which expand to 32.9 Å and 44.9 Å, respectively in the open one. As a consequence of the twist and blooming movement, the LBD-LBD interface area is increased by 183 Å 2 and the TMD-TMD interface is reduced dramatically by 832 Å 2 (Figure 5). Meanwhile, the LBD subunits move closer and the ion channel pore adopts a widely open configuration. It is evident that even when the widely open pore allows the passage of full hydrated sodium or potassium ions, the side chains of five Trp407 in the loop Ω within the LBD lumen occlude the ion channel (Figure 6). Local conformational change of Loop Ω regions can control ions access to the pore in the open state. Several studies have shown the key role of the LBD-TMD interface loops regions during the signal transduction from LBD to TMD. Besides the covalent Mapping the calcium binding sites in DeCLIC closed conformation. Examination of Fourier mFo-DFc difference maps derived from DeCLIC crystals grown in the presence of 200mM Ca 2+ reveals putative Calcium binding sites. To better visualize calcium locations in the closed form, we soaked the corresponding crystals into the mother liquor additionally containing 200mMBa2+ . The best diffraction data set was collected at 5 Å resolution at the absorption edge of barium. From the moderate-resolution isomorphous data set we can observe the strong anomalous signal coming from the barium, and therefore Ba 2+ ions can be readily located into the electron density to three distinct sets of sites in estimated five-symmetry related locations. Two sets of the Ba 2+ binding sites located at the interface of NTD1 and adjacent NTD2. The third set of sites locate at the LBD domain interface and are mainly coordinated by side-chain of acidic residues from both subunits. Those residues include Glu347 in β1-β2 loop and Glu437 at the end of β6 strand on the principle interface and Glu480 in loop F on the complementary interface. We observe no anomalous signal in the closed ion channel pore. It has been shown that Ca 2+ can inhibit members of pLGICs by occupying an extracellular site remote from the canonical ligand-binding cavity.

  and ELIC are blocked by divalent cations Ca 2+ , Zn 2+ as well as Ba 2+ ions at a micromolar concentration. Interestingly, mapping the Ba 2+ inhibitory sites in sTeLIC and ELIC give different results. In sTeLIC, we have mapped the Ba 2+ binding sites at the channel pore level. In ELIC, the mainly Ba 2+ inhibitory site locates near the Loop F region between the intersubunit interface. We propose that Ba 2+ can block the sTeLIC and ELIC via different binding sites and different mechanisms, especially considering the receptors can adopt different conformations. In the open conformation, Ba 2+ inhibit the agonist-induced current at the pore level. In the closed conformation, Ba 2+ inhibit the agonistinduced current by binding to the site near the Loop F. We also found a positive modulator 4-BrC, which can strongly potentiate the current of sTeLIC. In our study, we could locate the 4-BrC directly into the positive allosteric modulators (PAM) site, that is similar to the binding site of flurazepam (FZM) in ELIC2 . Based on the above analyses, we thus propose that ELIC and sTeLIC are representative members of a new subfamily of pLGICs, with specific characteristics for their vicinity of the Loop C, there is a cluster of positively charged residues. There is also a Histidine in Loop C and this Histidine is buried beneath the Loop C.

trophosome 8

 8 in the Tevina jerichonana, a tubeworm that colonizes the areas of post-eruptive basalt-hosted vents. The tubeworm is completely dependent on the symbionts for nutrition, since adult tubeworms lack a mouth and a digestive tract. The symbionts take up substrates such as oxygen, sulfide as well as carbon dioxide by diffusion from the flow fluids through the worm's plumes for chemosynthesis. The energy provided by sulfide oxidation of the symbionts is used to fix CO2 into organic matter. In this way, the enclosed symbionts provide nutrition to the worms. Interestingly, chemistry measurements of the plumes reveal that symbionts are exposed to a high sulfide concentration (~2 M) and quite low pH (5.4) environment9 . It should be noticed that symbionts of Tevnia jerichonana are highly similar with symbionts of Riftia pachyptila, which are exposed to neutral pH. sTeLIC and its homolog form symbiont of Riftia pachyptila share identical sequences. In our study, we show that sTeLIC starts to open at a pH above 7.5. It has been suggested that pLGICs from bacteria perform a role in chemotaxis10 , that guide the movement of the bacteria in response to a chemical stimulus. Since the symbionts are enclosed within the trophosome, their movements are limited. Is it possible that the symbionts of Tevnia jerichonana detect the environmental pH through sTeLIC and in turn give signal back to the host in order to maintain the pH constant or the concentration of some special substrates inside the host fluid. It has been shown that our gut microbiota contains trillions of microorganisms, and the relation between them

  through. Another study even suggests that this open state is a 'semi-open' conformation. Comparing to the channel conformation of sTeLIC, conclusion can be drawn that GLIC is not as widely open as sTeLIC, and then we may infer that this conformation of GLIC is only a partially open state. As discussed above, GLIC and sTeLIC may represent different subfamilies of pLGICs, their distinct behavior is experimentally demonstrated in the quaternary-ammonium cations trials. So a direct comparison of GLIC and sTeLIC should be handled with great care.

  conformations. One conformation displays a closed channel pore that is similar to ELIC, and another conformation displays a widely open channel pore that is similar to sTeLIC. The sodium ions can pass through the open channel without stripping off their hydration shells. In the open form, we also find a constriction ring in the LBD at the level of loop Ω contributed by side-chains of five Tryptophans. The same constriction ring is also observed in the open channel of

Table 1 : Diffraction data collection and models refinement statistics.

 1

			sTeLIC	sTeLIC+4-BrC	sTeLIC+Cs	+	sTeLIC+Ba	2+
	Data
	collection
	Beamlines		Soleil PX1	ESRF ID23_2	Soleil PX2		Soleil PX1
	Date		18/12/2015	29/08/2016	24/07/2016		12/06/2016
	Wavelength (Å)		0.978	0.872	2.100		2.000
	Oscillation range	0.10	0.15	0.2		0.1
	(°)
	Data
	processing
	Resolution (Å)		49.3-2.3	49.3-3	48.5-4.2		49.1-3.2
			(2.3-2.3)	(3.1-3.0)	(4.5-4.2)		(3.3-3.2)
	Space group		C 1 2 1	C 1 2 1	C 1 2 1		C 1 2 1
	Cell parameters		219.6 113.0 144.4	222.7 112.7 144.8	221.2 114.1 144.3	218.8 112.1 144.1
	(Å, °)		90 112.1 90	90 111.2 90	90 111.8 90		90 112.8 90
	Rmerge (%)		7.1(58.5)	7.1(93.5)	12.2(99.1)		5.2(34.2)
	Reflection		499194(22698)	444411(30905)	255049 (47002)	166241(14462)
	measured
	Reflection unique	144663(7079)	66506 (4454)	24499(4443)		52251(4511)
	I/sigma		6.4(1.4)	8.5(1.1)	6.3(1.2)		9.1(1.9)
	Multiplicity		3.4(3.2)	6.7(6.9)	10.4(10.6)		3.2(3.2)
	CC ½ (%)		99.7(42.6)	99.9(91.1)	99.9(92.1)		99.8(95.3)
	Completeness (%)	99.7(98.8)	99.3(99.0)	99.7(99.8)		98.4(99.1)
	Model
	refinement
	Resolution (Å)		20.0-2.3	20.0-3.0	20.0-4.2		20.0-3.2
	Rwork/Rfree (%)		0.21/0.24	0.19/0.22	0.24/0.24		0.21/0.23
	RSMD bond		0.010	0.010	0.008		0.010
	lengths (Å)
	RSMD bond		1.09	1.14	1.03		1.16
	angles (°)
	No. protein atoms	12830	12810	12830		12805
	No. of ligand		5		10		16			7
	No. of water		477	106	-			187
	molecules
	B factor overall (Å)	72.80	49.53	75.00		64.08
	B factor ligand (Å)	85.67	79.78	82.97		75.91
	B factor water (Å)	41.18	23.59	-			28.33
			97.7	97.4	99.0		99.0
	Ramachandran
	preferred (%)
			0		0		0			0
	Ramachandran
	outlier (%)
	Molprobity score	*	99	th	100	th	100	th		100	th
	*100 th percentile is the best among structure of comparable resolution; 0 th percentile is the worst.

Table 2 : Tested compounds with little or no effect.

 2 Compounds were tested at the maximal concentration indicated at pH 7.5 in the absence (left) or presence (right) of a positive allosteric modulator (PAM). No effect was observed except for compounds with a bolded font, signifying a small potentiating effect at the concentration indicated. Left: Standard solution (or ** CaCl 2 free standard solution). Right: Standard solution with 30 mM MES (or ** CaCl 2 free standard solution with 10 µM 4-BrC).

	Compound	[mM]	[mM] w/ PAM
	5-HT	5	5
	Acetylcholine β-Alanine	30 10	30 -
	Caffeic Acid	10**	-
	Citric Acid	1.8	-
	DMSO	(1% v/v)**	(1% v/v)**
	GABA	30	30
	L-Glutamate	30	30
	L-Glycine	30	30
	L-Histidine	30**	-
	Histamine	30**	10
	HEPES pH 7.5	30	30
	Ivermectin	0.5	0.5
	IPTG	1**	-
	L-Leucine	30**	30**
	Levodopa	10**	-
	L-Methionine	30**	30**
	Nicotine	0.35	-
	PEG 200	30**	30**
	Picrotoxin	0.1**	0.1**
	L-Serine	30**	30**
	Mixture of Sugars	5**	5**
	(Glucose,Sucrose,Maltose)
	Taurine	30	-
	Tris-HCl pH 7.5	30	-
	L-Tyrosine	1**	-

calculation for His and Tyr using FD/DH

  means!nonfunctional!receptor.!Significance!of!the!results!was! determined!as!values!larger!than!0.5!pH!units!for!the!mean!ΔpH50,!and!mean!pH50,!as!compared!to!Wt! (See!reference!Nemecz!et#al).!! .22 6.15 5.85 5.76 5.21 6.72 3.78 7.81 6.70 7.71 5.84 0.36 6.54 1.46 -0.71 H235 1.76 2.84 2.76 2.23 2.77 2.47 3.65 3.26 3.17 2.97 2.47 0.42 3.10 0.39 -0.63 H277 4.34 3.77 2.34 4.81 4.82 3.77 4.77 5.74 4.76 4.27 4.02 0.92 4.66 0.65 -0.65

	Mutant! WT! ! Table&2:&Electrophysiology:&Functional&characteristics&of&mutants.&pH50,&EC50&and& pH50! ΔpH50! Phenotype! Note! 5.2!±!0.4! ! nH&values&were&obtained&through&individual&fits&of&dose:response&current&curves&to&the& Hill&equation.&NF&stands&for&non:functional&and&was&used&for&currents&smaller&than&500& Table&3:&Diffraction&data&collection&and&model&refinement&statistics& ! Table&4:&Summary&of&Locally&closed&forms&of&different&GLIC&variants.& ! pKa Now we give the results for the Histidines
	nA.&n&represents&the&number&of&experiments.&For&all&the&data,&mean&values&and&the& corresponding&standard&deviations&are&presented.& Mutant& pH50& EC50&(µM)& nH& Imax&(µA)& Wild:type& 5.1&±&0.2& 8&±&3& 1.8&±&0.4& 5&±&1& C27S&T158C& 5.3&±&0.3& 6&±&3& 2.5&±&0.4& 7.2&±&1.8& C27S&G159C& NF& NF& NF& NF&(>&500&nA)& W160F& NF& NF& NF& NF&(>&50&nA)& C27S&Q193C& 5.0&±&0.1& 10&±&3& 1.8&±&0.4& 5&±&2& Q193L& 4.47&±&0.06& 34&±&4& 2.3&±&0.2& 1.1&±&0.4& Q193M& 4.5&±&0.1& 29&±&8& 2.1&±&0.3& 1.0&±&0.3& Y197A& NF& NF& NF& NF&(>&50&nA)& C27S&P250C& Y197F& 5.2&±&0.1& 6&±&2& 1.3&±&0.3& 4.1&±&0.1& 37.84! 53.57! 100.19! 84.19! 110.86! 73.64! B!factor!for!protein!(Å 2 )! ! 36.77! 53.10! 100.22! 84.25! 110.94! 73.71! B!factor!for!ligands!(Å 2 )! ! 62.52! 78.90! 82.38! 37.15! 59.09! 35.71! B!factor!for!solvent!(Å 2 )! ! 41.00! 41.00! C! 78.00! C! C! Ramachandran!outliers!(%)! 0! 0! 0! 0! 0! 0! ! B!factor!for!protein!(Å 2 )! ! 44.41! 46.49! 101.81! 70.87! 48.83! 42.03! T249A (T25'A) M2-M3 loop (Nonfunctional) (PDB ID: 4LMJ) n& 3& 4& 3& 3& 5& 4& 5& 2& 5& 49.86! 49.41! 65.73! 0! LC1 32.40! Data&processing& 47.02! 102.74! 71.30! 49.30! 41.97! Q193L Pre-M1 LC1 (Loss of function) This study Q193M Pre-M1 LC1 (Loss of function) This study Y197F-P250C Pre-M1 LC1 (Wild-type) This study I201W-E243C Pre-M1 LC2 (Nonfunction) This study H235F (H11'F) Upper M2 LC1 (Nonfunctional) (PDB ID: 3TLT) H235Q (H11'Q) Upper M2 LC (Loss of function) (PDB ID: 5NJY) N239C (N15'C) Upper M2 LC (Loss of function) (PDB ID: 5NKJ) E243G (E19'G) Upper M2 LC2 (Nonfunctional) This study E243P (E19'P) Upper M2 LC2 (Nonfunctional) (PDB ID: 3TLS) P247G (P23'G) M2-M3 loop LC2 (Loss of function) (PDB ID: 5HEG) ! 45.01! Q193C+MMTS Pre-M1 (Loss of fuction) This study LC1 & Wavelength!(Å)! 0.9771! 0.9785! 0.9660! 0.9789! 0.9789! 0.9790! 0.9677! Oscillation!range!(°)! 0.05! 0.20! 0.25! 0.10! 0.20! 0.10! 0.10! Wavelength!(Å)! 0.9785! 0.9771! 0.9801! 0.9801! 0.984! 0.9762! Oscillation!range!(°)! 0.20! 0.05! 0.10! 0.10! 0.10! 0.05! Variant Location Conformation (Phenotype) Res. O-1 O-2 O-3 O-4 O-5 C-1 C-2 C-3 C-4 C-5 O Rms C Rms Diff Note H127 6	44.61! 44.63! 0.9677! 0.10!
	RMSD!bond!lengths!(Å)! ! RMSD!bond!angles!(°)! B!factor!for!ligands!(Å 2 )! ! B!factor!for!solvent!(Å 2 )! Y251A (Y27'A) 0.010! 1.06! 59.33! 37.17! ! Molprobity!score a ! ! 100th! ! Ramachandran!outliers!(%)! 0! Wild type-10*His	0.010! 1.06! 64.40! 44.93! M2-M3 loop 0.008! 0.98! 132.13! 79.39! 100th! 100th! 0! 0! C-terminal 10*His	0.010! 1.10! 91.02! 58.86! LC1 (Nonfunctional) 0.010! 1.12! 67.90! 36.68! 100th! 100th! 0! 0! Co-exist LC and Open (PDB ID: 4NPP ) 0.010! 0.010! 1.08! 1.05! 31.22! 18.79! (PDB ID: 4MLM) 100th! 100th! 0!	74.99! 60.00! 0!
	RMSD!bond!lengths!(Å)! ! RMSD!bond!angles!(°)! ! Loop2-20' 0.010! 1.13! Molprobity!score a ! ! 100th! Loop2-21'	0.010! 1.06! Loop2 and M2-0.009! 1.13! M3 loop 100th! 100th! Loop2 and M2-M3 loop	! ! LC2 (Nonfunctional) 0.010! 0.009! 1.02! 1.03! LC1 (Nonfunctional) 100th! 100th!	!	0.009! 1.07! (PDB ID: 3UU3) 100th! (PDB ID: 3TLW)	0.010! 1.11! 100th!
	Loop2-22'	Loop2 and M2-M3 loop	LC3 (Loss of function)		(PDB ID: 3TLV)
	Loop2-24'	Loop 2 and M2-M3 loop	LC1 (Nonfunctional)		(PDB ID: 3TLU)
			!
			!
			!

  SUMMARY OF ANISOTROPY ANALYSIS AND MERGING STATISTICS FOR OBSERVED DATA Cell parameters: 159.415 337.295 111.835 90.00 90.00 90.00 Space group: P21 21 2

	Table&1&
	&
	DeCLIC_closed_20160612_punk5_position8_collect_2:&&&
		Overall	InnerShell	OuterShell
	Low resolution limit (Å)	48.177	48.177	3.722
	High resolution limit (Å)	3.501	10.336	3.501
	Rmerge	0.172	0.038	2.073
	Rmeas	0.179	0.041	2.153
	Rpim	0.049	0.013	0.579
	Total number of observations	852226	36501	43718
	Total number unique	63634	3179	3182
	Mean(I)/sd(I)	12.4	51.2	1.5
	Completeness (ellipsoidal)	94.6	99.5	60.3
	Multiplicity	13.4	11.5	13.7
	CC(1/2)	0.998	0.998	0.616
	Refinement
	Resolution (Å)	20.00 -3.5
	R work /R free	0.241/0.257
	DeCLIC_open&
	SUMMARY OF ANISOTROPY ANALYSIS AND MERGING STATISTICS FOR OBSERVED DATA:
	Cell Parameters: 141.066 116.030 169.635 90.00 109.17 90.00 P1 21 1
		Overall	InnerShell	OuterShell
	Low resolution limit (Å)	48.613	48.613	3.833
	High resolution limit (Å)	3.401	11.441	3.401
	Rmerge	0.081	0.028	1.252
	Rmeas	0.094	0.034	1.406
	Rpim	0.046	0.018	0.632
	Total number of observations	149387	6535	9131
	Total number unique	37907	1894	1896
	Mean(I)/sd(I)	9.5	35.2	1.4
	Completeness (ellipsoidal)	92.5	97.9	67.3
	Multiplicity	3.9	3.5	4.8
	CC(1/2)	0.998	0.997	0.467
	Refinement
	Resolution (Å)	20.00 -3.40
	R value	0.259/0.307
	!

& & &

& & & & & & &

Acknowledgments

We thank Anaïs Menny for sharing expertise on plasmid constructions. We thank the staff of the Crystallography Platform (PF6) in the Institut Pasteur for initial crystal screens. We thank Reinis Reinholds Ruza for help in data collection, Jérôme Loc'h for help during structural refinement and pictures preparation. We thank the staff of synchrotron beamlines in Soleil (Orsay) and ESRF (Grenoble) for data collection. We thank Pierre Legrand (Soleil synchrotron) for suggestions on refinement in Buster. We thank Marc Gielen for suggestions during the paper preparation. ÁN and ZFK were supported by ANR Grant 13-BSV8-0020 "Pentagate".

HH was sponsored by the China Scholarship Council and Institut Pasteur.

Acknowledgements

The authors would like to thank Marie Prevost for use of GLIC clones; Karima Medjebeur and Nathalie Barilone for molecular biology assistance; Frédéric Poitevin for aid with scripting and discussion about crystallographic analysis; Marc Gielen and Solène Lefebvre for critical reading of the manuscript. This study was supported by the French Agency for Research ANR-13-BSV8-0020.

Acknowledgements

We would like to thank the staff at crystallization platform (PF6) of Pasteur. We thank the staff at ESRF (European Synchrotron Radiation Facility, beamlines ID30, ID29 and ID23) and Synchrotron-Soleil (beamlines PX1 and PX2) for their help during data collection. We thank Jérôme Loc'h for his suggestions during the figures preparation. ZFK was supported by ANR. Haidai HU was sponsored by the China Scholarship Council and co-financed by Institut Pasteur.

Supplementary information

Summary

The Gloeobacter violaceus pentameric 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 pLIGC 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, along with E222, H235, E243, and H277 in the transmembrane domain 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 extracellular and transmembrane domain For clarity, the presentation of the results is organized according to five regions of a GLIC subunit: the apical top of the ECD, the Loop B and C on the principal (+) face of the interface, the basal ECD principal (+) and complementary (-) faces, and finally the TMD (Fig 1).

have the same marked effect, although T158 is the residue positioned closest to E35, indicating that the global environmental change in charge at position 35 is more important than the direct residue-residue interactions.

Subsequently, the additivity of the Asp/Glu mutations was also tested, however neither E82Q-D115N, E35Q-E82Q, nor E35Q-E82Q-D115N showed increased proton sensitivity as compared to E35Q. A combination of all other weak pH50-increasing mutations was performed with the addition of R179Q or E75Q-D97N-D136N to the triple mutant E35Q-E82Q-D115N, with neither of these mutants producing any shift greater than E35Q alone (Fig 4B).

The pH50 evaluation of this region effectively identified key residues involved in pH-sensing, but the results of the combined mutants demonstrates the complexity of the mechanism involved in GLIC-gating. interaction, has also previously been shown, using non-canonical amino acid substitution, to decrease the pH50 [38].

The structure of H277Q was performed and was also found to have no apparent deviation from the apparently open pH4 conformation further corroborating the several previously published works that show H277 does not appear to play a role in proton-gating (Supp Fig 1).

Materials and Methods

Residue numbering follows that which was established by Bocquet et. al. for consistency. This numbering is shifted by one from other reports (Baenziger/Dutzler), which use the correct GLIC numbering.

Two-electrode Voltage Clamp Electrophysiology

Xenopus laevis oocytes were obtained from the Centre de Ressources Biologiques-Rennes, France. Defolliculated oocytes were maintained at 4°C in a modified Barth's saline solution (88mM NaCl, 1mM KCl, 1mM MgSO4, 2.5mM NaHCO3, 5mM HEPES/Na pH7.3) with 0.7mM CaCl2. After intra-nucleus injection of ~30nL cDNA (80ng/µl specified clone cDNA with 20ng/µl of GFP cDNA), using a compressed air microinjection system, the oocytes were transferred to 18°C. 1-2d later they were evaluated for GFP expression, and subsequently maintained at 15°C. Recordings were made 1-5d after injection using low-resistance (0.2-2 MΩ) electrodes filled with 3M KCl, with a -40mV holding potential. The standard solution superfusing the oocyte during recording at room temperature was 100mM NaCl, 3mM KCl, 1mM MgCl2, 1mM CaCl2, 10mM MES at either pH7.3, 7.5, or 8 using NaOH. In order to maintain the desired pH and maintain an equivalent Na + concentration in all solutions, the stock solution was adjusted to the indicated pH using NaOH, and lower pH solutions were subsequently obtained using HCl and the addition of the stock solution.

Measurements were performed using pClamp 10.5 software, with a Digidata 1440A digitizer and GeneClamp 500 amplifier (Molecular Devices, LLC, Sunnyvale, CA), using an 8-valve (PS-8H) programmable gravity driven pinch valve perfusion system (Bioscience Tools, San Diego, CA). pH dependent responses were elicited by switching from pH7.3-pH8 to a series of pH values, with a minimal pH of 3.7, and a 0.5-log-unit increment from either pH6.5, or pH7.5 for elevated pH50 mutants. Perfusion times varied from 30s to 90s with equivalent recorded wash periods in the holding buffer. pH series were performed either in an ascending order directly followed by a descent, or a descending order directly followed by an ascent, in order to remove bias.

Evaluation of currents was done using Clampfit 10.5 (Molecular Devices, LLC, Sunnyvale, CA), with Imax (µA) reported as the peak amplitude of negative going

Crystallographic Analysis

Residue root-mean-squared deviation (RMSD) and Cα distance calculations were performed by aligning a given structure (M) using the two-subunit pair chains A+B, B+C, C+D, and D+E upon the chain pairs A+B, B+C, C+D, and D+E of a reference Wt structure at either pH 4 or 4.6 (Wt). The alignment of pair E+A was replaced with only an alignment of the E chain of M upon each chain of Wt, and each individual chain of M was aligned upon chain E of Wt as the Pymol structural alignment algorithm had difficulties doing an alignment with non-consecutive chain pairs. The pair-wise alignment method was chosen to include quaternary inter-subunit interface interactions which a simple single chain alignment would ignore. The RMSD of each residue, for which alternate side-chain conformations were removed, as well as the Cα atom distance was calculated between the 25 pair-wise alignments and subsequently averaged to yield VRMSD(M-Wt) and V ΔCα (M-Wt). The calculated intrinsic variation, VRMSD(Wt2-Wt1) and V ΔCα (Wt2-Wt1), between the two Wt structures at pH 4.6 (Wt1)

Summary

The proton-activated pentameric ligand-gated ion channel (pLGIC) from Gloeobacter violaceus (GLIC) can be used as a model system to study the structural and functional properties of its eukaryotic counterparts, which mediate signaling transduction in animal neuron cells by controlling permeation of ions flux gated by neurotransmitters binding. We used the Finite Difference Poisson-Boltzmann/Debye-Hückel (FD-DH) method to predict the pKas of all the Asp and Glu in GLIC both in the active and resting states. Those residues with a high deviation of predicted pKas in both forms were titrated by Attenuated Total Reflectance Fourier Transform Infra-Red Spectroscopy (ATR-FTIR) after reconstitution in lipid bilayers. The results indicate that E35 is the main protonsensor residue. Examination of the active form structure shows that E35 interacts with T158 from Loop F. We verified that breaking this interaction hinders the proton-elicited currents. We next probed the interfacial crevice immediately below E35, shaped by Loop F, Q193 (pre-M1) and the neighboring M2-M3 loop, where a previously unnoticed hydrogen bond network helps maintaining the channel open. We show that breaking this hydrophilic network originating from Q193 favors nonconductive conformations of the ion channel in the crystal states. Replacing side-chains of residues just below Q193, such as Y197 and I201, with other hydrophobic side-chains blocks the receptor in a locally-closed form. Two signal transduction networks are proposed, both originating from the key proton-sensing residue E35, loop F and the pre-M1 region, and separating at Y197: i) one hydrophilic network extends to the M2-M3 loop from the neighboring subunit and ii) one hydrophobic network interacts with the Cys-loop and the M2-M3 loop of the same subunit. Due to the strong structural conservation of these loop regions, this model can be proposed to be general for the entire pLGIC family. applied to characterize GLIC conformations in solution and they generally agree very well with the published crystal structures 30,31 .

GLIC's channel is gated by proton(s) at pH ranging between 7.0 and 4.0 (pH50 = 5.1 ± 0.2). This sharply contrasts with most human pLGICs, which are activated by binding a neurotransmitter to a well-characterized cavity in an inter-subunit interface in the ECD, mainly involving Loop B and Loop C (Figure 1A). However, various invertebrate pLGICs have been demonstrated to be directly responsive to pH [32][33][34] . Beyond the pLGIC family, some other ion channels are also gated by pH, such as acid-sensing ion channels (ASIC) and some potassium inwardly-rectifying channels (Kir) 35,36 . Recently, all Asp, Glu and His residues. Indeed the FD/DH method has been shown to be a powerful tool to filter candidate residues responsible for pH-induced channel gating, more powerful than classical FD-PB 37 . Then, we experimentally determined the pKas of those residues that possess a high deviation of predicted pKas between two states, in the vicinity of pH50, by employing Attenuated Total Reflectance Fourier Transform Infra-Red Spectroscopy (ATR-FTIR). As a result, residue E35 stands out as the main candidate for proton sensing. Site-directed mutagenesis, electrophysiology, and crystal structure analysis are then used to further explore the environment of residue E35, at the interface with Loop F of an adjacent monomer. In particular we show that an interfacial hydrogen network mediated by water molecules controls channel gating, in association with an intrasubunit hydrophobic cluster. Both networks are crucial for maintaining the channel open.

They branch out/separate from E35 and Q193 at the level of residue Y197, at the interface of the ECD and the TMD and propagate down to H235 in two adjacent subunits simultaneously.

The deviations of the pH titration curve become clear when the trace of wild-type is subtracted from each traces of the mutants (Figure 3). The deviation of the traces is in the range of 2 to 7 % of total intensity. Deviation of wild-type between two different experiments, which sets a basis of reproducibility error, does not exceed the range of ± 1%. Therefore, we only consider mutants whose deviations are larger than 2 %, namely E35A, E181A.

The other mutants, E26A, E75A and E104A, are below the criterion, thus we can consider that the effects of these mutations on the pH titration curve are not significant.

Note that the trace of E35A (red) exhibits a sigmoidal shape, as expected for a pH titration, while the curve for E181A (yellow) goes down below pH 4.2, decaying almost exponentially to zero until pH = 2.5. Although a slight decrease in the intensities for pH below 2.5 is observed, we consider these are mainly artificially caused by the instability of the lipid layer highly acidic environment (pH below 3.0). Since the structure of the lipid layer is relatively stable in this range of pH (4.2 to 3.0), this abrupt decrease is not caused by an artifact of the unstable lipid bilayer. Experimental pKa values of E35 and E181 residues were determined to be 5.9 and 5.0, respectively. S3 shows a plot of the normalized peak height of carboxylate groups at 1400 cm -1 versus pH for several other mutants, including E82A, D86A, D86N, D88A, D88N, D97A (for these mutants, the data sets were available only at the pH range between 4.0 and 7.0) as well as E26A, E26Q, E35A, E35Q, E75A, E104 and E181A. The intensities were normalized to zero at pH = 7.0 and -1 at pH = 4.0. Most of the data points converged into a single curve except those from E35 and possibly E181A. Clear deviations from wild type were not observed for other mutants (Suppl. Figure S3). From these results, we conclude that there is no significant shift of pKas for these residues.

Suppl. Figure

In addition, we tried to enhance the interaction of Q193C with M2-M3 loop by introducing a second cysteine residue (P250C), which may form a disulfide bridge with Q193C. As predicted, this double mutation shows a strong 'gain of function' phenotype. In oocytes expressing GLIC Q193C-P250C receptors, the recording shows a leak of current ≈ 1 µA at pH 7.3 that is blocked by 100 µM of picrotoxin, an open channel blocker (Suppl. Figure 5E). Thus, the interfacial hydrophilic residue Q193 fulfills several conditions expected for a key residue in the activation pathway.

Tyrosine mutations: coupling between the Pre-M1 region, the Pro-Loop and the M2-M3 loop is mediated by aliphatic/aromatic residues of the same subunit

Superimposing the LC form of Q193M/Q193L with the open-form of GLIC revealed that the pre-M1 region is also rearranged (Figure 5E). Interestingly, the side chain of Y194, which points toward the lipid bilayer in the open form, flips its orientation by almost 180°, becomes buried inside the inter-subunit cavity, and pushes the Y197 sidechain from an "inward" position to an "outward" position. This rearrangement of residues in the pre-M1 region also breaks the interaction of Y197 with R192. It is noteworthy that the equivalent position of Y197 is well conserved among members of the pLGIC family. To test whether both its hydroxyl group and aromatic group are required for proton-elicited channel currents, we mutated Y197 to phenylalanine and alanine separately.

The hydroxyl group of Y197 interacts with the Nη1 atom of R192 side chain by a hydrogen bond as revealed by an average distance between them around 3.4 Å. We replaced Y197 with phenylalanine in the context of P250C/C27S mutant and grew crystals of this mutant at pH 4 and determined its structure at 3.0 Å (Figure 8B and Table 3). The conformation of this mutant turned out to be identical to the LC form of Q193M/Q193L (Figure 8C), with the side chains of Y194 and Y197F adopting the same conformations as described in the model of Q193M/Q193L. Notably, the side chain of Q193 also flips and doesn't protrude into the ECD and TMD interfacial crevice anymore (Figure 8D).

We then evaluated the mutation of Y197F on the functional level to check whether this mutation causes a change in the dose-dependent channel activation in terms of pH50 as measured by electrophysiology. Surprisingly, Y197F doesn't show any decrease in proton sensitivity: indeed, the pH50 of Y197F shows that it has the same value as the wild-type GLIC (Figure 8A and Table 2). This suggests that even though the activation barrier between the open form and the LC form system might direct the transition towards the "dead-end" LC conformation while on top of the energy barrier, instead of going down to the open state.

Structural basis for an activation model with two alternating stabilization networks originating from the same ECD subunit interface

If we simply connect the dots for mutants adopting the LC-form together, we end up with a simple model for the coupling between proton binding to channel gating. This process can be described by the following steps (Figure 11B-D Several residues involved in the stabilization networks described here were also shown to predominantly affect the activation equilibrium constant in eukaryotic pLGICs 42 . Thus, the same activation model with two stabilization networks operating simultaneously at adjacent subunits, completely encircling the ECD-TMD interface of the entire pentamer and branching out at the level of Y197, can be also expected in the whole pLGIC family. Interestingly,, we, see, that, the, R1924D1224D32, triplet, is, also, connected, to, the, triplet, Y1194Y1974 K248,through,the,R1924Y197,interaction., Note,that,K248,has,a,gain4of4function,phenotype,,and,Y197,has,an,increased,kinetics,to,the,desen4, sitized,state.

Supplementary Informations

In the closed state, the pairs noted with * are conserved, the other are lost. The only "new" interacting pair in the closed state is the following: i-pr = 3 pair is A 64 LYS A 66 TYR coupling-energy = 8.31 kT but it is not conserved in bacterial sequences. Interestingly, most of the "lost/gained" interactions involve either LoopC or the ECD-TMD interface (Cys-Loop, pre-M1, M2-M3 loop) We note that in the closed state the R192-D122 and R192-D32 electrostatics interactions are lowered to about 6 kT each.

Supplementary Notes

S1: Band assignment of the pH induced different FT-IR spectra

The bands observed in the pH induced different spectra shown in Figure 2 consist of contributions not only from the protein but also from different background species as the lipid bilayer and the buffer solution. For the analysis of the spectral data, the bands have to be appropriately assigned to each species. Despite the fact that all samples were prepared in equivalent conditions, the obtained spectra show slightly different intensities for each band because of, for instance, differences of the protein concentration in the optical path, slight differences of the lipid/protein ratio, and instability due to the membrane swelling, etc. In order to compare these different data sets, the intensity normalization had been handled for each obtained bands. The νs(COO -) band at

Results

Structures determination and overall architectures of DeCLIC

The growth of good diffracting quality crystals from the full-length receptors required extensively seeding. DeCLIC was crystallized in its closed conformation of in the presence of 200mM of Ca 2+ at pH 7.5. This yielded crystals diffracting up to 3.50 Å resolution. The open form of DeCLIC was determined at 3.40 Å resolution at pH 6.5 (Table 1). Both forms were solved by molecular replacement. The non-crystallographic averaging significantly improved the electron density map of LBD and TMD regions and allowed us to trace the main chains without ambiguity and to build most of the side chains in the two regions.

However, the non-crystallographic symmetry is broken in the closed form in the

Tertiary changes at the LBD between the open and closed states

The β-sandwich folding of LBD monomer comprises two layers of βsheets (inner β-sheet labeled as the principal interface and outer β-sheet labeled as the complementary interface). We superimposed the LBD of single subunits between the two states to monitor the change of LBD at the tertiary level. A total of 186 Cα atoms were superimposed and yielded a root mean squared deviation

Materials and Methods

Cloning, protein expression and purification

The full-length DeCLIC contains 642 residues, where the N-terminal 33 residues consist of a signal peptide as predicted by the SignalP 4.1 Server. The DeCLIC gene coding for residues ranging from 33 to 642 was codon optimized for expression in E. coli and chemically synthesized. The N-terminus of this construct of DeCLIC was fused to an MBP tag with a thrombin protease cleavage site between them. The recombinant gene was subcloned into the pET20b vector.

The pET20b vectors containing the recombinant genes were introduced into E.

coli C43 competent cells and the protein was expressed in the 2YT medium.

During the culture, 1 mM ampicillin was added. When the OD reached to 0. DeCLIC protein was fresh frozen using liquid nitrogen and was stored at -80°C for further using.

Crystallization and data collection

All the crystallization trials were performed at 18°C using the hanging Å resolution, which is too low to solve the structre. However, the result is promising. We will continue to screen the conditions at lower pH to find better crystals that give better diffraction. As the Cryo-EM has proved to be a powerful method in recent years to determine the protein structures at the atomic level, we also wish to solve the structure of sTeLIC at acidic pH by Cryo-EM in order to capture the closed conformation of the receptor. Can we expect that the closed conformation of sTeLIC is similar to ELIC? The agonist of ELIC is GABA or cysteamine. Complex structures of ELIC with GABA or cysteamine adopt the same channel conformations 3 as the apo form. Other studies even suggest that ELIC conformation is a "uncoupled state" as observed in an uncoupled nAChR 4,5 .

So we expect some differences between the closed forms of sTeLIC and ELIC.

How does the basic pH trigger the sTeLIC channel open?

sTeLIC is activated at alkaline pH with the pH50 around 8.6 ± 0.4.

Understanding the molecular mechanism of sTeLIC signal-transduction thus requires identifying the locus where hydroxyl groups act and which titratable groups are crucial in this process. Each monomer of sTeLIC possesses 18 Arg, 10

His, 9 Lys. Most of them distribute at the ECD-ECD interface. Interestingly, in the sTeLIC and is contributed by side-chains of five Arginines. Interestingly, since this constriction is released in the closed form of DeCLIC, we can propose that the same thing could happen in the closed form of sTeLIC. This constriction ring may serve as a shut gate at the LBD to control the ions access to the open pore.

The N-terminal extension domain of DeCLIC

The largely N-terminal extension domain decorated at the top of LBD comprises NTD1 and NTD2 subdomains that form mobile regulatory structures by establishing extensive interactions with LBD domain. Because of its lower resolution and the observed high flexibility, the NTD1 domain is not well resolved in both conformations, especially most of its side-chains are not built properly. I tried to purify the NTD1 domain and to crystalize it independently. I managed to get crystals in the following conditions: 1.6M -2 M Ammonium sulfide, 0.1M NaCl, 0.1M Sodium Citrate pH 6.5. The best crystal diffracts to around 4 Å resolution. We are still trying to optimize the conditions to get better crystals.

The role of the N-terminal extension domain

Why are many bacterial pLGICs decorated with an N-terminal extension domain? According to sequence analysis and structure predictions, the Nterminal extension domains can be divided into several subfamilies: i) type 1 periplasmic binding proteins (PBP1). ii) Type 2 periplasmic binding proteins (PBP2). Both of them play a pivotal role in the transport of various low molecular weight compounds into cytoplasm of bacterial cells. iii) Cache domain that is predicted to have a role in small-molecule recognition in a wide range of proteins. The N-terminal extension domain we present here belongs to a new subfamily, the function of which is still a mystery. Indeed, we could find at least five pLGICs from different bacteria species decorated with such homologs extension. Can we infer their functions from the folding? Both of NTD1 and NTD2 share similar folding with collagenase, an enzyme that breaks the peptide bonds in collagen and assists in destroying extracellular structures in the pathogenesis of bacteria. However, the NTD1 and NTD2 lack the canonical active motif found in collagenases. Do they belong to a new collagenase subfamily? To assess this, we need to further investigate the function of the NTD domain.

N-terminal extension domain with eukaryotic pLGICs?

To date, we couldn't find any eukaryotic pLGICs decorated with an Nterminal extension domain. Interestingly, the NMDA-type receptors contain an N-terminal type I PBP domain and a C-terminal domain belonging to the type II PBP family. The metabotropic glutamate receptors as well as vertebrate taste receptors, which are GPCR family, contain an N-terminal type I PBP domain. We could expect that some eukaryotic pLGICs also contain such N-terminal domains or that some eukaryotic pLGICs can be modulated by N-terminal domains found from bacteria pLGICs. This can lead a new way to design therapeutic reagent targeting human pLGICs.