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Crystal structures of a pentameric ligand-gated ion channel provide a mechanism for activation Ludovic Sauguet a,1 , Azadeh Shahsavar a,1,2 , Frédéric Poitevin a,1 , Christèle Huon b , Anaïs Menny b , Àkos Nemecz b , Ahmed Haouz c , Jean-Pierre Changeux d,e , Pierre-Jean Corringer b , and Marc Delarue a,3 a Unité de Dynamique Structurale des Macromolécules, Unité Mixte de Recherche (UMR) 3528 du Centre National de la Recherche Scientifique (CNRS), Institut Pasteur, 75015 Paris, France; b Unité Récepteurs-Canaux, UMR 3571 du CNRS, Institut Pasteur, 75015 Paris, France; c Plateforme de Cristallogénèse, Institut Pasteur, 75015 Paris, France; d UMR 3571 du CNRS, Institut Pasteur, 75015 Paris, France; and e College de France, 75005 Paris, France Edited by Arthur Karlin, Columbia University College of Physicians and Surgeons, New York, NY, and approved November 22, 2013 (received for review August 8, 2013) Pentameric ligand-gated ion channels mediate fast chemical trans- mission of nerve signals. The structure of a bacterial proton-gated homolog has been established in its open and locally closed conformations at acidic pH. Here we report its crystal structure at neutral pH, thereby providing the X-ray structures of the two end-points of the gating mechanism in the same pentameric li- gand-gated ion channel. The large structural variability in the neu- tral pH structure observed in the four copies of the pentamer present in the asymmetric unit has been used to analyze the in- trinsic fluctuations in this state, which are found to prefigure the transition to the open state. In the extracellular domain (ECD), a marked quaternary change is observed, involving both a twist and a blooming motion, and the pore in the transmembrane do- main (TMD) is closed by an upper bend of helix M2 (as in locally closed form) and a kink of helix M1, both helices no longer inter- acting across adjacent subunits. On the tertiary level, detachment of inner and outer β sheets in the ECD reshapes two essential cavities at the ECDECD and ECDTMD interfaces. The first one is the ligand-binding cavity; the other is close to a known divalent cation binding site in other pentameric ligand-gated ion channels. In addition, a different crystal form reveals that the locally closed and open conformations coexist as discrete ones at acidic pH. These structural results, together with site-directed mutagenesis, physiological recordings, and coarse-grained modeling, have been integrated to propose a model of the gating transition pathway. X-ray crystallography | allostery | signal transduction | cys-loop receptor P entameric ligand-gated ion channels (pLGICs) are a super- family of membrane receptors that mediate fast chemical transmission of nerve signals in the central and peripheral ner- vous system (1). These allosteric receptors couple neurotrans- mitter (agonist) binding in the extracellular domain (ECD) to the opening of the ionic pore located in the transmembrane domain (TMD). There are two classes in pLGIC, with either cationic channels (acetylcholine nAChR and 5HT3 re- ceptors) or anionic ones (glycine and GABA receptors). The molecular understanding of their allosteric transitions is a cen- tral issue in the pharmacology of pLGICs, as it would allow the rational design of novel orthosteric and allosteric ligands. Re- cently reported full-length structures of several prokaryotic and eukaryotic members of the family have provided significant insights into the conserved architecture of these receptors (25). Nevertheless, little is known about the structural events that link the binding/unbinding of agonist to the opening/closure of the channel gate. To understand the gating mechanism, the structures of the same receptor in different allosteric states are needed at atomic resolution. Here we report two crystal structures of wild- type GLIC, a proton-gated bacterial ion channel from Gloeobacter violaceus (6) at two different pHs, above and below pH 50 . GLIC structure was initially solved at acid pH in its open conformation (4), and later in a locally closed (LC) conformation (7) displayed by six different mutants with little variations. The LC conformation shares several structural features with the open form but shows a closed pore as a result of a concerted bending of its M2 helices. Independently, recent progress has been made in understanding the structural mechanism of modulation by effectors such as general anesthetics (8) or ethanol (9) and the permeation process (10), using the same bacterial model system. Two other atomic X-ray structures of members of the pLGIC family have been described: an ion channel from the bacteria Erwinia chrysantemi (ELIC), in a closed pore conformation (2) and a eukaryotic glutamate-activated chloride channel (GluCl) in an open pore conformation similar to GLICs (5). However, the lack of strong sequence identity between the available structures prevents us from reliably deriving a mechanism of the gating process because it is impossible to disentangle sequence effects from functionally relevant conformational changes. Functional studies indicate that pLGIC can exist, in addition to an open and a resting form, in several closed forms, including desensitized and intermediate forms (11). In this study, we report a X-ray structure of GLIC at neutral pH in an apparently closed/resting-state.The resolution is moderate (4.35 Å), but noncrystallographic averaging over the four copies of the pen- tamer present in the asymmetric unit results in a much better electron density map than suggested by the nominal resolution. At this neutral pH the pore is closed by a tilt of the upper part of helix M2, as in the LC form, with an additional hinge movement of the beginning of helix M1. This is very different from what is Significance We describe the X-ray structures of the same pentameric li- gand-gated ion channel (pLGIC) in both its liganded or ligand- free conformations. This provides the molecular basis for un- derstanding the opening and closing (gating mechanism) of these key players in the fast transmission of chemical signals at synapses. As described with classical allosteric proteins, the tertiary changes of the subunits are linked together through the quaternary constraint by a marked reorganization of the interfaces between subunits and the associated binding pock- ets and cavities. The closed form displays a cavity that may allow a better understanding of the mechanism of action of pharmacological effectors of pentameric ligand-gated ion chan- nels and the rational design of new modulators. Author contributions: M.D. designed research; L.S., A.S., F.P., C.H., A.M., À.N., and A.H. performed research; L.S., A.S., F.P., P.-J.C., and M.D. analyzed data; and L.S., A.S., F.P., J.-P.C., and M.D. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: The crystallography, atomic coordinates, and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4NPP and 4NPQ). 1 L.S., A.S., and F.P. contributed equally to this work. 2 Present address: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2100 Copenhagen, Denmark. 3 To whom correspondence should be addressed. E-mail: [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1314997111/-/DCSupplemental. 966971 | PNAS | January 21, 2014 | vol. 111 | no. 3 www.pnas.org/cgi/doi/10.1073/pnas.1314997111 Downloaded by guest on July 29, 2021

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Page 1: Crystal structures of a pentameric ligand-gated ion ... · Crystal structures of a pentameric ligand-gated ion channel provide a mechanism for activation Ludovic Saugueta,1, Azadeh

Crystal structures of a pentameric ligand-gated ionchannel provide a mechanism for activationLudovic Saugueta,1, Azadeh Shahsavara,1,2, Frédéric Poitevina,1, Christèle Huonb, Anaïs Mennyb, Àkos Nemeczb,Ahmed Haouzc, Jean-Pierre Changeuxd,e, Pierre-Jean Corringerb, and Marc Delaruea,3

aUnité de Dynamique Structurale des Macromolécules, Unité Mixte de Recherche (UMR) 3528 du Centre National de la Recherche Scientifique (CNRS), InstitutPasteur, 75015 Paris, France; bUnité Récepteurs-Canaux, UMR 3571 du CNRS, Institut Pasteur, 75015 Paris, France; cPlateforme de Cristallogénèse, InstitutPasteur, 75015 Paris, France; dUMR 3571 du CNRS, Institut Pasteur, 75015 Paris, France; and eCollege de France, 75005 Paris, France

Edited by Arthur Karlin, Columbia University College of Physicians and Surgeons, New York, NY, and approved November 22, 2013 (received for reviewAugust 8, 2013)

Pentameric ligand-gated ion channels mediate fast chemical trans-mission of nerve signals. The structure of a bacterial proton-gatedhomolog has been established in its open and locally closedconformations at acidic pH. Here we report its crystal structureat neutral pH, thereby providing the X-ray structures of the twoend-points of the gating mechanism in the same pentameric li-gand-gated ion channel. The large structural variability in the neu-tral pH structure observed in the four copies of the pentamerpresent in the asymmetric unit has been used to analyze the in-trinsic fluctuations in this state, which are found to prefigure thetransition to the open state. In the extracellular domain (ECD),a marked quaternary change is observed, involving both a twistand a blooming motion, and the pore in the transmembrane do-main (TMD) is closed by an upper bend of helix M2 (as in locallyclosed form) and a kink of helix M1, both helices no longer inter-acting across adjacent subunits. On the tertiary level, detachmentof inner and outer β sheets in the ECD reshapes two essentialcavities at the ECD–ECD and ECD–TMD interfaces. The first one isthe ligand-binding cavity; the other is close to a known divalentcation binding site in other pentameric ligand-gated ion channels.In addition, a different crystal form reveals that the locally closedand open conformations coexist as discrete ones at acidic pH.These structural results, together with site-directed mutagenesis,physiological recordings, and coarse-grained modeling, have beenintegrated to propose a model of the gating transition pathway.

X-ray crystallography | allostery | signal transduction | cys-loop receptor

Pentameric ligand-gated ion channels (pLGICs) are a super-family of membrane receptors that mediate fast chemical

transmission of nerve signals in the central and peripheral ner-vous system (1). These allosteric receptors couple neurotrans-mitter (agonist) binding in the extracellular domain (ECD) tothe opening of the ionic pore located in the transmembranedomain (TMD). There are two classes in pLGIC, with eithercationic channels (acetylcholine — nAChR — and 5HT3 re-ceptors) or anionic ones (glycine and GABA receptors). Themolecular understanding of their allosteric transitions is a cen-tral issue in the pharmacology of pLGICs, as it would allow therational design of novel orthosteric and allosteric ligands. Re-cently reported full-length structures of several prokaryotic andeukaryotic members of the family have provided significantinsights into the conserved architecture of these receptors (2–5).Nevertheless, little is known about the structural events that linkthe binding/unbinding of agonist to the opening/closure of thechannel gate. To understand the gating mechanism, the structuresof the same receptor in different allosteric states are needed atatomic resolution. Here we report two crystal structures of wild-type GLIC, a proton-gated bacterial ion channel from Gloeobacterviolaceus (6) at two different pHs, above and below pH50.GLIC structure was initially solved at acid pH in its open

conformation (4), and later in a locally closed (LC) conformation(7) displayed by six different mutants with little variations. TheLC conformation shares several structural features with the open

form but shows a closed pore as a result of a concerted bendingof its M2 helices. Independently, recent progress has been madein understanding the structural mechanism of modulation byeffectors such as general anesthetics (8) or ethanol (9) and thepermeation process (10), using the same bacterial model system.Two other atomic X-ray structures of members of the pLGICfamily have been described: an ion channel from the bacteriaErwinia chrysantemi (ELIC), in a closed pore conformation (2)and a eukaryotic glutamate-activated chloride channel (GluCl)in an open pore conformation similar to GLIC’s (5). However,the lack of strong sequence identity between the availablestructures prevents us from reliably deriving a mechanism of thegating process because it is impossible to disentangle sequenceeffects from functionally relevant conformational changes.Functional studies indicate that pLGIC can exist, in addition

to an open and a resting form, in several closed forms, includingdesensitized and intermediate forms (11). In this study, we reporta X-ray structure of GLIC at neutral pH in an apparently“closed/resting-state.” The resolution is moderate (4.35 Å), butnoncrystallographic averaging over the four copies of the pen-tamer present in the asymmetric unit results in a much betterelectron density map than suggested by the nominal resolution.At this neutral pH the pore is closed by a tilt of the upper part ofhelix M2, as in the LC form, with an additional hinge movementof the beginning of helix M1. This is very different from what is

Significance

We describe the X-ray structures of the same pentameric li-gand-gated ion channel (pLGIC) in both its liganded or ligand-free conformations. This provides the molecular basis for un-derstanding the opening and closing (gating mechanism) ofthese key players in the fast transmission of chemical signals atsynapses. As described with classical allosteric proteins, thetertiary changes of the subunits are linked together throughthe quaternary constraint by a marked reorganization of theinterfaces between subunits and the associated binding pock-ets and cavities. The closed form displays a cavity that mayallow a better understanding of the mechanism of action ofpharmacological effectors of pentameric ligand-gated ion chan-nels and the rational design of new modulators.

Author contributions: M.D. designed research; L.S., A.S., F.P., C.H., A.M., À.N., and A.H.performed research; L.S., A.S., F.P., P.-J.C., and M.D. analyzed data; and L.S., A.S., F.P., J.-P.C.,and M.D. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The crystallography, atomic coordinates, and structure factors have beendeposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4NPP and 4NPQ).1L.S., A.S., and F.P. contributed equally to this work.2Present address: Department of Drug Design and Pharmacology, Faculty of Health andMedical Sciences, University of Copenhagen, DK-2100 Copenhagen, Denmark.

3To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1314997111/-/DCSupplemental.

966–971 | PNAS | January 21, 2014 | vol. 111 | no. 3 www.pnas.org/cgi/doi/10.1073/pnas.1314997111

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seen in ELIC. At the same time, the ECD has undergone significantstructural changes that lead to a profound rearrangement of boththe ECD–ECD and ECD–TMD interfaces. Strikingly, the largestructural variations observed in the four copies of the pentamer atpH 7 imply a rather flat energy landscape in the absence of ligand,whereas a crystal form grown at pH 4, also presented here, showsthat the LC form coexists with the open form, thus suggesting anenergy landscape made of two wells in the presence of the ligand.

ResultsStructural Variability and Intrinsic Fluctuations at Neutral pH. To getstructural information on the resting state of GLIC, crystals weregrown at neutral pH, which initially diffracted to 8.5 Å. Anelaborate dehydrating cryoprotection protocol and extensivescreening of the resulting crystals allowed us to collect diffractiondata at 4.35 Å (SI Appendix, Table S1). The crystals belong to thespace group P21 with four pentamers (about 6,200 residues) inthe asymmetric unit and a packing environment different fromthe one seen in the pH 4 structure (SI Appendix, Fig. S1).Noncrystallographic symmetry (NCS) averaging over the 20chains in the asymmetric unit together with the use of B-factorsharpening (12) significantly improved the quality of the initialelectron density map (Fig. 1A and SI Appendix, Fig. S2) andallowed the unambiguous reconstruction of the entire mainchain of the receptor, except for six out of 20 loop C (SI Ap-pendix, Fig. S3), and 65% of the side chains (Fig. 1A) with goodrefinement statistics (SI Appendix, Table S1).We observe that the TMD has lower B factors than the ECD

(SI Appendix, Fig. S4), which displays a large intrinsic structuralflexibility (Movie S1) and significant deviations from C5 sym-metry with a root-mean-square deviation (rmsd) of 1.2 ± 0.2 Åin the ECD compared with 0.2 Å in the TMD (SI Appendix, Fig.S5). The superimposition of the 20 different ECD structures ofthe pH 7 form shows that most of the variations on the tertiarylevel come from the loops: the β1–β2 loop and the region be-tween β2 and β5 (except for loop A), as well as β6–β7 (Cys loop),β8–β9 (loop F), and β9’–β10 (loop C), are more flexible than therest of the molecule (SI Appendix, Fig. S6A). However, the moststriking differences between the four pentamers come from theirquaternary structure: the rmsd between the six pairs of the fourcopies of the pH 7 pentamer is within 0.8–1.1 Å, much largerthan the rmsd of the open (Protein Data Bank ID code 4HFI)pentamer rotated upon itself using C5 symmetry (0.25 Å). Wewill return to a more detailed analysis of these structural fluc-tuations and their functional significance after the description ofthe quaternary changes between the neutral and acidic pH forms.

Quaternary Changes Between pH 4 and 7 in the ECD. Compared withthe open form, a concerted quaternary reorganization is observedin the ECD of the pH 7 structure, whereupon the separate mo-nomeric domains have undergone rigid-bodymotions with respectto the C5 symmetry axis (SI Appendix, Fig. S7A). The ECD–ECDinterface is reduced in the neutral pH conformation by about 180Å2 and the ECD–TMD interface by about 50 Å2 (SI Appendix,Table S2). Meanwhile the solvent exposure is increased by about80Å2 per monomer in the pore when it opens. The resulting effectis best described in terms of a differential radial (blooming) andtangential (twist) motions (Fig. 1 B and C) at different heightsabove the membrane plane (SI Appendix, Fig. S8A). These motionsare also revealed by coarse-grained Normal Mode Analysis (NMA)(SI Appendix, Fig. S8B): the third lowest frequency mode of theneutral pH form shows a combination of blooming and twist andaccounts for 47% of the difference vectors between the two forms,whereas the pH 4 form has two separate modes (again twist andbloom, but separated) that account for, respectively, 22%and25%ofthe transition. These modes have already been described as an im-portant componentof theallosteric transitionofpLGICs,on thebasisof NMA on only one form (13, 14) or by using ELIC as a model ofthe closed form (4). Here, however, the relative contribution of theblooming motion is markedly different. The remaining part of the

transition is explained by more local modes of higher frequency thataccount for the closure of the pore (Fig. 1 B andD).Comparing each pair of adjacent monomers of the pH 7 GLIC

structure after superimposition of only one monomer revealsthat the position of the next-neighbor ECD monomers differ by1.32 ± 0.6 Å (SI Appendix, Fig. S9) compared with 0.31 ± 0.16 Å inGLIC open state (pH 4), further illustrating the greater flex-ibility in the neutral pH structure than in the open structure ofGLIC (Movie S1). This plasticity in the ECD–ECD interactionswas already suggested by earlier studies on the isolated GLIC–ECD that crystallizes as a hexamer (15). Having at our disposala structural ensemble composed of 20 different dimer interfaces,we asked whether the conformational changes of one monomercorrelate with those of the neighboring subunit. Pair-wise cross-

7-8 loop (Loop B)

loop (Loop C)

6-7 loop (Cys-loop)

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Fig. 1. Superimposition of the pH 4 and pH 7 GLIC structures. (A) The Cαtrace of a full-length monomer of GLIC is represented in gray. The blue meshis the 2mFo–DFc NCS-averaged electron density map contoured at a level of1.5 σ. (B) Side view of a full-length monomer; superimposition of the openpH 4 (green) and closed pH 7 (red) GLIC structures; the black arrows show thedirection of the motion observed from the closed form to the open form. (C)Upper view of ECDs. (D) Upper view of TMDs. (E) Enlarged view of theorthosteric agonist binding site at the interface.

Sauguet et al. PNAS | January 21, 2014 | vol. 111 | no. 3 | 967

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correlation analysis reveals that the tip of loop C follows therigid-body motion of the inner β sheet of the neighboringmonomer, together with an interaction between the Cys loop andloop F (SI Appendix, Fig. S9). In the pH 4 form, loop C elicitsmore contacts with loop B of the same subunit than in the pH 7form, through a network of interactions between the side chainsof Arg77, Arg133, and Glu181 that is disrupted in the neutral pHform. Superimposition of the two dimers observed at pH 4 andpH 7 using all C-alpha atoms shows that the rearrangement ofthe interface of the orthosteric pocket (Fig. 1E) involves thesliding (translation) of the complementary subunit by about oneinterstrand distance, highlighting the importance of subunit in-terface plasticity in this allosteric transition. As a consequence,the change from pH 7 to pH 4 results in a constriction of theorthosteric pocket (SI Appendix, Fig. S10) and a better definedstructure of loop C. To analyze the potential functional signifi-cance of the fluctuations in the neutral pH crystal form, wecalculated the principal components (PCs) of the correspondingcovariance matrix, averaged over the 20 monomers. We thenprojected the difference vectors between the acidic and neutralpH forms onto the first PC. For the subunit–subunit fluctuations,the first PC has an overlap of 0.88 with the quaternary transitionin the ECD (SI Appendix, Fig. S11). This is consistent with a verypowerful idea in statistical physics (16) that states that the fluc-tuations of an unperturbed system prefigure the response of thissystem to an external perturbation (here the binding of the li-gand). Strikingly, we also found that the first PC of the monomerfluctuations in the pH 7 form has an overlap of 0.75 with the tertiarytransition in the ECD. We now describe these tertiary transitions.

Tertiary Changes Between pH 4 and pH 7 in the ECD. Like othermembers of pLGICs and acetylcholine binding proteins (AChBPs),the ECD of GLIC adopts a highly conserved Ig-like β sandwichcomposed of an inner and an outer β sheet, whose connectingloops play a critical role in channel function (1). Concerning theloops, the rmsd of each neutral pH monomer with the acidic pHform (SI Appendix, Fig. S6B) indicates that both loop C and loopF significantly differ in the two forms, whereas differences in theβ1–β2 loop and the region between β2 and β5 are less pronounced(Figs. 1B and 2A). This is consistent with the changes of both loopC and loop F occurring in the agonist-bound form, as described inboth AChBP and other pLGICs (17, 18). In addition, there aresome differences in the Cys loop and β10–M1 (pre-M1) regions,which play a critical role at the ECD–TMD interface.

Compared with the open form, we observe a tertiary structuralchange of the outer β sheet (Fig. 2A) that is characterized by theloss of the hydrogen bonding pattern typical of a β strand in β9’and β10 strands, accompanied by a vertical shift of loop C (Fig.2B). The relative movement of the inner and outer β sheetscauses the loss of a conserved salt bridge between residues Asp32in the β1–β2 loop and Arg192 in β10–M1 loop, as well as a re-distribution of the network of hydrophobic interactions betweenthe β1–β2 loop, loop F, the pre-M1, and the Cys loop (Fig. 2C).The disruption of the D32–R192 salt bridge by site-directedmutagenesis has been shown to drastically reduce the open–closed probability of the channel in nAChR and other pLGICs(19, 20). The β sheet’s movement was further analyzed to identifyrigid body parts and the rotation axis that relates them (SI Ap-pendix, Fig. S7B). It is necessary to distinguish between lowerouter β sheet and the rest—namely, the upper outer β sheet andthe inner β sheet. The rotation axis that describes their relativemovement goes through the hydrophobic core of the ECD, remi-niscent of, but not identical to the one determined by the super-position of alpha (ligand-bound) and nonalpha (unbound) subunit(s) structures obtained by cryo-EM on the muscle nAChR (21).Upon rotation of the inner β sheet with respect to the lower

outer β sheet, these two β sheets detach from each other, at thelevel of the ECD–TMD interface. This detachment createsa cavity involving 12 residues belonging to the Cys loop, the pre-M1, and loop F. These residues delineate a hydrophobic pocketin the core of the ECD (SI Appendix, Fig. S12), close to its in-terface with the TMD, and display a high level of conservationwithin the superfamily (22). In particular, the pocket is borderedon one side by two small hydrophobic residues, Cys27 andLeu30, and on the side of loop F (Leu157) by a larger aromaticresidue, either a conserved Phe/Tyr in the anionic subfamily orTrp160 in the cationic one. Interestingly, the latter Trp residue ispart of the conserved GEW sequence motif (SI Appendix, Fig.S13), which has been shown to be implicated in the binding ofregulatory Ca2+ ions (23). Strikingly, this region has also beenidentified as a binding site for other regulatory divalent ions inELIC (24), as well as a Bromoform molecule (25).

Changes in the TMD. In the pH 7 structure the radius of thetransmembrane pore is not compatible with (hydrated) ion flowand is thus in a nonconductive conformation (SI Appendix, Fig.S14). This is due to a concerted bending of the upper part of theM2 helices of all five subunits that obstructs the pore by forming

1-2 loop (Loop 2)

6 7 10

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Fig. 2. Tertiary reorganization observed in an ECDmonomer. (A) The structural elements that differ be-tween the pH 4 and pH 7 ECD GLIC structures areshown in green for pH 4 and in red for pH 7 GLIC.Other parts of the structures are in gray. ResiduesAsp32 and Arg192 are shown as sticks. The dashedline delineates the inner and outer β sheets. (B) En-larged view of the orthosteric agonist binding site il-lustrating the detachment of loop C from loop B. Theblue mesh is the 2mFo–DFc NCS-averaged electrondensity map contoured at a level of 1.5 σ. (C) Theinterface between the ECD and the TMD at the levelof the β1–β2, β6–β7, and β10–M1 loops. The dashedline indicates the distance between the Cα atoms ofAsp32 and Arg192.

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a tightly packed bundle, along with a revolving motion of theM2–3 loop that is similar to what is observed in the LC con-formation (7) (Fig. 1D). However, there are some differences withthe LC form, especially a 10° hinge motion of the N terminus ofM1 occurring at the level of a conserved proline residue (Pro204in GLIC). This hinge motion could be used, in conjunction withthe rotation described above, as collective variables to analyze thetransition pathway. In the pLGIC family this canonical proline inthe top of M1 disrupts the normal α-helix hydrogen bondingnetwork (26). The critical role of this portion of M1 in gating,along with M2, is supported by the role previously assigned to themain-chain carbonyl oxygen at position n-4 to this proline forbinding allosteric modulators such as ivermectin in GluCl (5) andethanol in GLIC (9). These movements of M1 and M2 result ina very different TMD–TMD interface, where now M2 helixinteracts more closely with the other subunit through helix M1rather than helix M2, in the open form (Fig. 1D and 4C).

Critical Residues at the ECD–TMD Interface. In the GLIC openconformation the C-terminal end of the M2–3 loop interacts withpre-M1 through hydrophobic interactions including side chainsof Phe195 and Met252. These interactions are lost in the GLICneutral pH structure due to the tilt of pre-M1 following thetertiary structural change in the ECD; in this process, Phe195 Cαposition changes, whereas Met252 does not (SI Appendix, Fig.S15). We tested the F195A mutant by electrophysiology andobserved a marked loss of function, despite normal expression inthe membrane, suggesting that this aromatic side chain con-tributes to the gating (Table 1 and SI Appendix, Fig. S16). Themutants Y194A and M252A are comparatively almost wild-typelike (SI Appendix, Fig. S16 and Table 1).In the open conformation Leu246 interacts with the ECD

through a hydrophobic cleft formed by the side chains of Phe116and Tyr119 from the β6–β7 loop and the backbone of the β1–β2loop (upward binding mode), whereas it interacts with the TMDthrough a hydrophobic cleft formed upon the relative detach-ment of M2 and M3 helices in the neutral pH conformation(downward binding mode). We generated the L246A mutationand observed a striking loss-of-function, as expected (Table 1). Itshould be noted that this position is highly conserved within thefamily (SI Appendix, Fig. S13) and site-directed mutagenesisexperiments performed on various pLGICs yield either stronggain or loss-of-function phenotypes (7).The loss of the Asp32–Arg192 salt bridge in the pH 7 form,

together with the redistribution of hydrophobic interactions be-tween the β1–β2 loop, loop F, the pre-M1, and the β6–β7 loop(Cys loop), locally destabilize the β1–β2 and β6–β7 loops, thusdiscouraging the upward binding mode of Leu246. We per-formed two mutants to probe the importance of the D32–R192

interaction for stabilizing the open form: D32N has a significantlyshifted pH50, whereas the double-mutant D32N–R192Q has amarked loss-of-function (SI Appendix, Fig. S16 and Table 1).Finally, there is a shift of the backbone in the loop F region that

causes a change of environment for Leu157 from a hydrophobicone to a more exposed one. We thus constructed the mutantL157A and tested it by electrophysiology; a marked loss of func-tion was observed, as predicted (SI Appendix, Fig. S16 and Table1). Overall there is a very good agreement between the predictedphenotypes of the designed mutants and the observed ones.

Open and LC Forms of GLIC Coexist in Crystals at pH 4.GLIC fused toa C-terminal tag made of 10 histidines (GLICHis10) was producedin insect cells, purified, and crystallized at pH 4. This resulted ina P212121 crystal form (SI Appendix, Fig. S1), distinct from thepreviously described C2 crystal form that contains the open struc-ture of GLIC (4), and this structure was solved at 3.35 Å resolution(SI Appendix, Table S1). Surprisingly, the electron density revealsthat both the open and the LC conformations coexist in the unitcell (Fig. 3A). Indeed, neither the open form alone nor the LC formalone fully satisfied the diffraction data, as strong peaks in theFourier difference map clearly indicated the occurrence of morethan one conformation of the upper half of M2 helices and theM2–M3 loops (SI Appendix, Fig. S17). After incorporation of thesetwo alternative conformations in the model, it was found they weresufficient to explain the data (there was no unexplained density leftin the Fo–Fc map). In a homopentamer, the LC and open formsare exclusive from each other, as neighboring chains displayingdifferent (open and LC) conformations would sterically clash. It istherefore likely that the crystal contains a mixture of pentamersadopting either the open or the LC conformations.To summarize, the unique structure of GLIC at low pH with the

LC and open forms coexisting as discrete forms in a crystal sug-gests that both conformations are in equilibrium when the ECD isin an agonist-bound conformation, displaying fluctuations that aresignificantly different in nature from the ones observed in theresting state (Fig. 3B).

Gating Transition Pathway: A Plausible Model. Previous attempts tomodel the gating transition assume that ELIC is a good modelfor the resting form of GLIC (27–29). Here we provide a modelbased on the crystal structure of the two end points of thetransition of the same pLGIC. To generate physically plausibletrajectories we used iENM (30), a coarse-grained method basedon a simplified energy landscape with two harmonic wells cen-tered on the start and end points of the transition. It was foundto predict the expected sequence of structural events on hemo-globin (31). It was used here to generate plausible coarse-grained(CA atoms only) trajectories between GLICpH7, GLICLC, andGLICOpen (Movies S2 and S3). Analysis of these trajectoriesreveals that the conformational changes at the tertiary andquaternary level happen roughly at the same pace (SI Appendix,Fig. S18A): the ECD transitions first, and the TMD follows (SIAppendix, Fig. S18B). Three main events can be distinguished,involving the sequential transition of three groups: (i) the innerand outer β sheets, most of their connecting loops, and the pre-M1; (ii) the Cys loop, loop M2–3, and the most extracellular partof the transmembrane helices; and (iii) the rest of the TMDlocated below the level of the conserved Proline in M1. Thecorresponding motions—in the reference frame of the full-lengthpentamer—are (i) an unblooming of the ECD, whose amplitudedecreases as the twisting motion increases, resulting in thereshaping of the ECD–ECD interfaces; (ii) initiation of channelopening at the level of the hydrophobic gate in the trans-membrane pore; and (iii) rigid-body rotation of the TMD, cor-responding to the end of the twisting motion (Fig. 4).When transitions from GLICpH7 toward either GLICLC or

GLICopen are compared, they appear to share the same initialconformational changes (SIAppendix, Fig. S18C): it is onlywhen theECD has experienced about 70% of its transition that both trajec-tories split apart and the TMD either stays closed or opens up. The

Table 1. Electrophysiology of GLIC mutants, showing the pH50,Hill-slope (nH), and lower and upper bound of Imax found for nreplicate oocytes (measured from at least three separateinjections)

Construct pH50 nH Imax, μA n

GLIC WT 5.3 ± 0.2 1.29 ± 0.06 −1.8<>−7.5 7D32E 4.6 ± 0.1 1.6 ± 0.2 −0.4<>−1.1 4D32N ≤4.0 2 ± 0.6 −0.4<>−1.7 7D32N/R192Q ND ND −0.2<>−0.3 4L157A ND ND −0.1<>−0.2 4Y194A 5.2 ± 0.3 1.7 ± 0.3 −3.7<>−7.8 4F195A ND ND −0.4<>−1.0 3L246A ND ND −0.1<>−0.3 3M252A 5.3 ± 0.4 1.2 ± 0.2 −1.9<>−7.8 4

ND represents nondeterminable pH50 as currents only appeared at pH 4,usually with very low amplitudes, and no dose–response curve could berecorded. Because fits for D32N did not plateau and resulted in many in-complete curves, the pH50 is not certain at 4.0 ± 0.1 and listed as ≤4.0.

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gating mechanism that emerges is thus concerted. It starts at theextracellular tip and propagates toward the cytoplasmic end. Thispredicted sequence of gating events is consistent with a wealth ofexperimental results collected on the nicotinic receptor eukaryotichomolog (32), even though details of the scenariomight differ fromone member of the family to another.

DiscussionThe structural reorganization observed upon pH activation inGLIC can now be analyzed in view of numerous functionalstudies accumulated over decades of research on other pLGICs.Upon activation, we observe a contraction of the orthosteric siteand a more extensive interface between neighboring subunits.This observation is in line with many studies on AChBP struc-tures that correlate agonist and antagonist binding to a contrac-tion or an expansion of the orthosteric site, respectively (17, 33).However, both the amplitude and the mechanism of this ex-pansion/contraction in GLIC differ from what is usually de-scribed in AChBP structures, especially because there is noquaternary rearrangement in the latter structures, whereas thereis a profound one in GLIC. It should also be mentioned that,based on the structure of muscle nAChR obtained by electronmicroscopy (21), a closure of loops B and C was also describedand suggested to occur upon activation, as observed by com-parison of alpha versus nonalpha subunits. Judging from ourstructures, a tighter association of loops A and B, which are lo-cated at the junction of the inner and outer β sheets, with loop C,whose tip remains attached to the complementary subunit in both

forms, may indeed constitute one of the keys of the local re-organization due to the agonist binding.The pore conformation observed in the neutral form of GLIC

is consistent with molecular dynamics (MD) studies of the TMDalone of GLIC (34), which showed closing events of the poreinvolving the bending of the top of helix M2. Furthermore,crosslinking M2 and M3 in their upper parts as in the open form(and not as in the resting form) results in a marked gain-of-function phenotypes (7). Also, it is striking that the M2–M1–M2interface specific to the pH 4 form coincides with the recentlyidentified binding site of a positive modulator (9) (Fig. 4).In addition, recent experimental studies on GLIC, obtained by

other groups, are also in line with the gating mechanism derivedfrom the GLIC X-ray structures presented here. Indeed, thesurface cysteine accessibility method (35), as well as site-directedspin labeling and electron paramagnetic resonance spectros-copy (36, 37), revealed that activation involves an outwardtranslational movement of the tip of M2 helices, whereas thelower part of these helices remains relatively immobile. The

M3

M2

M2-3 loopA B

C

Fig. 3. GLIC open and LC forms coexist at pH 4. (A) Ribbon representationof the GLIC receptor showing the equilibrium of the M2 helices and M2–M3loops corresponding to the coexisting LC (brown) and open (magenta)conformations in GLICHis10 structure. The rest of the structure is colored ingray. The receptor is viewed from the side, and the two front subunits areremoved for clarity. (B) Enlarged view showing the 2mFo–DFc electrondensity map surrounding the M2 helix and the M2–M3 loop contoured ata level of 1 σ (blue mesh). (C) Energy landscape inferred from the pH 7 crystalform (Upper, pink) and the pH 4 form (Lower, green) described here, foreach domain (TMD and ECD).

Fig. 4. A sequential model for the activation of pLGICs. (A) The reducedflexibility in the ECD when going from the pH 7 form (Left, pink) to the pH 4form (Right, green) is illustrated with superimposed atomic models. The qua-ternary associated twist and bloom motions are indicated with black arrows.The binding site of the agonist is shown in red. (B) Changes of the ECD–TMDinterface in both states are shown in a schematic way. Residues shown ex-perimentally to be involved in these interactions are indicated. The top of M2helix is in blue, and the N terminus of M1 is in yellow. (C) Schematic view fromthe top and down the C5 axis of the TMD conformation in both states. Thebinding site for a positive modulator (anesthetics) is shown in green.

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tilting of the top of helix M2 was also invoked in a recent paper asthe primary mechanism for the closure of the pore (36), ina manner similar to what we observe in the neutral pH form ofGLIC. Therefore, we conclude that the neutral pH GLIC structurepresented here possesses all of the structural features expectedfrom the resting-state form. We stress that the closure of the porein GLIC pH 7 is different from the one observed in ELIC in whichM2 and M3 move as a block and M4 appears decoupled from therest of the TMD. Actually, agonist binding in ELIC is accompa-nied by no significant structural change (38, 39), as would beexpected for a desensitized form. Further studies will beneeded to see if a crystal structure of GLIC can also be solvedin such a conformation.We note that other known allosteric systems (40), including

membrane receptors (41) also display a marked structural flex-ibility in the resting state and that some models of the voltage-gated ion channels postulate diffusion in a flat energy landscapefor this form (42). In the large-scale allosteric transition ofadenylate kinase, it was recently found that the conformationalbasin of the closed form is much larger than previously assumedand wider than in the open state (43), as inferred here (Fig. 3).This would also imply a large entropy in the number of preex-isting transition pathways, which was predicted to lower thefree-energy barrier of allosteric transitions (44). Furthermore,the analysis of the trends of the fluctuations of the resting forms

in GLIC explains a large part of the transition toward the openform, as predicted by linear response theory (16). On the otherhand, the coexistence of the LC and open forms in the samecrystal as discrete states fits well with the allosteric schemepostulated long ago for these receptors (1, 45). Here we pro-vide a plausible pathway based on a coarse-grained model (30).More detailed models of the transition using all-atoms modelswill be needed to understand the exact role of pH in thetransition and will be reported elsewhere. The validation ofthese possible pathways will require experimental data such asthose available on nAChR (32).

Materials and MethodsGLIC and GLICHis10 were expressed and purified as in ref. 8. Crystals underwenta special dehydration protocol before cryocooling. Electrophysiology record-ings were performed by expressing GLIC and its variants in Xenopus laevisoocytes as in ref. 10. Full methods are provided in SI Appendix.

ACKNOWLEDGMENTS. We thank L. Malherbe for preparing some of themutants. We thank J. Gouge for help in data collection, M. Prevost for sharingdata on mutants, and G. Bricogne (Global Phasing) for sharing expertise onrefinement in Buster. We thank the Platform for Protein Expression of theProteopole in the Institut Pasteur (respectively, J. Bellalou and S. Petres) as wellas the staff of synchrotron beamlines in both ESRF (Grenoble) and Soleil (Orsay).We gratefully acknowledge the financial support of ANR (Grant NicoChimera)andDARRI (Institut Pasteur). L.S. was funded by a postdoctoral Roux fellowship.

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