Allosteric binding site in a Cys-loop receptorligand-binding domain unveiled in the crystal structureof ELIC in complex with chlorpromazineMieke Nysa, Eveline Wijckmansa, Ana Farinhaa, Özge Yolukb,c, Magnus Anderssonb,c, Marijke Bramsa,Radovan Spurnya,1, Steve Peigneurd, Jan Tytgatd, Erik Lindahlb,c,e, and Chris Ulensa,2

aLaboratory of Structural Neurobiology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium; bScience for Life Laboratory, Stockholm and Uppsala,SE-17121 Stockholm, Sweden; cTheoretical and Computational Biophysics, Department of Theoretical Physics, Kungliga Tekniska Högskolan Royal Instituteof Technology, SE-17121 Stockholm, Sweden; dLaboratory of Toxicology and Pharmacology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium; andeDepartment of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, SE-17121 Stockholm, Sweden

Edited by Jean-Pierre Changeux, CNRS, Institut Pasteur, Paris, France, and approved August 22, 2016 (received for review February 24, 2016)

Pentameric ligand-gated ion channels or Cys-loop receptors areresponsible for fast inhibitory or excitatory synaptic transmission.The antipsychotic compound chlorpromazine is a widely usedtool to probe the ion channel pore of the nicotinic acetylcholinereceptor, which is a prototypical Cys-loop receptor. In this study,we determine the molecular determinants of chlorpromazinebinding in the Erwinia ligand-gated ion channel (ELIC). We reportthe X-ray crystal structures of ELIC in complex with chlorpromazineor its brominated derivative bromopromazine. Unexpectedly, wedo not find a chlorpromazine molecule in the channel pore of ELIC,but behind the β8–β9 loop in the extracellular ligand-binding do-main. The β8–β9 loop is localized downstream from the neurotrans-mitter binding site and plays an important role in coupling of ligandbinding to channel opening. In combination with electrophysiolog-ical recordings from ELIC cysteine mutants and a thiol-reactive de-rivative of chlorpromazine, we demonstrate that chlorpromazinebinding at the β8–β9 loop is responsible for receptor inhibition.We further use molecular-dynamics simulations to support theX-ray data and mutagenesis experiments. Together, these dataunveil an allosteric binding site in the extracellular ligand-bind-ing domain of ELIC. Our results extend on previous observationsand further substantiate our understanding of a multisite modelfor allosteric modulation of Cys-loop receptors.

ligand-gated ion channel | X-ray crystallography | allosteric modulation |Cys-loop receptor | nicotinic acetylcholine receptor

Chlorpromazine (CPZ) (Fig. 1), a phenothiazine-derived an-tipsychotic drug, was introduced in psychiatry in the early

1950s, revolutionizing the treatment of psychotic disorders (1, 2).The main mechanism of action of CPZ consists in the blockageof dopamine receptors (2–4), but the numerous side effects as-sociated with this drug indicate that it interacts with other phys-iologically relevant targets. CPZ was indeed shown to interferewith several voltage- and ligand-gated channels: it inhibits neu-ronal voltage-gated K+ channels (5–7), BKCa channels (8), andthe human α1E subunit-mediated Ca2+ channels (9); CPZ wasalso shown to inhibit GABAergic currents (10, 11), specificallythrough GABAA receptors (GABAARs) (12), and to inhibit se-rotonin type-3 receptors (5-HT3Rs) (13, 14) and nicotinic ace-tylcholine receptors (nAChRs) (15, 16), members of the Cys-loopreceptor family.The Cys-loop receptor family is composed of membrane-

spanning ligand-gated ion channels that are responsible for fastexcitatory or inhibitory synaptic neurotransmission. They arecomposed of five identical or nonidentical subunits, each of themcomprising an N-terminal extracellular domain, which containsthe neurotransmitter binding site, four transmembrane helices,that when assembled allow ions to pass through the membrane,and an intracellular domain, responsible for channel conductance,receptor modulation, and trafficking (17, 18). Initial structural

insight into the mechanism of Cys-loop receptor function derivesfrom cryo-EM images of the Torpedo marmorata nAChR (19–22)as well X-ray crystal structures of the acetylcholine binding protein(AChBP) (23, 24). AChBPs are water-soluble homologs of theextracellular ligand-binding domain of the nAChR and lack thepore-forming transmembrane domain. To date, more than 100cocrystal structures of AChBP in complex with different agonists,partial agonists, antagonists, and allosteric modulators have beendetermined, creating a wealth of information on the moleculardeterminants of ligand recognition in nAChRs (25). Subsequently,the identification of Cys-loop receptors in prokaryotes (26)allowed the first X-ray structure determination of integral Cys-loop receptors Erwinia ligand-gated ion channel (ELIC) (27) andGloeobacter ligand-gated ion channel (GLIC) (28, 29), which likelyrepresent a nonconducting and conducting conformation of thechannel pore, respectively. Later on, X-ray crystal structures weredetermined for the first eukaryote Cys-loop receptors, includingthe Caenorhabditis elegans glutamate-gated chloride channel GluCl(30, 31), the human β3 GABAAR (32), and the mouse 5-HT3AR

Significance

Cys-loop receptors belong to a family of ion channels that areinvolved in fast synaptic transmission. Allosteric modulators ofCys-loop receptors hold therapeutic potential as they tweakreceptor function while preserving the normal fluctuationsin neurotransmitter signaling at the synapse. Here, we takeadvantage of a model Cys-loop receptor, the Erwinia ligand-gated ion channel (ELIC). We determined cocrystal structuresof ELIC in complex with chlorpromazine (IC50, ∼160 μM) andits brominated derivative bromopromazine, which unveil anallosteric binding site localized at the interface between theextracellular ligand-binding domain and the pore-formingtransmembrane domain. Our results demonstrate that thedifferent allosteric binding sites present in Cys-loop receptorsform an almost continuous path stretching from top to bottomof the receptor.

Author contributions: C.U. designed research; E.W., A.F., Ö.Y., M.A., M.B., R.S., S.P., and C.U.performed research; M.N., E.W., A.F., Ö.Y., M.A., M.B., R.S., S.P., J.T., E.L., and C.U. analyzeddata; and M.N., E.W., A.F., Ö.Y., M.A., M.B., J.T., E.L., and C.U. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org [PDB ID codes 5LG3 (ELIC+CPZ) and 5LID (ELIC+BrPZ)].1Present address: Structural Virology, Central European Institute of Technology, MasarykUniversity, 62500 Brno, Czech Republic.

2To whom correspondence should be addressed. Email: chris.ulens@kuleuven.be.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1603101113/-/DCSupplemental.

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(33). More recently, the cryo-EM structure of the α1 GlyR wasdetermined in closed, open, and desensitized conformations (34).Finally, the X-ray crystal structure of the α3 GlyR was determinedin a strychnine-bound state (35).In this study, we take advantage of the ELIC, a prokaryote

homolog of vertebrate Cys-loop receptors, which is activatedby primary amine molecules, including GABA (36, 37). Theavailability of several relatively high-resolution X-ray cocrystalstructures of ELIC in complex with known ligands renders thischannel a relevant model for the study of Cys-loop receptormodulation (36, 38–42).CPZ, referred to as a noncompetitive antagonist of nAChRs

(16), was also found to inhibit GABA-evoked responses in ELIC—although with low potency (IC50, >100 μM)—suggesting a dis-tinct pharmacology (43). Initial studies aimed at identifying themolecular determinants of CPZ binding in nAChRs showedthat [3H]CPZ binds to the channel pore (44). Subsequently,[3H]CPZ became a widely used tool to probe the channel pore ofnAChRs in closed, open, and desensitized states (16, 45–50).Initial photoaffinity labeling studies on the Torpedo nAChR inthe desensitized state revealed that CPZ binds to a high-affinitybinding site near the cytoplasmic end of the channel pore, com-prising the 2′, 6′, and 9′ positions of the pore-lining M2-helix (46–49). Recently, Chiara et al. (50) extended on these observationsand identified an additional binding site in the desensitized statefor [3H]CPZ near the extracellular end of the channel pore,comprising the 16′, 17′, and 20′ positions of the M2 segment. Inthe closed state, [3H]CPZ labeling was observed at 5′, 6′, and 9′,with no labeling at 2′ (50). Additionally, a binding site for CPZwas identified in the intracellular domain, as photoaffinity la-beling was also observed for residues αMet-386 and αSer-393,which are localized in the intracellular MA-helices (50). In con-trast, in 5-HT3Rs CPZ acts directly on the neurotransmitterbinding site (13, 14) and competitively antagonizes the actionof serotonin.In this study, we set out to investigate the structural deter-

minants of CPZ binding in ELIC. To facilitate structural studies,we used a brominated derivative of CPZ, termed bromoproma-zine (BrPZ) (Fig. 1B). Here, we report the X-ray crystal struc-

tures of ELIC in complex with CPZ or BrPZ at 3.7 Å resolution.We further characterize this interaction using two-electrode voltage-clamp (TEVC) recordings with a thiol-reactive methanethiosulfo-nate analog of CPZ (MTS-PZ) (Fig. 1C) on ELIC expressed inXenopus oocytes and also perform molecular-dynamics simula-tions of the complex. Together, our results expand our currentunderstanding of allosteric modulation in the family of pen-tameric ligand-gated ion channels.

ResultsX-Ray Crystal Structures of ELIC in Complex with CPZ or BrPZ. Inagreement with previous observations (43), we determined thatCPZ inhibits ELIC expressed in Xenopus oocytes with an IC50value of 158 ± 37 μM and a Hill coefficient of 1.6 ± 0.5 (n = 3–13; Fig. 1 D and E). To investigate the structural determinants ofCPZ recognition in ELIC, we determined the X-ray cocrystalstructures of ELIC in complex with CPZ or BrPZ (crystallo-graphic statistics are reported in Table S1). We obtained dif-fraction data to a resolution of 3.7 Å and took advantage of thebromine atom in BrPZ to collect anomalous diffraction data,which allowed us to calculate a so-called anomalous differencedensity map and identify the density of the anomalously scat-tering electrons around the bromine atoms even at mediumresolution. Both the fivefold averaged simple electron differencedensity maps (Fo-Fc) as well as the fivefold averaged anomalousdifference density map allowed us to localize two distinct loca-tions for the binding of CPZ or BrPZ (Fig. 2). Unexpectedly, wedo not observe any electron density in the pore domain of thechannel. Instead, we observe simple difference density in theextracellular ligand binding domain of ELIC at a site that is lo-cated near to the β8–β9 loop (Fig. 2 A–C). In eukaryote recep-tors, this loop together with the Cys-loop, the M2–M3 loop, andthe pre-M1 region form the interface between the ligand bindingdomain and the pore domain of the channel. At each of the fivesites in the pentamer, the simple difference density displays acurved shape (6σ) consistent with the curvature of the tricyclic10H-phenothiazine ring in CPZ. The electron density for thedimethylpropylamino-moiety is visible only at lower σ levels,indicating it is more disordered in the crystal structure. Thebinding location of CPZ at this site is further substantiated by thepresence of a strong anomalous peak (10σ) at each of the fivesites of the pentamer in the ELIC+BrPZ cocrystal structure(Fig. 2 D–F). Importantly, the β8–β9 loop undergoes a confor-mational rearrangement to accommodate CPZ at this site, whichwill be discussed in further detail below. Additionally, in theELIC+BrPZ cocrystal structure, we also observe anomalousdifference density at a second location in the extracellular ligandbinding domain, namely at the agonist binding site, which is lo-cated at the interface between each of two subunits (Fig. 2G).This site is lined by highly conserved aromatic residues localizedon historically designated “loops,” termed loops A–B–C on theprincipal face of the binding site and loops D–E–F on thecomplementary face of the binding site. The highly conservedaromatic residues at this site form a so-called aromatic box,which creates an electronegative environment for agonist rec-ognition. In ELIC, this site includes Y38 (loop D), F133 (loopB), and Y175 and F188 (both loop C). In the ELIC+BrPZstructure, the anomalous difference density at this site is slightlyoffset toward Y38 and F133, suggesting a possible location of thebromine atom in BrPZ. Importantly, the binding of BrPZ at thissite is consistent with the earlier observation that CPZ bindsdirectly at the neurotransmitter binding site in the related 5-HT3receptor (13, 14). Together, the ELIC X-ray crystal structuresreveal that CPZ and BrPZ bind at two distinct sites in the ex-tracellular domain, but not in the pore-forming transmembranedomain. The two binding sites are localized at functionally im-portant domains, namely the β8–β9 loop, which contributes tocoupling of ligand binding to channel opening, and the agonist

Fig. 1. Structure and function of chlorpromazine (CPZ) and analogs. (A–C)Chemical structures of CPZ, bromopromazine (BrPZ), and methanethiosul-fonate-promazine (MTS-PZ), respectively. (D) Electrophysiological recordingsfrom Xenopus oocytes expressing ELIC. Channels were activated by the ap-plication of the agonist GABA at the EC50 (20 mM). In the presence of 30 μMCPZ, this response was reduced. (E) Concentration–inhibition curve for CPZon ELIC. Averaged data ± SEM are shown for three to nine different oocytes.

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binding site, which contains the structural determinants for ag-onist recognition. The lack of CPZ binding in the pore domain ofELIC can likely be explained by the presence of an unusual andbulky phenylalanine residue at the extracellular end (F16′) of theELIC channel pore that restricts pore access of known poreblockers, including memantine (40) and likely also CPZ.

Conformational Change of the β8–β9 Loop. The CPZ-bound ELICstructure superimposes well with the apo form of ELIC [ProteinData Bank (PDB) ID code 2VL0] with a rmsd of 0.9 Å for 2,954out of 3,070 aligned residues. This suggests that the conforma-tional state of ELIC remains unaltered after CPZ binding andcorresponds to a closed nonconductive conformation of the re-ceptor (27). However, detailed inspection of the simple electrondensity map (2Fo-Fc) reveals structural differences in the β8–β9loop of the CPZ-bound structure, which was manually rebuiltand refined (Fig. 3A). A detailed view of a monomeric subunitsuperimposed for apo ELIC (yellow) and ELIC+CPZ (blue) isshown in Fig. 3B. As stated above, the overall structure ofELIC+CPZ is nearly identical to apo ELIC, except for a changein the β8–β9 loop. Most of the conformational change can beobserved in the descending part of the β8–β9 loop, includingresidues Y148 to E155. Residues involved in forming the interfacewith the M2–M3 loop of the neighboring subunit as well as the

ascending part of the β8–β9 loop remain unaltered. Detailedanalysis of the interactions between CPZ and residues of the β8–β9 pocket reveals a wide range of mostly hydrophobic interactions(Fig. 3C). These include I20, N21, and I23 on the β1-strand, F126on the β7-strand, and V147, T149, E150, E155, D158, W160, andI162 on the β8–β9 loop. Weak hydrogen bonds are formed be-tween the dimethylamino-moiety of CPZ and the side-chain oxy-gen atoms of D158 (indicated with dashed lines in Fig. 3C) andbetween the 10H-phenothiazine nitrogen and the main-chaincarbonyl atom of I23.

Cysteine-Scanning Mutagenesis of the β8–β9 Loop Binding Site. Toexplore the contribution of individual amino acids in the β8–β9loop binding site to molecular recognition of CPZ, we in-dividually mutated each residue involved in the CPZ interactionto a cysteine residue in the background of a Cys-less ELIC var-iant, which is functionally identical to wild-type ELIC (Table 1).To determine the effect of CPZ binding at this specific site, andnot elsewhere in the protein, we used a thiol-reactive analog ofCPZ termed MTS-PZ.First, we investigated the effect of the cysteine mutation alone

on the function of ELIC by expressing each mutant in Xenopusoocytes and investigating the response to the agonist GABAusing TEVC. All mutants were functional and responded to

Fig. 2. X-ray crystal structures of ELIC in complex with chlorpromazine (CPZ) and bromopromazine (BrPZ). Side view (A) and top view (B) of ELIC in complexwith CPZ in blue ribbon representation. The green mesh represents fivefold averaged Fo-Fc difference electron density contoured at a level of 6σ. The Inset (C)shows a detailed view of the β8–β9 loop binding site and its location relative to the Cys-loop, the M2–M3 loop, and the pre-M1 region. CPZ is shown in stickrepresentation. Yellow is carbon, blue is nitrogen, green is chlorine, and orange is sulfur. Side view (D) and top view (E) of ELIC in complex with BrPZ in blueribbon representation. The red mesh represents fivefold averaged anomalous difference electron density contoured at a level of 10σ. The Insets show adetailed view of the β8–β9 loop binding site (F) and the agonist binding site (G).

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application of GABA. For each mutant, we determined a GABAconcentration–activation curve and calculated EC50 values (Table 1).We observe that EC50 values varied from a threefold decrease inmutant I20C, to a fivefold increase in mutant W160C. EC50 valueswere statistically compared between Cys-less ELIC and all of theCys-mutants (Table 1): for mutants I20C, F126C, T149C, D158C,and W160C, EC50 values were significantly different from Cys-lessELIC; the Hill coefficients of the entire set of mutants were notsignificantly different from Cys-less ELIC. These results pointtoward a functional role of β8–β9 loop residues to channel gat-ing, which is consistent with the β8–β9 loop’s established con-tribution to channel gating. In addition, we observe that allmutants express at levels that are comparable to wild-type ELICexcept for W160C, which expresses severalfold lower, sug-gesting that this mutation critically affects protein folding and/or trafficking.Next, we investigated the functional effect of MTS-PZ binding

at each of the individual Cys-mutants. To accomplish this, weused a protocol in which ELIC displayed stable channel activa-tion following two consecutive applications of GABA at theEC50. Next, we perfused the oocyte with 200 μM MTS-PZ for2 min and washed out unreacted MTS-PZ during a 30-s washout.The change of channel activation after MTS-PZ modificationwas then measured with a third application of GABA at theEC50. As expected, MTS-PZ did not affect the amplitude ofthe GABA response in Cys-less ELIC (Fig. 4). We observed thatthe GABA response was significantly reduced in four mutants,namely, E150C (34.0% ± 10.0, n = 3), D158C (52.0% ± 6.9, n =3), W160C (55.0% ± 6.2, n = 3), and I162 (41.0% ± 13.0, n = 3).This is consistent with the ligand-binding pose in the ELICcocrystal structure, which puts the thiol-reactive moiety of MTS-PZ (equivalent to the dimethylamino-moiety in CPZ) in closeproximity to these residues. For the seven other mutants, MTS-PZ application did not affect the GABA response. This indicatesthat either the mutant did not react with MTS-PZ due to the

increased distance between the MTS moiety and the sulfhydrylside chain, or that the mutant reacted with MTS-PZ but did notfunctionally affect the channel. In conclusion, we demonstratethat covalent modification of residues E150C, D158C, W160C,and I162 in Cys-less ELIC with MTS-PZ results in functionalinhibition of the GABA-evoked channel response. This resultsuggests that CPZ binding at the β8–β9 loop binding site is in-volved in negative allosteric modulation of ELIC.

Binding Stability of CPZ. To characterize the stability of CPZ bindingand its effect on surrounding loops, two molecular-dynamicssimulations were performed with and without CPZ bound to thestructure (labels CPZ and apo, respectively).Except for one subunit, the binding of CPZ was stable and the

molecule remained within the allosteric binding site as measuredby the distance from F126 and W160 to the C11 atom of CPZ(Fig. 5 A and B). The cavity volume, on the other hand, increasedby 250 Å3 in CPZ-bound simulations compared with the crystalstructure (893 Å3) (Fig. 5C), whereas the volume of the samecavity in apo simulations decreased by 50 Å3. The increase incavity volume did not disrupt the weak hydrogen bond interac-tions of CPZ with the surrounding loops. The CPZ moleculesspent on average ∼12% of the simulation time in contact withthe β8–β9 loop (residues 148–162) and the β1-strand (residues22–23) and ∼2% with the Cys-loop (residues 113, 126) (Fig. 5D).The predicted hydrogen bond interactions of CPZ with the β8–β9 loop include two residues, T149 and E155, that were not la-beled by MTS-PZ when mutated to cysteine. Although theseresidues are in proximity of the bound CPZ, the conformationssampled by the side chains were not as favorable for interactionsas other residues, that is, E150 and D158 (Fig. 5 E and F). To-gether, the molecular-dynamics simulations support a ligand-induced conformational change of the β8–β9 loop forming anallosteric binding site for CPZ. The reactivity of the specific sidechains identified in the cysteine-scanning mutagenesis experi-ments is consistent with the rotamers sampled by these residuesin simulations.Because the molecular-dynamic simulations indicate an in-

crease in the allosteric CPZ-binding pocket, which is larger thannecessary to adapt CPZ, additional electrophysiological experi-ments were conducted to exclude the possibility of nonspecificCPZ-binding. IC50 values were determined for 12 additionalphenothiazine analogs, and some of these compounds indeedexhibited significantly different IC50 values, indicating that thebinding of phenothiazine analogs occurs in a structure-dependentmanner (Table S2). Additionally, we observed that the presenceof a piperazine group at position R1 gave rise to more potent

Fig. 3. Conformational change of the β8–β9 loop in ELIC. (A) Stereo rep-resentation of the β8–β9 loop in ELIC. The ELIC backbone is shown as greenribbon. Chlorpromazine (CPZ) is shown in yellow sticks. The blue mesh issimple electron density (2Fo-Fc) contoured at a level of 1.4σ. (B) Superposi-tion of a single monomer of apo ELIC in yellow (PDB ID code 2VL0) and ELICin complex with CPZ in cartoon representation. The Inset shows a detailedview of the β8–β9 loop. CPZ is shown in sphere representation. White iscarbon, blue is nitrogen, green is chlorine, and orange is sulfur. (C) Detailedview of amino acids involved in ligand interactions with CPZ. Dashed linesindicate hydrogen bonds. CPZ is shown in stick representation.

Table 1. Summary of the functional characterization of WT andmutant ELIC: GABA EC50 and nH and Imax ± SEM

ELIC construct EC50, mM nH Imax, μA n

Wild-type ELIC 21 ± 1.0 2.1 ± 0.20 21 ± 2.4 4Cys-less ELIC 23 ± 3.2 2.3 ± 0.70 16 ± 1.9 4I20C 62 ± 8.0* 2.0 ± 0.40 3.8 ± 0.70 5N21C 30 ± 5.3 2.1 ± 1.0 1.3 ± 0.10 3I23C 24 ± 3.6 2.3 ± 0.80 4.9 ± 2.90 2–3F126C 14 ± 0.5* 2.2 ± 0.10 6.9 ± 3.60 3–5V147C 41 ± 12.2 1.7 ± 0.90 21 ± 5.8 3T149C 16 ± 1.1* 2.4 ± 0.30 31 ± 3.1 3E150C 20 ± 7.8 2.0 ± 1.30 6.2 ± 1.40 3E155C 20 ± 2.7 2.7 ± 0.70 11 ± 1.9 3–5D158C 8.5 ± 2.00* 3.0 ± 2.30 6.6 ± 1.30 2–4W160C 3.5 ± 0.30* 3.1 ± 0.80 0.20 ± 0.07 3–4I162C 22 ± 5.5 2.3 ± 1.10 6.6 ± 3.20 3

*P < 0.05, significantly different from Cys-less ELIC, Student’s t test.

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inhibitors, most likely due to an increase of the interacting in-terface with the expanded CPZ-binding pocket.

DiscussionIn the present work, we identify an allosteric binding site in theextracellular domain of the Erwinia pentameric ligand-gated ionchannel ELIC using X-ray crystallography. In combination withcysteine-scanning mutagenesis and electrophysiological record-ings of ELIC expressed in Xenopus oocytes, we demonstrate thatthe identified β8–β9 loop site is involved in negative allostericmodulation of ELIC. These results are further supported withmolecular-dynamics simulations, which confirm our observationsin the crystal structure and the mutagenesis experiments. Theseresults extend on previous observations of allosteric binding sitesin different Cys-loop receptors and substantiate our understandingof a multisite model of allosteric modulation in this family ofion channels.We here discuss our results in the context of previously de-

termined Cys-loop receptor crystal structures in which allostericbinding sites were revealed (Fig. 6 A–D). To enhance clarityin this figure several structures were grouped and classified as“closed,” “open,” and “desensitized,” although we emphasizethat subtle and important differences exists in, for example, theGLIC locally closed state (51) and the GluCl apo state (31),which we both classified as closed structures, but most likelyrepresent “intermediate” conformational states (31, 51). For adetailed discussion of the known conformational differences incurrently available Cys-loop receptor structures, we refer to re-cent reviews (52, 53). Notably, several additional sites have beenidentified using other methods, such as photoaffinity labelingand mutagenesis, but due to space limitation these results extendbeyond the scope of the current discussion. Finally, it should benoted that the agonist binding site loop F, which precedes theβ8–β9 loop site, adopts significantly different conformations in

different Cys-loop receptors, including ELIC and different formsof GLIC.First, we discuss allosteric binding sites unveiled in a chimera

of the Lymnaea AChBP and the ligand binding domain of the α7nAChR, α7-AChBP (green ribbon, Fig. 6A). Using a fragment-based screening approach, Spurny et al. (54) discovered threeallosteric binding sites in α7-AChBP, which are remote from theorthosteric binding site occupied by the agonist lobeline in thesestructures (yellow spheres, Fig. 6A), and also the agonist epi-batidine (55) or the competitive antagonist α-bungarotoxin inother α7-AChBP cocrystal structures (56). One fragment mole-cule, fragment 1, was identified that binds at the interface be-tween the N-terminal α-helix and a loop that corresponds to themain immunogenic region (MIR) in the α1 muscle nAChR(white spheres, Fig. 6A). This site was termed the “top site” andis involved in negative modulation of the α7 nAChR (54). Thesame fragment molecule also occupies an allosteric binding sitethat is located just below the orthosteric binding site and that wastermed the “agonist subsite” (pink spheres, Fig. 6A) (54). Thissite corresponds to the ketamine binding site reported in theGLIC (57), where ketamine also binds just below the orthostericagonist binding site and is involved in inhibition of GLIC (pinkspheres, Fig. 6C). Another fragment molecule, fragment 4, wasidentified that occupies an allosteric binding site accessible fromthe vestibule of the receptor and was termed the “vestibule site”(firebrick spheres, Fig. 6A) (54). This fragment is involved in

Fig. 5. Binding stability of CPZ in simulations. (A and B) Binding of CPZmeasured by the distance from F126 and W160 to the C11 atom of CPZ (solidand dashed lines, respectively) per subunit. (C) Allosteric cavity volume inpresence and absence of CPZ; the crystal structure value is indicated by thedashed line. (D) Average hydrogen bond interactions of CPZ. (E) Probabilitydistribution of side-chain angles relative to CPZ; measured from Cβ–Cα–NC2atom positions. (F) MTS-PZ–mediated inhibition (purple, no significant ef-fect; green, inhibition). Outliers (T149, E155) are marked with black arrows.

Fig. 4. Cysteine-scanning mutagenesis of the β8–β9 loop in ELIC. (A) Elec-trophysiological recordings of Cys-less ELIC in response to repetitive pulses ofGABA at the EC50 (=20 mM) and application of 200 μM of a thiol-reactive CPZderivative termed MTS-PZ. (B) Example traces of a Cys mutant, E155C,showing no effect of MTS-PZ. (C) Example traces of a Cys mutant, D158C,showing an inhibitory effect of MTS-PZ. (D) Summary of MTS-PZ–mediatedchannel inhibition on the different Cys mutants. Data represent the mean ±SEM of three to five experiments. *P < 0.05, significantly different from Cys-less ELIC, Student’s t test; **P < 0.01, significantly different from Cys-lessELIC, Student’s t test.

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negative modulation of the α7 nAChR (54). The same site wasunveiled in the crystal structure of the ELIC in complex withflurazepam (firebrick spheres, closed state, Fig. 6B), which isinvolved in positive modulation of ELIC (36). The importance ofthe same site was also confirmed in the crystal structure of GLIC incomplex with acetate (firebrick spheres, open state, Fig. 6C) (58).Second, we discuss allosteric binding sites unveiled in the closed

state of Cys-loop receptors, and we show the ELIC structure as arepresentative example because most of the available modulatorcocrystal structures have been determined with this ion channel(green ribbon, Fig. 6B) (27). Similar to other Cys-loop receptors,the orthosteric binding site in ELIC is occupied by the partialagonist GABA (36) or the competitive antagonist bromo-flur-azepam (yellow spheres, Fig. 6B) (36). The orthosteric site is alsooccupied by the competitive antagonist strychnine in the closed α3GlyR (35) and closed α1 GlyR structures (34). The occupancy ofthe vestibule site by flurazepam in ELIC was already mentioned inthe previous paragraph (firebrick spheres, Fig. 6B) (36). It wasreported that ELIC is negatively modulated by divalent cations,including Ca2+ and Ba2+, and it was demonstrated that thesecations bind at three distinct binding sites (39) (red spheres,Fig. 6B). The first Ba2+ site is located at the outer rim of the“vestibule pocket” where it is coordinated by two residues at theend of the β4-strand, namely S84 on the principal subunit and D86

on the complementary subunit (39) (red spheres, Fig. 6B). Thissite overlaps with a Cs+ binding site in the GLIC A13′F mutant(59) and a Ni2+ binding site in wild-type GLIC (60) (red spheres,open state, Fig. 6C). A second Ba2+ site is located at the subunitinterface about 15 Å below the orthosteric agonist site (redspheres, Fig. 6B). This site is formed by residues at the end of theβ6-strand on the principal subunit and the loop connecting the β8-and β9-strand on the complementary subunit (39). This site is just8 Å distant from the CPZ binding site (β8–β9 loop) reported inthis paper (cyan spheres, Fig. 6B). The third Ba2+ site is located atthe extracellular entrance of the channel pore where it binds atthe 20′ position of the M2-helix (39) (red sphere, Fig. 6D). An-other allosteric binding site near to the CPZ binding site is oc-cupied by either bromoform in ELIC (magenta sphere, Fig. 6B)(41) or xenon in locally closed GLIC (gray sphere, Fig. 6B) (61).Additional binding sites for xenon in locally closed GLIC arelocated at the intrasubunit general anesthetic binding site (bluesphere, Fig. 6B), the inner-interfacial sites (gray spheres, Fig. 6B),and the outer-interfacial sites (gray spheres, Fig. 6B) (61). Finally,xenon also occupies the 9′ pore site in locally closed GLIC (graysphere, Fig. 6B) (61). Two additional binding sites for bromoformhave been localized in the ELIC cocrystal structure, namely at anintersubunit transmembrane site (magenta spheres, Fig. 6B) andat the 13′ pore site (magenta sphere, Fig. 6B) (41). The 13′ pore

Fig. 6. Overview of allosteric binding sites in different conformational states of Cys-loop receptors. Overview of allosteric binding sites in the closed, open,and desensitized states of Cys-loop receptors. (A) Green ribbon representation of α7-AChBP structure as an example of “ligand binding domain-only”structures (54). (B) Green ribbon presentation of the ELIC ion channel as a representative example of a closed state (27). (C) Green ribbon presentation of theGluCl+ivermectin ion channel structure as a representative example of an open state (30). (D) Green ribbon presentation of the β3 GABAA receptor structureas a representative example of a desensitized state (32). Allosteric modulators identified in the different conformational states are shown in sphere rep-resentation. Identical color codes have been used for overlapping sites in the different states, for example, orthosteric site in yellow, vestibule site in firebrick,etc. Detailed explanation of PDB ID codes, allosteric modulator color codes, and references for all structures used in this figure are given in Table S3.

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site for bromoform overlaps with one of the pore sites (13′) of theanesthetic isoflurane site in ELIC (light blue spheres, Fig. 6B)(42). Isoflurane simultaneously occupies the 6′ pore site in ELIC.The pore blocker memantine has been identified at the 16′ poreposition in ELIC F16′S (orange spheres, Fig. 6B) (40).Third, we discuss allosteric binding sites identified in the open

state of Cys-loop receptors, and we show the GluCl+ivermectincrystal structure as a representative example (green ribbon,Fig. 6C). In GluCl, the orthosteric binding site is occupied byglutamate (yellow spheres, Fig. 6C) (30), and in open GLIC, thissite is occupied by acetate (58). Allosteric binding sites for xenonhave been determined for open GLIC, which overlap with thosedescribed in locally closed GLIC (gray spheres, Fig. 6B), exceptfor the pore site, and these were omitted in Fig. 6C for clarity.The allosteric binding sites in the extracellular domain for Cs+

(red spheres, Fig. 6C) (59), ketamine (pink spheres, Fig. 6C)(57), and acetate (firebrick spheres, Fig. 6C) (58) were alreadymentioned in the previous paragraphs. In the transmembranedomain of open GLIC, an intrasubunit binding site has beenidentified for general anesthetics, including propofol (bluespheres, Fig. 6C) (62), desflurane (62), and bromoform (63), andis localized at the upper half of the interface between the M1-and M3-transmembrane helix. In an engineered F14′A mutant ofGLIC, the ethanol binding site was identified (light orangespheres, Fig. 6C) (63), which localizes at the upper half of thetransmembrane domain at the interface between two neighbor-ing M2-subunits (63). The ethanol binding site partially overlapswith the ivermectin binding site in open GluCl (violet spheres,Fig. 6C), but in the latter ivermectin wedges in between the M1-helix of one subunit and the M3-helix of a neighboring subunit(30). The frontal ivermectin molecule in Fig. 6C was omitted forclarity because it obscures view on the pore. The ivermectinbinding site was also found in the structure of α1 GlyR in com-plex with ivermectin and glycine (34). Different pore blockersites have been identified in open GLIC, including at the 13′position for bromo-lidocaine (brown sphere, Fig. 6C) (64), at the6′ position for tetraethylarsonium (light blue sphere, Fig. 6C)(64), at the 2′ position for Cs+ (red sphere, Fig. 6C) (59, 64), andat −2′ position for Zn2+ and Cd2+ (wheat sphere, Fig. 6C) (64).The −2′ pore site, which forms the ion selectivity filter, alsooverlaps with the picrotoxinin binding site in open GluCl (30).Fourth, we discuss allosteric binding sites identified in the

desensitized state of Cys-loop receptors and the β3 GABAARstructure is shown as a representative example (green ribbon,Fig. 6D) (32). In this structure, the orthosteric binding site isoccupied by the β3-agonist benzamidine (yellow spheres, Fig.6D). This site is also occupied by the agonist 3-bromopropyl-amine (3-BrPPA) in the ELIC complex with 3-BrPPA and iso-flurane (42). Isoflurane occupies the 13′ and 6′ pore sites (lightblue spheres, Fig. 6D), which are identical to those for the closedstate (Fig. 6B). Finally, ivermectin in desensitized α1 GlyR oc-cupies the same site as in the open GluCl structure (violetspheres, Fig. 6D) (34). The ivermectin site overlaps with thebinding site for the lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phos-phocholine (POPC) in an intermediate GluCl structure (31).An important question concerns the relevance of the ELIC+

CPZ cocrystal structure reported in this paper in relation to

other prokaryote and eukaryote Cys-loop receptors. First, theexistence of the CPZ binding site near the β8–β9 loop was pre-dicted in the crystal structure of GLIC at neutral pH, whichrepresents a “closed/resting state” (60). It was recently confirmedas a possible xenon binding site in GLIC (60). Importantly, ahighly conserved aromatic residue of the β8–β9 loop, namely,Trp in cationic receptors (W160 in ELIC) or Phe/Tyr in anionicreceptors, forms part of the conserved GEW sequence motif,which was previously demonstrated to be implicated in the posi-tive allosteric modulation of the neuronal α7 nAChR by regula-tory Ca2+ ions (65). This β8–β9 loop site is distinct from thewidely known pore blocker site of CPZ, which has been exten-sively studied in the Torpedo nAChR using electrophysiological,mutagenesis, and photoaffinity labeling studies (16, 45–50). In thepresent study, we could not observe CPZ binding in the ELICpore, and this can be likely explained by the unusual and bulkyPhe residue at the 16′ pore position in ELIC, which prevents poreaccess to noncompetitive pore blockers such as memantine (40)and probably also CPZ. Therefore, it is possible that the β8–β9loop site identified in our study on ELIC corresponds to an“external” binding site for CPZ described in one of the pio-neering studies on mouse C2 muscle-type nAChRs and that isdistinct from the high-affinity “internal” pore blocker site (66).Collectively, the results from these structural studies offer a

landscape view of different allosteric binding sites in Cys-loopreceptors with different sites localized at the extracellular ligandbinding domain, the pore domain and the transmembrane do-main. The different allosteric sites form an almost continuouspath stretching from one extreme end at the top of the N-ter-minal α-helix to the bottom of the intracellular entrance of thechannel pore. With the structure determination of ELIC incomplex with CPZ, an important and missing gap is filled,namely, at a site that forms the interface between the ligandbinding domain and the pore-forming transmembrane domain.The β8–β9 loop site is structurally and functionally important asit affects coupling between ligand binding and channel opening.

MethodsELICwas expressed as a N-terminal fusionwithmaltose-binding protein (MBP)in C43 Escherichia coli cells. The fusion protein was purified on amylose resin(New England Biolabs), and ELIC was cleaved off with C3V protease. Con-centrated protein (10 mg/mL) was supplemented with E. coli lipids andcocrystallized with 1–10 mM CPZ or BrPZ using the vapor diffusion crystal-lization technique. The X-ray cocrystal structures of ELIC were solved usingmolecular replacement. Cysteine-scanning mutagenesis and current record-ings were carried out on ELIC mutants expressed in Xenopus oocytes usingthe TEVC technique. Details on protein purification, X-ray crystallography,electrophysiological recordings, and molecular-dynamics simulations arereported in SI Methods.

ACKNOWLEDGMENTS. We are grateful for the support from beamlinescientists at the X06A station of the Swiss Light Source and the PROXIMA-Istation of SOLEIL. Dr. Pierre Legrand at the PROXIMA-I beam stationassisted with data collection and processing of multiple merged crystals.This work was supported by Onderzoekstoelage Grant OT/13/095 andFonds voor Wetenschappelijk Onderzoek–Vlaanderen Grants G.0939.11and G.0762.13 (to C.U.). E.W. was supported by a fellowship from Agent-schap voor Innovatie door Wetenschap en Technologie (131118). E.L. wassupported by Vetenskapsrådet and computing time from Swedish NationalInfrastructure for Computing.

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