Allosteric binding site in a Cys-loop PNAS PLUS ligand-binding domain unveiled in the crystal structure of ELIC in complex with chlorpromazine

Mieke 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 Institute of Technology, SE-17121 Stockholm, Sweden; dLaboratory of Toxicology and Pharmacology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium; and eDepartment 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 are insight into the mechanism of Cys-loop receptor function derives responsible for fast inhibitory or excitatory synaptic transmission. from cryo-EM images of the Torpedo marmorata nAChR (19–22) The antipsychotic compound chlorpromazine is a widely used as well X-ray crystal structures of the acetylcholine binding protein tool to probe the pore of the nicotinic acetylcholine (AChBP) (23, 24). AChBPs are water-soluble homologs of the receptor, which is a prototypical Cys-loop receptor. In this study, extracellular ligand-binding domain of the nAChR and lack the we determine the molecular determinants of chlorpromazine pore-forming transmembrane domain. To date, more than 100 binding in the Erwinia ligand-gated ion channel (ELIC). We report cocrystal structures of AChBP in complex with different agonists, the X-ray crystal structures of ELIC in complex with chlorpromazine partial agonists, antagonists, and allosteric modulators have been or its brominated derivative bromopromazine. Unexpectedly, we determined, creating a wealth of information on the molecular do not find a chlorpromazine molecule in the channel pore of ELIC, determinants of ligand recognition in nAChRs (25). Subsequently, but behind the β8–β9 loop in the extracellular ligand-binding do- the identification of Cys-loop receptors in prokaryotes (26)

main. The β8–β9 loop is localized downstream from the neurotrans- PHARMACOLOGY allowed the first X-ray structure determination of integral Cys- mitter binding site and plays an important role in coupling of ligand Erwinia binding to channel opening. In combination with electrophysiolog- loop receptors ligand-gated ion channel (ELIC) (27) and ical recordings from ELIC cysteine mutants and a thiol-reactive de- Gloeobacter ligand-gated ion channel (GLIC) (28, 29), which likely rivative of chlorpromazine, we demonstrate that chlorpromazine represent a nonconducting and conducting conformation of the binding at the β8–β9 loop is responsible for receptor inhibition. channel pore, respectively. Later on, X-ray crystal structures were We further use molecular-dynamics simulations to support the determined for the first Cys-loop receptors, including X-ray data and mutagenesis experiments. Together, these data the Caenorhabditis elegans glutamate-gated chloride channel GluCl β unveil an allosteric binding site in the extracellular ligand-bind- (30, 31), the human 3GABAAR (32), and the mouse 5-HT3AR ing domain of ELIC. Our results extend on previous observations and further substantiate our understanding of a multisite model Significance for allosteric modulation of Cys-loop receptors. Cys-loop receptors belong to a family of ion channels that are ligand-gated ion channel | X-ray crystallography | allosteric modulation | involved in fast synaptic transmission. Allosteric modulators of Cys-loop receptor | nicotinic acetylcholine receptor Cys-loop receptors hold therapeutic potential as they tweak receptor function while preserving the normal fluctuations hlorpromazine (CPZ) (Fig. 1), a phenothiazine-derived an- in neurotransmitter signaling at the synapse. Here, we take Ctipsychotic drug, was introduced in psychiatry in the early advantage of a model Cys-loop receptor, the Erwinia ligand- 1950s, revolutionizing the treatment of psychotic disorders (1, 2). gated ion channel (ELIC). We determined cocrystal structures The main mechanism of action of CPZ consists in the blockage of ELIC in complex with chlorpromazine (IC50, ∼160 μM) and of dopamine receptors (2–4), but the numerous side effects as- its brominated derivative bromopromazine, which unveil an sociated with this drug indicate that it interacts with other phys- allosteric binding site localized at the interface between the iologically relevant targets. CPZ was indeed shown to interfere extracellular ligand-binding domain and the pore-forming with several voltage- and ligand-gated channels: it inhibits neu- transmembrane domain. Our results demonstrate that the + ronal voltage-gated K channels (5–7), BKCa channels (8), and different allosteric binding sites present in Cys-loop receptors 2+ the human α1E subunit-mediated Ca channels (9); CPZ was form an almost continuous path stretching from top to bottom also shown to inhibit GABAergic currents (10, 11), specifically of the receptor. through GABAA receptors (GABAARs) (12), and to inhibit se- Author contributions: C.U. designed research; E.W., A.F., Ö.Y., M.A., M.B., R.S., S.P., and C.U. rotonin type-3 receptors (5-HT3Rs) (13, 14) and nicotinic ace- tylcholine receptors (nAChRs) (15, 16), members of the Cys-loop 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. analyzed data; and M.N., E.W., A.F., Ö.Y., M.A., M.B., J.T., E.L., and C.U. wrote the paper. receptor family. The Cys-loop receptor family is composed of membrane- The authors declare no conflict of interest. spanning ligand-gated ion channels that are responsible for fast This article is a PNAS Direct Submission. excitatory or inhibitory synaptic neurotransmission. They are Freely available online through the PNAS open access option. composed of five identical or nonidentical subunits, each of them Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 5LG3 (ELIC+CPZ) and 5LID (ELIC+BrPZ)]. comprising an N-terminal extracellular domain, which contains 1Present address: Structural Virology, Central European Institute of Technology, Masaryk the neurotransmitter binding site, four transmembrane helices, University, 62500 Brno, Czech Republic. that when assembled allow ions to pass through the membrane, 2To whom correspondence should be addressed. Email: [email protected]. and an intracellular domain, responsible for channel conductance, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. receptor modulation, and trafficking (17, 18). Initial structural 1073/pnas.1603101113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1603101113 PNAS Early Edition | 1of8 Downloaded by guest on October 6, 2021 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 in Xenopus oocytes and also perform molecular-dynamics simula- tions of the complex. Together, our results expand our current understanding of allosteric modulation in the family of pen- tameric ligand-gated ion channels. Results X-Ray Crystal Structures of ELIC in Complex with CPZ or BrPZ. In agreement with previous observations (43), we determined that CPZ inhibits ELIC expressed in Xenopus oocytes with an IC50 value 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 of CPZ recognition in ELIC, we determined the X-ray cocrystal structures 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 the bromine atom in BrPZ to collect anomalous diffraction data, – which allowed us to calculate a so-called anomalous difference Fig. 1. Structure and function of chlorpromazine (CPZ) and analogs. (A C) density map and identify the density of the anomalously scat- Chemical structures of CPZ, bromopromazine (BrPZ), and methanethiosul- fonate-promazine (MTS-PZ), respectively. (D) Electrophysiological recordings tering electrons around the bromine atoms even at medium from Xenopus oocytes expressing ELIC. Channels were activated by the ap- resolution. Both the fivefold averaged simple electron difference

plication of the agonist GABA at the EC50 (20 mM). In the presence of 30 μM density maps (Fo-Fc) as well as the fivefold averaged anomalous CPZ, this response was reduced. (E) Concentration–inhibition curve for CPZ difference density map allowed us to localize two distinct loca- on ELIC. Averaged data ± SEM are shown for three to nine different oocytes. tions for the binding of CPZ or BrPZ (Fig. 2). Unexpectedly, we do not observe any electron density in the pore domain of the channel. Instead, we observe simple difference density in the α (33). More recently, the cryo-EM structure of the 1GlyRwas extracellular ligand binding domain of ELIC at a site that is lo- determined in closed, open, and desensitized conformations (34). cated near to the β8–β9 loop (Fig. 2 A–C). In eukaryote recep- Finally, the X-ray crystal structure of the α3 GlyR was determined tors, this loop together with the Cys-loop, the M2–M3 loop, and in a strychnine-bound state (35). the pre-M1 region form the interface between the ligand binding In this study, we take advantage of the ELIC, a prokaryote domain and the pore domain of the channel. At each of the five homolog of vertebrate Cys-loop receptors, which is activated sites in the pentamer, the simple difference density displays a by primary amine molecules, including GABA (36, 37). The curved shape (6σ) consistent with the curvature of the tricyclic availability of several relatively high-resolution X-ray cocrystal 10H-phenothiazine ring in CPZ. The electron density for the structures of ELIC in complex with known ligands renders this dimethylpropylamino-moiety is visible only at lower σ levels, channel a relevant model for the study of Cys-loop receptor indicating it is more disordered in the crystal structure. The modulation (36, 38–42). binding location of CPZ at this site is further substantiated by the CPZ, referred to as a noncompetitive antagonist of nAChRs presence of a strong anomalous peak (10σ) at each of the five (16), was also found to inhibit GABA-evoked responses in ELIC— sites of the pentamer in the ELIC+BrPZ cocrystal structure > μ — although with low potency (IC50, 100 M) suggesting a dis- (Fig. 2 D–F). Importantly, the β8–β9 loop undergoes a confor- tinct pharmacology (43). Initial studies aimed at identifying the mational rearrangement to accommodate CPZ at this site, which molecular determinants of CPZ binding in nAChRs showed will be discussed in further detail below. Additionally, in the 3 that [ H]CPZ binds to the channel pore (44). Subsequently, ELIC+BrPZ cocrystal structure, we also observe anomalous 3 [ H]CPZ became a widely used tool to probe the channel pore of difference density at a second location in the extracellular ligand nAChRs in closed, open, and desensitized states (16, 45–50). binding domain, namely at the agonist binding site, which is lo- Initial photoaffinity labeling studies on the Torpedo nAChR in cated at the interface between each of two subunits (Fig. 2G). the desensitized state revealed that CPZ binds to a high-affinity This site is lined by highly conserved aromatic residues localized binding site near the cytoplasmic end of the channel pore, com- on historically designated “loops,” termed loops A–B–C on the prising the 2′,6′,and9′ positions of the pore-lining M2-helix (46– principal face of the binding site and loops D–E–F on the 49). Recently, Chiara et al. (50) extended on these observations complementary face of the binding site. The highly conserved and identified an additional binding site in the desensitized state aromatic residues at this site form a so-called aromatic box, for [3H]CPZ near the extracellular end of the channel pore, which creates an electronegative environment for agonist rec- comprising the 16′,17′,and20′ positions of the M2 segment. In ognition. In ELIC, this site includes Y38 (loop D), F133 (loop the closed state, [3H]CPZ labeling was observed at 5′,6′,and9′, B), and Y175 and F188 (both loop C). In the ELIC+BrPZ with no labeling at 2′ (50). Additionally, a binding site for CPZ structure, the anomalous difference density at this site is slightly was identified in the intracellular domain, as photoaffinity la- offset toward Y38 and F133, suggesting a possible location of the beling was also observed for residues αMet-386 and αSer-393, bromine atom in BrPZ. Importantly, the binding of BrPZ at this which are localized in the intracellular MA-helices (50). In con- site is consistent with the earlier observation that CPZ binds trast, in 5-HT3Rs CPZ acts directly on the neurotransmitter directly at the neurotransmitter binding site in the related 5-HT3 binding site (13, 14) and competitively antagonizes the action receptor (13, 14). Together, the ELIC X-ray crystal structures of serotonin. reveal that CPZ and BrPZ bind at two distinct sites in the ex- In this study, we set out to investigate the structural deter- tracellular domain, but not in the pore-forming transmembrane minants of CPZ binding in ELIC. To facilitate structural studies, domain. The two binding sites are localized at functionally im- we used a brominated derivative of CPZ, termed bromoproma- portant domains, namely the β8–β9 loop, which contributes to zine (BrPZ) (Fig. 1B). Here, we report the X-ray crystal struc- coupling of ligand binding to channel opening, and the agonist

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.1603101113 Nys et al. Downloaded by guest on October 6, 2021 PNAS PLUS PHARMACOLOGY

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 complex

with 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 stick representation. 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 blue ribbon representation. The red mesh represents fivefold averaged anomalous difference electron density contoured at a level of 10σ. The Insets show a detailed view of the β8–β9 loop binding site (F) and the agonist binding site (G).

binding site, which contains the structural determinants for ag- ascending part of the β8–β9 loop remain unaltered. Detailed onist recognition. The lack of CPZ binding in the pore domain of analysis of the interactions between CPZ and residues of the β8– ELIC can likely be explained by the presence of an unusual and β9 pocket reveals a wide range of mostly hydrophobic interactions bulky phenylalanine residue at the extracellular end (F16′) of the (Fig. 3C). These include I20, N21, and I23 on the β1-strand, F126 ELIC channel pore that restricts pore access of known pore on the β7-strand, and V147, T149, E150, E155, D158, W160, and blockers, including memantine (40) and likely also CPZ. I162 on the β8–β9 loop. Weak hydrogen bonds are formed be- tween the dimethylamino-moiety of CPZ and the side-chain oxy- Conformational Change of the β8–β9 Loop. The CPZ-bound ELIC gen atoms of D158 (indicated with dashed lines in Fig. 3C)and structure superimposes well with the apo form of ELIC [Protein between the 10H-phenothiazine nitrogen and the main-chain Data Bank (PDB) ID code 2VL0] with a rmsd of 0.9 Å for 2,954 carbonyl atom of I23. out of 3,070 aligned residues. This suggests that the conforma- tional state of ELIC remains unaltered after CPZ binding and Cysteine-Scanning Mutagenesis of the β8–β9 Loop Binding Site. To corresponds to a closed nonconductive conformation of the re- explore the contribution of individual amino acids in the β8–β9 ceptor (27). However, detailed inspection of the simple electron loop binding site to molecular recognition of CPZ, we in- density map (2Fo-Fc) reveals structural differences in the β8–β9 dividually mutated each residue involved in the CPZ interaction loop of the CPZ-bound structure, which was manually rebuilt to a cysteine residue in the background of a Cys-less ELIC var- and refined (Fig. 3A). A detailed view of a monomeric subunit iant, which is functionally identical to wild-type ELIC (Table 1). superimposed for apo ELIC (yellow) and ELIC+CPZ (blue) is To determine the effect of CPZ binding at this specific site, and shown in Fig. 3B. As stated above, the overall structure of not elsewhere in the protein, we used a thiol-reactive analog of ELIC+CPZ is nearly identical to apo ELIC, except for a change CPZ termed MTS-PZ. in the β8–β9 loop. Most of the conformational change can be First, we investigated the effect of the cysteine mutation alone observed in the descending part of the β8–β9 loop, including on the function of ELIC by expressing each mutant in Xenopus residues Y148 to E155. Residues involved in forming the interface oocytes and investigating the response to the agonist GABA with the M2–M3 loop of the neighboring subunit as well as the using TEVC. All mutants were functional and responded to

Nys et al. PNAS Early Edition | 3of8 Downloaded by guest on October 6, 2021 increased distance between the MTS moiety and the sulfhydryl side chain, or that the mutant reacted with MTS-PZ but did not functionally affect the channel. In conclusion, we demonstrate that covalent modification of residues E150C, D158C, W160C, and I162 in Cys-less ELIC with MTS-PZ results in functional inhibition of the GABA-evoked channel response. This result suggests 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 binding and its effect on surrounding loops, two molecular-dynamics simulations were performed with and without CPZ bound to the structure (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 measured by the distance from F126 and W160 to the C11 atom of CPZ (Fig. 5 A and B). The cavity volume, on the other hand, increased by 250 Å3 in CPZ-bound simulations compared with the crystal structure (893 Å3) (Fig. 5C), whereas the volume of the same cavity in apo simulations decreased by 50 Å3. The increase in cavity volume did not disrupt the weak hydrogen bond interac- Fig. 3. Conformational change of the β8–β9 loop in ELIC. (A) Stereo rep- tions of CPZ with the surrounding loops. The CPZ molecules resentation of the β8–β9 loop in ELIC. The ELIC backbone is shown as green spent on average ∼12% of the simulation time in contact with ribbon. Chlorpromazine (CPZ) is shown in yellow sticks. The blue mesh is the β8–β9 loop (residues 148–162) and the β1-strand (residues σ simple electron density (2Fo-Fc) contoured at a level of 1.4 .(B) Superposi- 22–23) and ∼2% with the Cys-loop (residues 113, 126) (Fig. 5D). tion of a single monomer of apo ELIC in yellow (PDB ID code 2VL0) and ELIC The predicted hydrogen bond interactions of CPZ with the β8– in complex with CPZ in cartoon representation. The Inset shows a detailed β view of the β8–β9 loop. CPZ is shown in sphere representation. White is 9 loop include two residues, T149 and E155, that were not la- carbon, blue is nitrogen, green is chlorine, and orange is sulfur. (C) Detailed beled by MTS-PZ when mutated to cysteine. Although these view of amino acids involved in ligand interactions with CPZ. Dashed lines residues are in proximity of the bound CPZ, the conformations indicate hydrogen bonds. CPZ is shown in stick representation. sampled by the side chains were not as favorable for interactions as other residues, that is, E150 and D158 (Fig. 5 E and F). To- gether, the molecular-dynamics simulations support a ligand- application of GABA. For each mutant, we determined a GABA induced conformational change of the β8–β9 loop forming an – concentration activation curve and calculated EC50 values (Table 1). allosteric binding site for CPZ. The reactivity of the specific side We observe that EC50 values varied from a threefold decrease in chains identified in the cysteine-scanning mutagenesis experi- mutant I20C, to a fivefold increase in mutant W160C. EC50 values ments is consistent with the rotamers sampled by these residues were statistically compared between Cys-less ELIC and all of the in simulations. Cys-mutants (Table 1): for mutants I20C, F126C, T149C, D158C, Because the molecular-dynamic simulations indicate an in- and W160C, EC50 values were significantly different from Cys-less crease in the allosteric CPZ-binding pocket, which is larger than ELIC; the Hill coefficients of the entire set of mutants were not necessary to adapt CPZ, additional electrophysiological experi- significantly different from Cys-less ELIC. These results point ments were conducted to exclude the possibility of nonspecific β –β toward a functional role of 8 9 loop residues to channel gat- CPZ-binding. IC50 values were determined for 12 additional ing, which is consistent with the β8–β9 loop’s established con- phenothiazine analogs, and some of these compounds indeed tribution to channel gating. In addition, we observe that all exhibited significantly different IC50 values, indicating that the mutants express at levels that are comparable to wild-type ELIC binding of phenothiazine analogs occurs in a structure-dependent except for W160C, which expresses severalfold lower, sug- manner (Table S2). Additionally, we observed that the presence gesting that this mutation critically affects protein folding and/ of a piperazine group at position R1 gave rise to more potent or trafficking. Next, we investigated the functional effect of MTS-PZ binding at each of the individual Cys-mutants. To accomplish this, we Table 1. Summary of the functional characterization of WT and n I ± used a protocol in which ELIC displayed stable channel activa- mutant ELIC: GABA EC50 and H and max SEM

tion following two consecutive applications of GABA at the ELIC construct EC50,mM nH Imax, μA n EC50. Next, we perfused the oocyte with 200 μM MTS-PZ for 2 min and washed out unreacted MTS-PZ during a 30-s washout. Wild-type ELIC 21 ± 1.0 2.1 ± 0.20 21 ± 2.4 4 The change of channel activation after MTS-PZ modification Cys-less ELIC 23 ± 3.2 2.3 ± 0.70 16 ± 1.9 4 was then measured with a third application of GABA at the I20C 62 ± 8.0* 2.0 ± 0.40 3.8 ± 0.70 5 EC . As expected, MTS-PZ did not affect the amplitude of N21C 30 ± 5.3 2.1 ± 1.0 1.3 ± 0.10 3 50 ± ± ± – the GABA response in Cys-less ELIC (Fig. 4). We observed that I23C 24 3.6 2.3 0.80 4.9 2.90 2 3 ± ± ± – the GABA response was significantly reduced in four mutants, F126C 14 0.5* 2.2 0.10 6.9 3.60 3 5 ± ± ± namely, E150C (34.0% ± 10.0, n = 3), D158C (52.0% ± 6.9, n = V147C 41 12.2 1.7 0.90 21 5.8 3 ± ± ± 3), W160C (55.0% ± 6.2, n = 3), and I162 (41.0% ± 13.0, n = 3). T149C 16 1.1* 2.4 0.30 31 3.1 3 ± ± ± This is consistent with the ligand-binding pose in the ELIC E150C 20 7.8 2.0 1.30 6.2 1.40 3 E155C 20 ± 2.7 2.7 ± 0.70 11 ± 1.9 3–5 cocrystal structure, which puts the thiol-reactive moiety of MTS- ± ± ± – PZ (equivalent to the dimethylamino-moiety in CPZ) in close D158C 8.5 2.00* 3.0 2.30 6.6 1.30 2 4 W160C 3.5 ± 0.30* 3.1 ± 0.80 0.20 ± 0.07 3–4 proximity to these residues. For the seven other mutants, MTS- I162C 22 ± 5.5 2.3 ± 1.10 6.6 ± 3.20 3 PZ application did not affect the GABA response. This indicates that either the mutant did not react with MTS-PZ due to the *P < 0.05, significantly different from Cys-less ELIC, Student’s t test.

4of8 | www.pnas.org/cgi/doi/10.1073/pnas.1603101113 Nys et al. Downloaded by guest on October 6, 2021 different Cys-loop receptors, including ELIC and different forms PNAS PLUS of GLIC. First, we discuss allosteric binding sites unveiled in a chimera of the Lymnaea AChBP and the ligand binding domain of the α7 nAChR, α7-AChBP (green ribbon, Fig. 6A). Using a fragment- based screening approach, Spurny et al. (54) discovered three allosteric binding sites in α7-AChBP, which are remote from the orthosteric binding site occupied by the agonist lobeline in these structures (yellow spheres, Fig. 6A), and also the agonist epi- batidine (55) or the competitive antagonist α-bungarotoxin in other 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 the main immunogenic region (MIR) in the α1 muscle nAChR (white spheres, Fig. 6A). This site was termed the “top site” and is involved in negative modulation of the α7 nAChR (54). The same fragment molecule also occupies an allosteric binding site that is located just below the orthosteric binding site and that was termed the “agonist subsite” (pink spheres, Fig. 6A) (54). This site corresponds to the ketamine binding site reported in the GLIC (57), where ketamine also binds just below the orthosteric Fig. 4. Cysteine-scanning mutagenesis of the β8–β9 loop in ELIC. (A)Elec- agonist binding site and is involved in inhibition of GLIC (pink trophysiological recordings of Cys-less ELIC in response to repetitive pulses of spheres, Fig. 6C). Another fragment molecule, fragment 4, was = μ GABA at the EC50 ( 20 mM) and application of 200 M of a thiol-reactive CPZ identified that occupies an allosteric binding site accessible from derivative termed MTS-PZ. (B) Example traces of a Cys mutant, E155C, the vestibule of the receptor and was termed the “vestibule site” showing no effect of MTS-PZ. (C) Example traces of a Cys mutant, D158C, (firebrick spheres, Fig. 6A) (54). This fragment is involved in showing an inhibitory effect of MTS-PZ. (D) Summary of MTS-PZ–mediated channel inhibition on the different Cys mutants. Data represent the mean ± SEM of three to five experiments. *P < 0.05, significantly different from Cys- PHARMACOLOGY less ELIC, Student’s t test; **P < 0.01, significantly different from Cys-less ELIC, Student’s t test.

inhibitors, most likely due to an increase of the interacting in- terface with the expanded CPZ-binding pocket. Discussion In the present work, we identify an allosteric binding site in the extracellular domain of the Erwinia pentameric ligand-gated ion channel ELIC using X-ray crystallography. In combination with cysteine-scanning mutagenesis and electrophysiological record- ings of ELIC expressed in Xenopus oocytes, we demonstrate that the identified β8–β9 loop site is involved in negative allosteric modulation of ELIC. These results are further supported with molecular-dynamics simulations, which confirm our observations in the crystal structure and the mutagenesis experiments. These results extend on previous observations of allosteric binding sites in different Cys-loop receptors and substantiate our understanding of a multisite model of allosteric modulation in this family of ion channels. We here discuss our results in the context of previously de- termined Cys-loop receptor crystal structures in which allosteric binding sites were revealed (Fig. 6 A–D). To enhance clarity in this figure several structures were grouped and classified as “closed,”“open,” and “desensitized,” although we emphasize that subtle and important differences exists in, for example, the GLIC locally closed state (51) and the GluCl apo state (31), which we both classified as closed structures, but most likely represent “intermediate” conformational states (31, 51). For a detailed discussion of the known conformational differences in currently available Cys-loop receptor structures, we refer to re- Fig. 5. Binding stability of CPZ in simulations. (A and B) Binding of CPZ cent reviews (52, 53). Notably, several additional sites have been measured by the distance from F126 and W160 to the C11 atom of CPZ (solid identified using other methods, such as photoaffinity labeling and dashed lines, respectively) per subunit. (C) Allosteric cavity volume in and mutagenesis, but due to space limitation these results extend presence and absence of CPZ; the crystal structure value is indicated by the dashed line. (D) Average hydrogen bond interactions of CPZ. (E) Probability beyond the scope of the current discussion. Finally, it should be distribution of side-chain angles relative to CPZ; measured from Cβ–Cα–NC2 noted that the agonist binding site loop F, which precedes the atom positions. (F) MTS-PZ–mediated inhibition (purple, no significant ef- β8–β9 loop site, adopts significantly different conformations in fect; green, inhibition). Outliers (T149, E155) are marked with black arrows.

Nys et al. PNAS Early Edition | 5of8 Downloaded by guest on October 6, 2021 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 the

GluCl+ivermectin ion channel structure as a representative example of an open state (30). (D) Green ribbon presentation of the β3 GABAA receptor structure as 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.

negative modulation of the α7 nAChR (54). The same site was on the complementary subunit (39) (red spheres, Fig. 6B). This + unveiled in the crystal structure of the ELIC in complex with site overlaps with a Cs binding site in the GLIC A13′F mutant + flurazepam (firebrick spheres, closed state, Fig. 6B), which is (59) and a Ni2 binding site in wild-type GLIC (60) (red spheres, + involved in positive modulation of ELIC (36). The importance of open state, Fig. 6C). A second Ba2 site is located at the subunit the same site was also confirmed in the crystal structure of GLIC in interface about 15 Å below the orthosteric agonist site (red complex with acetate (firebrick spheres, open state, Fig. 6C) (58). spheres, Fig. 6B). This site is formed by residues at the end of the Second, we discuss allosteric binding sites unveiled in the closed β6-strand on the principal subunit and the loop connecting the β8- state of Cys-loop receptors, and we show the ELIC structure as a and β9-strand on the complementary subunit (39). This site is just representative example because most of the available modulator 8 Å distant from the CPZ binding site (β8–β9 loop) reported in + cocrystal structures have been determined with this ion channel this paper (cyan spheres, Fig. 6B). The third Ba2 site is located at (green ribbon, Fig. 6B) (27). Similar to other Cys-loop receptors, the extracellular entrance of the channel pore where it binds at the orthosteric binding site in ELIC is occupied by the partial the 20′ position of the M2-helix (39) (red sphere, Fig. 6D). An- agonist GABA (36) or the competitive antagonist bromo-flur- other allosteric binding site near to the CPZ binding site is oc- azepam (yellow spheres, Fig. 6B) (36). The orthosteric site is also cupied by either bromoform in ELIC (magenta sphere, Fig. 6B) occupied by the competitive antagonist strychnine in the closed α3 (41) or xenon in locally closed GLIC (gray sphere, Fig. 6B)(61). GlyR (35) and closed α1 GlyR structures (34). The occupancy of Additional binding sites for xenon in locally closed GLIC are the vestibule site by flurazepam in ELIC was already mentioned in located at the intrasubunit general anesthetic binding site (blue the previous paragraph (firebrick spheres, Fig. 6B) (36). It was sphere, Fig. 6B), the inner-interfacial sites (gray spheres, Fig. 6B), reported that ELIC is negatively modulated by divalent cations, and the outer-interfacial sites (gray spheres, Fig. 6B) (61). Finally, + + including Ca2 and Ba2 , and it was demonstrated that these xenon also occupies the 9′ pore site in locally closed GLIC (gray cations bind at three distinct binding sites (39) (red spheres, sphere, Fig. 6B) (61). Two additional binding sites for bromoform + Fig. 6B). The first Ba2 site is located at the outer rim of the have been localized in the ELIC cocrystal structure, namely at an “vestibule pocket” where it is coordinated by two residues at the intersubunit transmembrane site (magenta spheres, Fig. 6B)and end of the β4-strand, namely S84 on the principal subunit and D86 at the 13′ pore site (magenta sphere, Fig. 6B) (41). The 13′ pore

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.1603101113 Nys et al. Downloaded by guest on October 6, 2021 site for bromoform overlaps with one of the pore sites (13′)ofthe other prokaryote and eukaryote Cys-loop receptors. First, the PNAS PLUS anesthetic isoflurane site in ELIC (light blue spheres, Fig. 6B) existence of the CPZ binding site near the β8–β9 loop was pre- (42). Isoflurane simultaneously occupies the 6′ pore site in ELIC. dicted in the crystal structure of GLIC at neutral pH, which The pore blocker memantine has been identified at the 16′ pore represents a “closed/resting state” (60). It was recently confirmed position in ELIC F16′S (orange spheres, Fig. 6B)(40). as a possible xenon binding site in GLIC (60). Importantly, a Third, we discuss allosteric binding sites identified in the open highly conserved aromatic residue of the β8–β9 loop, namely, state of Cys-loop receptors, and we show the GluCl+ivermectin Trp in cationic receptors (W160 in ELIC) or Phe/Tyr in anionic crystal structure as a representative example (green ribbon, receptors, forms part of the conserved GEW sequence motif, Fig. 6C). In GluCl, the orthosteric binding site is occupied by which was previously demonstrated to be implicated in the posi- glutamate (yellow spheres, Fig. 6C) (30), and in open GLIC, this tive allosteric modulation of the neuronal α7 nAChR by regula- + site is occupied by acetate (58). Allosteric binding sites for xenon tory Ca2 ions (65). This β8–β9 loop site is distinct from the have been determined for open GLIC, which overlap with those widely known pore blocker site of CPZ, which has been exten- described in locally closed GLIC (gray spheres, Fig. 6B), except sively studied in the Torpedo nAChR using electrophysiological, for the pore site, and these were omitted in Fig. 6C for clarity. mutagenesis, and photoaffinity labeling studies (16, 45–50). In the + The allosteric binding sites in the extracellular domain for Cs present study, we could not observe CPZ binding in the ELIC (red spheres, Fig. 6C) (59), ketamine (pink spheres, Fig. 6C) pore, and this can be likely explained by the unusual and bulky (57), and acetate (firebrick spheres, Fig. 6C) (58) were already Phe residue at the 16′ pore position in ELIC, which prevents pore mentioned in the previous paragraphs. In the transmembrane access to noncompetitive pore blockers such as memantine (40) domain of open GLIC, an intrasubunit binding site has been and probably also CPZ. Therefore, it is possible that the β8–β9 identified for general anesthetics, including propofol (blue loop site identified in our study on ELIC corresponds to an spheres, Fig. 6C) (62), desflurane (62), and bromoform (63), and “external” binding site for CPZ described in one of the pio- is localized at the upper half of the interface between the M1- neering studies on mouse C2 muscle-type nAChRs and that is and M3-transmembrane helix. In an engineered F14′A mutant of distinct from the high-affinity “internal” pore blocker site (66). GLIC, the ethanol binding site was identified (light orange Collectively, the results from these structural studies offer a spheres, Fig. 6C) (63), which localizes at the upper half of the landscape view of different allosteric binding sites in Cys-loop transmembrane domain at the interface between two neighbor- receptors with different sites localized at the extracellular ligand ing M2-subunits (63). The ethanol binding site partially overlaps binding domain, the pore domain and the transmembrane do-

with the ivermectin binding site in open GluCl (violet spheres, main. The different allosteric sites form an almost continuous PHARMACOLOGY Fig. 6C), but in the latter ivermectin wedges in between the M1- path stretching from one extreme end at the top of the N-ter- helix of one subunit and the M3-helix of a neighboring subunit minal α-helix to the bottom of the intracellular entrance of the (30). The frontal ivermectin molecule in Fig. 6C was omitted for channel pore. With the structure determination of ELIC in clarity because it obscures view on the pore. The ivermectin complex with CPZ, an important and missing gap is filled, binding site was also found in the structure of α1 GlyR in com- namely, at a site that forms the interface between the ligand plex with ivermectin and glycine (34). Different pore blocker binding domain and the pore-forming transmembrane domain. sites have been identified in open GLIC, including at the 13′ The β8–β9 loop site is structurally and functionally important as position for bromo-lidocaine (brown sphere, Fig. 6C) (64), at the it affects coupling between ligand binding and channel opening. 6′ position for tetraethylarsonium (light blue sphere, Fig. 6C) + (64), at the 2′ position for Cs (red sphere, Fig. 6C) (59, 64), and Methods + + at −2′ position for Zn2 and Cd2 (wheat sphere, Fig. 6C) (64). ELIC was expressed as a N-terminal fusion with maltose-binding protein (MBP) The −2′ pore site, which forms the ion selectivity filter, also in C43 Escherichia coli cells. The fusion protein was purified on amylose resin overlaps with the picrotoxinin binding site in open GluCl (30). (New England Biolabs), and ELIC was cleaved off with C3V protease. Con- Fourth, we discuss allosteric binding sites identified in the centrated protein (10 mg/mL) was supplemented with E. coli lipids and β cocrystallized with 1–10 mM CPZ or BrPZ using the vapor diffusion crystal- desensitized state of Cys-loop receptors and the 3 GABAAR lization technique. The X-ray cocrystal structures of ELIC were solved using structure is shown as a representative example (green ribbon, molecular replacement. Cysteine-scanning mutagenesis and current record- Fig. 6D) (32). In this structure, the orthosteric binding site is ings were carried out on ELIC mutants expressed in Xenopus oocytes using occupied by the β3-agonist benzamidine (yellow spheres, Fig. the TEVC technique. Details on protein purification, X-ray crystallography, 6D). This site is also occupied by the agonist 3-bromopropyl- electrophysiological recordings, and molecular-dynamics simulations are amine (3-BrPPA) in the ELIC complex with 3-BrPPA and iso- reported in SI Methods. flurane (42). Isoflurane occupies the 13′ and 6′ pore sites (light blue spheres, Fig. 6D), which are identical to those for the closed ACKNOWLEDGMENTS. We are grateful for the support from beamline B α scientists at the X06A station of the Swiss Light Source and the PROXIMA-I state (Fig. 6 ). Finally, ivermectin in desensitized 1 GlyR oc- station of SOLEIL. Dr. Pierre Legrand at the PROXIMA-I beam station cupies the same site as in the open GluCl structure (violet assisted with data collection and processing of multiple merged crystals. spheres, Fig. 6D) (34). The ivermectin site overlaps with the This work was supported by Onderzoekstoelage Grant OT/13/095 and binding site for the lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phos- Fonds voor Wetenschappelijk Onderzoek–Vlaanderen Grants G.0939.11 phocholine (POPC) in an intermediate GluCl structure (31). and 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. was An important question concerns the relevance of the ELIC supported by Vetenskapsrådet and computing time from Swedish National CPZ cocrystal structure reported in this paper in relation to Infrastructure for Computing.

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