Allosteric binding site in a Cys-loop receptor 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 ion channel 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 eukaryote 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,