title: Allosteric binding site in a Cys-loop receptor ligand-binding domain unveiled in the crystal structure of ELIC in complex with chlorpromazine.
abstract: Pentameric ligand-gated ion channels or Cys-loop receptors are responsible for fast inhibitory or excitatory synaptic transmission. The antipsychotic compound chlorpromazine is a widely used tool to probe the ion channel pore of the nicotinic acetylcholine receptor, which is a prototypical Cys-loop receptor. In this study, we determine the molecular determinants of chlorpromazine binding in the Erwinia ligand-gated ion channel (ELIC). We report the X-ray crystal structures of ELIC in complex with chlorpromazine or its brominated derivative bromopromazine. Unexpectedly, we do not find a chlorpromazine molecule in the channel pore of ELIC, but behind the β8-β9 loop in the extracellular ligand-binding domain. The β8-β9 loop is localized downstream from the neurotransmitter binding site and plays an important role in coupling of ligand binding to channel opening. In combination with electrophysiological recordings from ELIC cysteine mutants and a thiol-reactive derivative of chlorpromazine, we demonstrate that chlorpromazine binding at the β8-β9 loop is responsible for receptor inhibition. We further use molecular-dynamics simulations to support the X-ray data and mutagenesis experiments. Together, these data unveil an allosteric binding site in the extracellular ligand-binding domain of ELIC. Our results extend on previous observations and further substantiate our understanding of a multisite model for allosteric modulation of Cys-loop receptors.
authors: Laboratory of Structural Neurobiology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
authors: Mieke
authors: M
authors: Nys
authors: Laboratory of Structural Neurobiology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
authors: Eveline
authors: E
authors: Wijckmans
authors: Laboratory of Structural Neurobiology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
authors: Ana
authors: A
authors: Farinha
authors: Science for Life Laboratory, Stockholm and Uppsala, SE-17121 Stockholm, Sweden; Theoretical and Computational Biophysics, Department of Theoretical Physics, Kungliga Tekniska Högskolan Royal Institute of Technology, SE-17121 Stockholm, Sweden.
authors: Özge
authors: Ö
authors: Yoluk
authors: Science for Life Laboratory, Stockholm and Uppsala, SE-17121 Stockholm, Sweden; Theoretical and Computational Biophysics, Department of Theoretical Physics, Kungliga Tekniska Högskolan Royal Institute of Technology, SE-17121 Stockholm, Sweden.
authors: Magnus
authors: M
authors: Andersson
authors: Laboratory of Structural Neurobiology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
authors: Marijke
authors: M
authors: Brams
authors: Laboratory of Structural Neurobiology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
authors: Radovan
authors: R
authors: Spurny
authors: Laboratory of Toxicology and Pharmacology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
authors: Steve
authors: S
authors: Peigneur
authors: Laboratory of Toxicology and Pharmacology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
authors: Jan
authors: J
authors: Tytgat
authors: Science for Life Laboratory, Stockholm and Uppsala, SE-17121 Stockholm, Sweden; Theoretical and Computational Biophysics, Department of Theoretical Physics, Kungliga Tekniska Högskolan Royal Institute of Technology, SE-17121 Stockholm, Sweden; Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, SE-17121 Stockholm, Sweden.
authors: Erik
authors: E
authors: Lindahl
authors: Laboratory of Structural Neurobiology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium; chris.ulens@kuleuven.be.
authors: Chris
authors: C
authors: Ulens
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D014150:Antipsychotic Agents / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002746:Chlorpromazine / Q000031:analogs & derivatives*; D018360:Crystallography, X-Ray; D058466:Cysteine Loop Ligand-Gated Ion Channel Receptors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004885:Erwinia / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D054879:Halogenation; D007700:Kinetics; D008958:Models, Molecular; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D014150:Antipsychotic Agents; D001426:Bacterial Proteins; D058466:Cysteine Loop Ligand-Gated Ion Channel Receptors; D011994:Recombinant Proteins; D002746:Chlorpromazine
keywords: Cys-loop receptor; X-ray crystallography; allosteric modulation; ligand-gated ion channel; nicotinic acetylcholine receptor