Bertozzi C , Zimmermann I , Engeler S , Hilf RJ , Dutzler R .

	Fig 1. Domain Interface of pLGICs.(A) Aligned sequences of the domain interface of GLIC and ELIC with residue numbering shown above and below, respectively. Secondary structure elements are indicated. Identical residues are colored in blue. Residues mutated in this study in either protein are highlighted in yellow. (B) Domain interface of ELIC (Protein Data Bank entry 2VL0). The protein is displayed as Cα-trace, mutated regions are shown in unique colors, the views are indicated. Left: View at the interface from the outside. Right: View from the pore region on two adjacent subunits. (C) Domain interface of GLIC (Protein Data Bank entry 3EHZ). The protein is displayed as Cα-trace, with mutated regions shown in unique colors. The views are as in B.
	Fig 2. Nonactivating mutations at the domain interface.Cα-trace of parts of the domain interface in a single subunit of (A), ELIC and (B), GLIC with sidechains of selected nonactivating mutants shown as CPK models. The views are from within the pore (as in Fig 1B, right panel). Residues in ELIC for which alanine mutants were characterized in detail (Phe116 and Tyr258) and the corresponding residues in GLIC (Phe115 and Tyr250) are colored in red. (C–D), Surface expression of nonactivating mutants of (C), ELIC and (D), GLIC. Data show averages of 7–14 different oocytes and are normalized to wild type (WT). Background of noninjected (non inj.) oocytes was not subtracted. Errors are standard error of the mean (SEM). (E–F), Current response of a representative oocyte at high agonist concentration that was subsequently used for the analysis of surface expression. Currents were recorded at −40 mV. (E) Response in ELIC after application of 25 mM cysteamine. (F) Response in GLIC after change to pH 4. Application of agonist is indicated by a red bar. (See S1 Data for the raw data used to generate plots shown in panels C and D).
	Fig 3. Characterization of two nonactivating mutants of ELIC.(A) Refined structure of the ELIC mutant F116A. Left, Cα-trace of part of a single subunit of F116A (grey) showing the domain interface superimposed on ELIC WT (orange). Right, interface of F116A with sidechains of selected residues shown as CPK models. View and selection are as in Fig 2A. (B) Analogous representation of the refined structure of the mutant Y258A. In A and B, the site of mutation is indicated by an asterisk. (C) Macroscopic currents recorded from representative patches of ELIC WT upon fast application and washout of agonist. Left, current response of a membrane patch of X. laevis oocytes expressing ELIC upon application of 20 mM cysteamine. Right, current recorded from a membrane patch of ELIC expressed in human embryonic kidney 293 (HEK293) cells upon application of 25 mM propylamine. The difference in magnitude of the currents is due to the higher expression of ELIC in HEK293 cells. (D) Representative current response of a membrane patch of the ELIC mutant F116A expressed in X. laevis oocytes upon application of 20 mM cysteamine. (E) Representative current response of a membrane patch of the ELIC mutant Y258A expressed in HEK293 cells upon application of 25 mM propylamine. C–E, Currents were recorded from excised patches in the outside-out configuration at a potential of −50 mV for HEK cell and −80 mV for X. laevis oocyte patches. Application of agonist by fast solution exchange is indicated by a black bar. (F–I), ITC experiments. Agonist and antagonist binding to (F), ELIC WT, (G), the ligand binding site mutant R91A, the double mutants (H), R91A/F116A and (I), R91A/Y258A. Graphs show a fit of the integrated and corrected heat upon addition of the agonist propylamine (left) and the antagonist acetylcholine (right) to a binding isotherm (red line). Experiments were repeated twice with similar results. (See S1 Data for the raw data used to generate plots shown in panels F–I).
	Fig 4. ELIC mutant with strongly increased potency for the agonist.Structure of the interaction region between the β1-β2 turn, a conserved arginine the end of β-10 and the β6-β7 loop in (A), ELIC and (B), GLIC. The view is from the extracellular side. Side-chains of selected residues forming a branched salt bridge in GLIC and their equivalent residues in ELIC are shown as CPK models. (C) Dose-response relationships of X. laevis oocytes expressing the ELIC mutants T28A and T28D measured by two-electrode voltage clamp electrophysiology. Current response of WT is shown for comparison. All currents were recorded at an outside Ca2+ concentration of 0.5 mM. Panels show averages of 4–6 independent measurements, solid lines show a fit to a Hill equation, errors are standard deviation (SD). (D) Agonist and antagonist binding to the ELIC mutant T28D as determined by ITC. Graphs show a fit of the integrated and corrected heat upon addition of the agonist propylamine (left) and the antagonist acetylcholine (right) to a binding isotherm (red line). (E) Patch clamp recording of the ELIC mutant T28D expressed in X. laevis oocytes. Currents were measured from excised patches in the outside-out configuration at −80 mV. Application of 20 mM cysteamine by fast solution exchange is indicated by a black bar. The recording shows considerable basal activity in the absence of the ligand. (See S1 Data for the raw data used to generate plots shown in panels C and D).
	Fig 5. Interaction between the β1-β2 turn and the pore domain.Relationship between the residue at the tip of the β1-β2 turn and a conserved proline at the M2-M3 loop in (A), ELIC and (B), GLIC. The view is parallel to the membrane plane. Side chains of selected residues are shown as CPK models. (C–F), Dose-response relationships of X. laevis oocytes expressing mutants of either ELIC or GLIC measured by two-electrode voltage clamp electrophysiology. Current response of the respective WT is shown for comparison. For ELIC, currents were recorded at an outside Ca2+ concentration of 0.5 mM. Panels show averages of 3–5 independent measurements, solid lines are a fit to a Hill equation, errors are SD. Mutants of the β1-β2 turn: (C), ELIC mutant L29A and the channel carrying a deletion of Leu29 (ΔL29). (D), GLIC mutant K32A. Mutants of the M2-M3 loop: (E), ELIC mutants P254A and P254G. (F), GLIC mutants P246A and P246G. For ELIC P254G, ligand was not removed following application due to the very slow deactivation of the channels. (G) Refined structure of the ELIC mutant P254G. Left, Cα-trace of part of a single subunit of P254G (grey) showing the domain interface superimposed on ELIC WT (orange). An asterisk marks the site of mutation. Right, structure of the M2-M3 loop of the mutant P254G and of WT with side chains of R255A and E155 from the adjacent subunit shown as CPK model. (H), Structure of the GLIC mutant P246G. Left, Cα-trace of part of a single subunit of P246G (grey) showing the domain interface and part of the pore region superimposed on GLIC WT (green). An asterisk marks the site of mutation. Right, Pore radius of GLIC (blue) and P246G (red) along the channel axis. The membrane boundary is indicated. (I) Macroscopic currents recorded from representative patches of the ELIC mutant P254G (left) and the double mutant P254G/R255A (right) upon fast application and washout of agonist. Currents were recorded from membrane patches of the respective ELIC mutants expressed in HEK293 cells in response to application of 25 mM propylamine. The difference in the deactivation rate in both mutants is apparent. (See S1 Data for the raw data used to generate plots shown in panels C-F).
	Fig 6. Role of the domain interface in channel activation.Interactions between residues at the domain interface in different channel conformations. (A) Sequence alignment of regions of the domain interface. Selected residues and their interactions are highlighted and schematically depicted above. Solid lines indicate interactions that are present in all conformations and dashed line interactions that are only formed in the presumably open GLIC-like conformation. Numbering below the sequences indicates the position of residues in the pore. Sequences are GLIC (UniProt: Q7NDN8), ELIC (GB: WP_013319743), GluCl (GB: AAA50785.1), GlyR (GB: NP_571477.1), GabaA (GB: M82919), 5-HT3 (GB: NM_001099644.1), AChRα (UniProt: P04756), AChRα7 (UniProt: P49582). Cα-trace of the interface of a single subunit of (B) GLIC in a presumably open conformation (GLIC-like), (C) GLIC at pH 7.0 (locally-closed), (D) ELIC (ELIC-like). The view is from within the membrane (left) and from the extracellular side (right). The pore domain is colored in blue and the extracellular domain in green. The positions of selected residues whose mutation exerts a strong effect on activation in ELIC and GLIC are shown as spheres. The residue at the tip of the β1-β2 turn and a conserved proline in the M2-M3 loop are colored in red. Selected interactions (as in A) are indicated by dashed lines.

, Correction: Signal Transduction at the Domain Interface of Prokaryotic Pentameric Ligand-Gated Ion Channels. 2016, Pubmed

, Correction: Signal Transduction at the Domain Interface of Prokaryotic Pentameric Ligand-Gated Ion Channels. 2016, Pubmed
Absalom, Role of charged residues in coupling ligand binding and channel activation in the extracellular domain of the glycine receptor. 2003, Pubmed
Adams, PHENIX: building new software for automated crystallographic structure determination. 2002, Pubmed
Althoff, X-ray structures of GluCl in apo states reveal a gating mechanism of Cys-loop receptors. 2014, Pubmed
Auerbach, Thinking in cycles: MWC is a good model for acetylcholine receptor-channels. 2012, Pubmed
Bafna, Gating at the mouth of the acetylcholine receptor channel: energetic consequences of mutations in the alphaM2-cap. 2008, Pubmed
Bartos, Structural basis of activation of cys-loop receptors: the extracellular-transmembrane interface as a coupling region. 2009, Pubmed
Bocquet, A prokaryotic proton-gated ion channel from the nicotinic acetylcholine receptor family. 2007, Pubmed , Xenbase
Bocquet, X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation. 2009, Pubmed
Bondarenko, NMR structure of the transmembrane domain of the n-acetylcholine receptor beta2 subunit. 2010, Pubmed
Bouzat, Coupling of agonist binding to channel gating in an ACh-binding protein linked to an ion channel. 2004, Pubmed
Bouzat, The interface between extracellular and transmembrane domains of homomeric Cys-loop receptors governs open-channel lifetime and rate of desensitization. 2008, Pubmed
Campos-Caro, A single residue in the M2-M3 loop is a major determinant of coupling between binding and gating in neuronal nicotinic receptors. 1996, Pubmed , Xenbase
Chakrapani, Gating dynamics of the acetylcholine receptor extracellular domain. 2004, Pubmed
Collaborative Computational Project, Number 4, The CCP4 suite: programs for protein crystallography. 1994, Pubmed
Dellisanti, Crystal structure of the extracellular domain of nAChR alpha1 bound to alpha-bungarotoxin at 1.94 A resolution. 2007, Pubmed
Dellisanti, Site-directed spin labeling reveals pentameric ligand-gated ion channel gating motions. 2013, Pubmed , Xenbase
Du, Glycine receptor mechanism elucidated by electron cryo-microscopy. 2015, Pubmed
Edelstein, Allosteric transitions of the acetylcholine receptor. 1998, Pubmed
Emsley, Features and development of Coot. 2010, Pubmed
Geertsma, A versatile and efficient high-throughput cloning tool for structural biology. 2011, Pubmed
Gonzalez-Gutierrez, Mutations that stabilize the open state of the Erwinia chrisanthemi ligand-gated ion channel fail to change the conformation of the pore domain in crystals. 2012, Pubmed , Xenbase
Gonzalez-Gutierrez, Gating of the proton-gated ion channel from Gloeobacter violaceus at pH 4 as revealed by X-ray crystallography. 2013, Pubmed
Groot-Kormelink, Achieving optimal expression for single channel recording: a plasmid ratio approach to the expression of alpha 1 glycine receptors in HEK293 cells. 2002, Pubmed
Grosman, The extracellular linker of muscle acetylcholine receptor channels is a gating control element. 2000, Pubmed
Grutter, Molecular tuning of fast gating in pentameric ligand-gated ion channels. 2005, Pubmed
Hassaine, X-ray structure of the mouse serotonin 5-HT3 receptor. 2014, Pubmed
Hibbs, Principles of activation and permeation in an anion-selective Cys-loop receptor. 2011, Pubmed
Hilf, A prokaryotic perspective on pentameric ligand-gated ion channel structure. 2009, Pubmed
Hilf, Structural basis of open channel block in a prokaryotic pentameric ligand-gated ion channel. 2010, Pubmed , Xenbase
Hilf, X-ray structure of a prokaryotic pentameric ligand-gated ion channel. 2008, Pubmed
Hilf, Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel. 2009, Pubmed
Huang, Crystal structure of human glycine receptor-α3 bound to antagonist strychnine. 2015, Pubmed
Jha, Acetylcholine receptor gating at extracellular transmembrane domain interface: the cys-loop and M2-M3 linker. 2007, Pubmed
Karlin, On the application of "a plausible model" of allosteric proteins to the receptor for acetylcholine. 1967, Pubmed
Kash, Coupling of agonist binding to channel gating in the GABA(A) receptor. 2003, Pubmed
Keller, High-precision isothermal titration calorimetry with automated peak-shape analysis. 2012, Pubmed
Laha, Macroscopic kinetics of pentameric ligand gated ion channels: comparisons between two prokaryotic channels and one eukaryotic channel. 2013, Pubmed , Xenbase
Lee, Binding to gating transduction in nicotinic receptors: Cys-loop energetically couples to pre-M1 and M2-M3 regions. 2009, Pubmed
Lee, Principal pathway coupling agonist binding to channel gating in nicotinic receptors. 2005, Pubmed
Lester, Cys-loop receptors: new twists and turns. 2004, Pubmed
Lewis, Properties of human glycine receptors containing the hyperekplexia mutation alpha1(K276E), expressed in Xenopus oocytes. 1998, Pubmed , Xenbase
Lorenz, Heteromultimeric CLC chloride channels with novel properties. 1996, Pubmed , Xenbase
Lynch, Identification of intracellular and extracellular domains mediating signal transduction in the inhibitory glycine receptor chloride channel. 1997, Pubmed
Marabelli, Mechanism of activation of the prokaryotic channel ELIC by propylamine: a single-channel study. 2015, Pubmed
McCoy, Phaser crystallographic software. 2007, Pubmed
Miller, Crystal structure of a human GABAA receptor. 2014, Pubmed
Miyazawa, Structure and gating mechanism of the acetylcholine receptor pore. 2003, Pubmed
Nury, Crystal structure of the extracellular domain of a bacterial ligand-gated ion channel. 2010, Pubmed
Pan, Structure of the pentameric ligand-gated ion channel ELIC cocrystallized with its competitive antagonist acetylcholine. 2012, Pubmed
Prevost, A locally closed conformation of a bacterial pentameric proton-gated ion channel. 2012, Pubmed
Price, Transducing agonist binding to channel gating involves different interactions in 5-HT3 and GABAC receptors. 2007, Pubmed , Xenbase
Purohit, Acetylcholine receptor gating at extracellular transmembrane domain interface: the "pre-M1" linker. 2007, Pubmed
Purohit, Functional anatomy of an allosteric protein. 2013, Pubmed
Rienzo, Structural requirements in the transmembrane domain of GLIC revealed by incorporation of noncanonical histidine analogs. 2014, Pubmed , Xenbase
Sauguet, Crystal structures of a pentameric ligand-gated ion channel provide a mechanism for activation. 2014, Pubmed , Xenbase
Schofield, Alanine-scanning mutagenesis in the signature disulfide loop of the glycine receptor alpha 1 subunit: critical residues for activation and modulation. 2004, Pubmed
Shen, Mutation causing severe myasthenia reveals functional asymmetry of AChR signature cystine loops in agonist binding and gating. 2003, Pubmed
Sine, Recent advances in Cys-loop receptor structure and function. 2006, Pubmed
Sixma, Acetylcholine binding protein (AChBP): a secreted glial protein that provides a high-resolution model for the extracellular domain of pentameric ligand-gated ion channels. 2003, Pubmed
Smart, HOLE: a program for the analysis of the pore dimensions of ion channel structural models. 1996, Pubmed
Taly, Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system. 2009, Pubmed
Tasneem, Identification of the prokaryotic ligand-gated ion channels and their implications for the mechanisms and origins of animal Cys-loop ion channels. 2005, Pubmed
Unwin, Refined structure of the nicotinic acetylcholine receptor at 4A resolution. 2005, Pubmed
Velisetty, Structural basis for allosteric coupling at the membrane-protein interface in Gloeobacter violaceus ligand-gated ion channel (GLIC). 2014, Pubmed
Wang, Intramembrane proton binding site linked to activation of bacterial pentameric ion channel. 2012, Pubmed
Xiu, A unified view of the role of electrostatic interactions in modulating the gating of Cys loop receptors. 2005, Pubmed , Xenbase
Zerangue, A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels. 1999, Pubmed , Xenbase
Zimmermann, Ligand activation of the prokaryotic pentameric ligand-gated ion channel ELIC. 2011, Pubmed
Zimmermann, Inhibition of the prokaryotic pentameric ligand-gated ion channel ELIC by divalent cations. 2012, Pubmed , Xenbase
daCosta, Gating of pentameric ligand-gated ion channels: structural insights and ambiguities. 2013, Pubmed