Schmandt N , Velisetty P , Chalamalasetti SV , Stein RA , Bonner R , Talley L , Parker MD , Mchaourab HS , Yee VC , Lodowski DT , Chakrapani S .

R00EY019718 NEI NIH HHS , R01DK81567 NIDDK NIH HHS , P41 GM103403 NIGMS NIH HHS , S10 RR027091 NCRR NIH HHS , R00 EY019718 NEI NIH HHS , R01 DK081567 NIDDK NIH HHS , 1R01GM108921 NIGMS NIH HHS , U54 GM087519 NIGMS NIH HHS , U54-GM087519 NIGMS NIH HHS , S10RR027091 NCRR NIH HHS , R01 GM108921 NIGMS NIH HHS

	Figure 1. Agonist- and antagonist-evoked conformational changes in the ELIC-ECD. (A) Superimposition of apo-ELIC (PDB accession no. 3RQU) and ACh-ELIC (PDB accession no. 3RQW) structures shows an inward (counterclockwise) motion of loop C. (B) Concentration-dependent changes in the EPR line shape and amplitude measured at position Ser180 in the presence and absence of 3 mM ACh. In each case, the spectra are normalized to the total number of spin (see Materials and methods). A plot of peak amplitude (of the central line) as a functional 3-AP concentration (right). (C) Spin-normalized CW-EPR spectra of representative loop C residues in apo state and in the presence of ACh or 3-AP. (D) Changes in probe mobility of loop C positions in the three conditions. The gray boxes highlight regions displaying prominent changes.
	Figure 2. Functional characterization of the ELIC-GLIC chimera. (A) A schematic representation of the chimera based on GLIC (PDB accession no. 4HFI) and ELIC (PDB accession no. 3RQU) structures, and the sequence alignment highlighting the region where the ELIC-ECD was fused to the GLIC-TMD (top). Agonist-induced (25 mM 3-AP) 13-min current response in oocytes measured by two-electrode voltage clamp (bottom). Inset shows (B) current traces for the chimera 9′Ala mutant in response to activation by acid (pH 5.0), agonist (25 mM GABA), and a combination of both. The plot shows peak response for the three conditions. The error bars denote standard deviation (n = 3). (C) Dose–response curves for the 9′Ala mutant (solid line) in the presence of 3-AP (dashed line, compared with ELIC) or acid (dashed line, comparison with GLIC). Currents are expressed as a fraction of the maximal response. The error bars denote standard deviation (n > 5). The EC50 (3-AP) and pH50 values are 32.8 ± 9.1 mM and 4.22 ± 0.11, and the corresponding Hill coefficients are 1.1 ± 0.2 and 9.1 ± 2.1, respectively. The EC50 (3-AP) and Hill coefficient for WT ELIC are 10.02 ± 0.05 mM and 2.5 ± 0.3. The EC50 (pH) and Hill coefficient for WT GLIC are 4.89 ± 0.06 mM and 2.0 ± 0.1. The error bars for WT denote standard deviation (n > 3).
	Figure 3. Structural differences in the ELIC-GLIC chimera compared with ELIC and GLIC. (A) Top view of TMDs comparing the ELIC-GLIC chimera with GLIC structures in the closed (pH 7.0; PDB accession no. 4NPQ) and open (pH 4.0; PDB accession no. 4HFI) forms. Red arrows highlight positional differences. (B) Superimposition of the ELIC (PDB accession no. 2YN6), GLIC (pH 7.0; PDB accession no. 4NPQ), and ELIC-GLIC structures at the level of the ECD. The ECD pentamer comprising of residues 11–200 was superimposed at the Cα level. Black arrows show the direction of twist from ELIC to ELIC-GLIC (left) and from ELIC to GLIC (right). Key regions showing difference in position are marked by black circles.
	Figure 4. Pore profile of the ELIC-GLIC chimera and GLIC structures. (A) Water-accessible region of the pore as determined by the MOLE PyMOL plugin (Petřek et al., 2007). Two subunits are shown, with residues lining the ion permeation pathway represented as sticks. (B) A close-up view of diagonal M2 helices. (C) Pore radius along the channel axis in the ELIC-GLIC chimera and in the GLIC-closed (PDB accession no. 4NPQ) and -open (PDB accession no. 4HFI) forms calculated using MOLE software (Petřek et al., 2007).
	Figure 5. Loop C movements in the ELIC-GLIC chimera. (A) An overlay of ELIC-ECD in the apo (PDB accession no. 3RQU)- and GABA-flurazepam (PDB accession no. 4A96)–bound forms, with residues Asn184 and Ser189 highlighted (left). CW-EPR line shapes for the loop C positions in the apo conformation, and in the presence of 20 mM 3-AP, or at pH 3.0. The light and dark blue arrows highlight the immobile and mobile components of the spectra, respectively. (B) ELIC structure showing the location of Ser189 and corresponding cβ-cβ distances for the adjacent and nonadjacent subunits (left). Background-subtracted DEER echo intensity is plotted against evolution time and fit using model-free Tikhonov regularization. The corresponding interspin distance distribution (right). The arrows highlight the direction of change (orange, 3-AP; green, pH 3.0).
	Figure 6. 3-AP– and pH-induced conformational changes in loop F of the ELIC-GLIC chimera. (A) Locations of examined residues in ELIC-GLIC (left) and spin-normalized CW-EPR spectra of the residues in the apo state, with 20 mM 3-AP, and at pH 3.0 (right). (B) The corresponding distances mapped on the ELIC structure (PDB accession no. 3RQU; left). The DEER echo and the distance distribution under the three conditions (right). The peak values for distance distribution are marked for the apo state. The arrows highlight the direction of change (orange, 3-AP; green, pH 3.0).
	Figure 7. Structural rearrangements at the domain interface in the ELIC-GLIC chimera. (A) Overlays of Apo-ELIC (PDB accession no. 3RQU) and ELIC-GABA-flurazepam (PDB accession no. 4A96) (left) and spin-normalized CW spectra of interface domain residues L29R1 (β1–β2 loop), V82R1 (β4–β5 loop), M114R1, and F116R1 (β6–β7 loop) in the apo state, in the presence of 20 mM 3-AP, or at pH 3.0 (right). (B) Overlay of GLIC structures in the closed (PDB accession no. 4NPQ) and open conformations (PDB accession no. 4HFI; left). CW-EPR spectra of K255R1 (M2–M3 linker) and F322R1 (M4) in the apo state, in the presence of 20 mM 3-AP, and at pH 3.0 (right).

Adams, PHENIX: a comprehensive Python-based system for macromolecular structure solution. 2010, Pubmed

Adams, PHENIX: a comprehensive Python-based system for macromolecular structure solution. 2010, Pubmed
Altenbach, Accessibility of nitroxide side chains: absolute Heisenberg exchange rates from power saturation EPR. 2005, Pubmed
Blanc, Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. 2004, 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
Bourne, Crystal structure of a Cbtx-AChBP complex reveals essential interactions between snake alpha-neurotoxins and nicotinic receptors. 2005, 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
Brams, A structural and mutagenic blueprint for molecular recognition of strychnine and d-tubocurarine by different cys-loop receptors. 2011, Pubmed
Celie, Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an alpha-conotoxin PnIA variant. 2005, Pubmed , Xenbase
Celie, Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. 2004, Pubmed
Chakrapani, A quantitative description of KcsA gating I: macroscopic currents. 2007, Pubmed
Chakrapani, EPR Studies of Gating Mechanisms in Ion Channels. 2015, Pubmed
Chen, MolProbity: all-atom structure validation for macromolecular crystallography. 2010, Pubmed
Chiang, The determination of pair distance distributions by pulsed ESR using Tikhonov regularization. 2005, Pubmed
Collaborative Computational Project, Number 4, The CCP4 suite: programs for protein crystallography. 1994, Pubmed
Cooper, Up-regulation of cell-surface alpha4beta2 neuronal nicotinic receptors by lower temperature and expression of chimeric subunits. 1999, Pubmed
Corringer, Structure and pharmacology of pentameric receptor channels: from bacteria to brain. 2012, Pubmed
Cortes, Molecular architecture of full-length KcsA: role of cytoplasmic domains in ion permeation and activation gating. 2001, Pubmed
Delcour, Modified reconstitution method used in patch-clamp studies of Escherichia coli ion channels. 1989, Pubmed
Dellisanti, Site-directed spin labeling reveals pentameric ligand-gated ion channel gating motions. 2013, Pubmed , Xenbase
Duret, Functional prokaryotic-eukaryotic chimera from the pentameric ligand-gated ion channel family. 2011, Pubmed , Xenbase
Eiselé, Chimaeric nicotinic-serotonergic receptor combines distinct ligand binding and channel specificities. 1993, Pubmed , Xenbase
Emsley, Features and development of Coot. 2010, Pubmed
Farahbakhsh, Spin labeled cysteines as sensors for protein-lipid interaction and conformation in rhodopsin. 1992, Pubmed
Gonzalez-Gutierrez, Bridging the gap between structural models of nicotinic receptor superfamily ion channels and their corresponding functional states. 2010, 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, The atypical cation-conduction and gating properties of ELIC underscore the marked functional versatility of the pentameric ligand-gated ion-channel fold. 2015, Pubmed
Gross, Identification of protein side chains near the membrane-aqueous interface: a site-directed spin labeling study of KcsA. 2002, Pubmed
Grutter, Molecular tuning of fast gating in pentameric ligand-gated ion channels. 2005, Pubmed
Hansen, Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations. 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, 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
Jeschke, Direct conversion of EPR dipolar time evolution data to distance distributions. 2002, Pubmed
Kabsch, XDS. 2010, Pubmed
Khatri, Structural rearrangements in loop F of the GABA receptor signal ligand binding, not channel activation. 2009, Pubmed , Xenbase
Kinde, Conformational Changes Underlying Desensitization of the Pentameric Ligand-Gated Ion Channel ELIC. 2015, Pubmed
Krissinel, Inference of macromolecular assemblies from crystalline state. 2007, Pubmed
Lape, On the nature of partial agonism in the nicotinic receptor superfamily. 2008, Pubmed
Lee, Principal pathway coupling agonist binding to channel gating in nicotinic receptors. 2005, Pubmed
Li, Ligand-binding domain of an α7-nicotinic receptor chimera and its complex with agonist. 2011, Pubmed
McCoy, Solving structures of protein complexes by molecular replacement with Phaser. 2007, Pubmed
Mchaourab, Motion of spin-labeled side chains in T4 lysozyme. Correlation with protein structure and dynamics. 1996, Pubmed
Miller, Crystal structure of a human GABAA receptor. 2014, Pubmed
Moraga-Cid, Allosteric and hyperekplexic mutant phenotypes investigated on an α1 glycine receptor transmembrane structure. 2015, Pubmed
Mukhtasimova, Detection and trapping of intermediate states priming nicotinic receptor channel opening. 2009, Pubmed
Mukhtasimova, Initial coupling of binding to gating mediated by conserved residues in the muscle nicotinic receptor. 2005, Pubmed
Murshudov, Refinement of macromolecular structures by the maximum-likelihood method. 1997, Pubmed
Pan, Structure of the pentameric ligand-gated ion channel ELIC cocrystallized with its competitive antagonist acetylcholine. 2012, Pubmed
Pannier, Dead-time free measurement of dipole-dipole interactions between electron spins. 2000, Pubmed
Petrek, MOLE: a Voronoi diagram-based explorer of molecular channels, pores, and tunnels. 2007, Pubmed
Polyhach, Rotamer libraries of spin labelled cysteines for protein studies. 2011, Pubmed
Polyhach, Spin pair geometry revealed by high-field DEER in the presence of conformational distributions. 2007, Pubmed
Prevost, A locally closed conformation of a bacterial pentameric proton-gated ion channel. 2012, Pubmed
Purohit, Loop C and the mechanism of acetylcholine receptor-channel gating. 2013, Pubmed
Rienzo, Structural requirements in the transmembrane domain of GLIC revealed by incorporation of noncanonical histidine analogs. 2014, Pubmed , Xenbase
Sauguet, Structural basis for ion permeation mechanism in pentameric ligand-gated ion channels. 2013, Pubmed , Xenbase
Sauguet, Crystal structures of a pentameric ligand-gated ion channel provide a mechanism for activation. 2014, Pubmed , Xenbase
Sine, Recent advances in Cys-loop receptor structure and function. 2006, Pubmed
Spurny, Pentameric ligand-gated ion channel ELIC is activated by GABA and modulated by benzodiazepines. 2012, Pubmed , Xenbase
Thompson, The structural basis of function in Cys-loop receptors. 2010, Pubmed
Tillman, ELIC-α7 Nicotinic acetylcholine receptor (α7nAChR) chimeras reveal a prominent role of the extracellular-transmembrane domain interface in allosteric modulation. 2014, Pubmed
Ulens, The prokaryote ligand-gated ion channel ELIC captured in a pore blocker-bound conformation by the Alzheimer's disease drug memantine. 2014, Pubmed , Xenbase
Unwin, Gating movement of acetylcholine receptor caught by plunge-freezing. 2012, Pubmed
Velisetty, Structural basis for allosteric coupling at the membrane-protein interface in Gloeobacter violaceus ligand-gated ion channel (GLIC). 2014, Pubmed
Velisetty, Conformational transitions underlying pore opening and desensitization in membrane-embedded Gloeobacter violaceus ligand-gated ion channel (GLIC). 2012, Pubmed
Velisetty, Desensitization mechanism in prokaryotic ligand-gated ion channel. 2012, 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
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
Zou, Increased sensitivity and extended range of distance measurements in spin-labeled membrane proteins: Q-band double electron-electron resonance and nanoscale bilayers. 2010, Pubmed
daCosta, Gating of pentameric ligand-gated ion channels: structural insights and ambiguities. 2013, Pubmed