1. Crystal structures of a pentameric ion channel gated by alkaline pH show a widely open pore and identify a cavity for modulation
- Author
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Marc Delarue, Catherine Van Renterghem, Zaineb Fourati, Pierre-Jean Corringer, Ludovic Sauguet, Haidai Hu, Ákos Nemecz, Dynamique structurale des Macromolécules / Structural Dynamics of Macromolecules, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Sorbonne Université (SU), Récepteurs Canaux - Channel Receptors, Á.N. and Z.F. were supported by Agence Nationale de Recherches Grant 13-BSV8-0020 'Pentagate.' H.H. was sponsored by the China Scholarship Council and Institut Pasteur., We thank Anaïs Menny for sharing expertise on plasmid constructions, the staff of the Crystallography Platform (PF6) in the Institut Pasteur for initial crystal screens, Reinis Reinholds Ruza for help in data collection, Jérôme Loc’h for help during structural refinement and picture preparation, Stacy Gellenoncourt for help in purification and crystallization of the sTeLIC R86A mutant, the staff of synchrotron beamlines in Soleil (Orsay) and European Synchrotron Radiation Facility (Grenoble) for data collection, Pierre Legrand (Soleil synchrotron) for suggestions on refinement in Buster, and Marc Gielen for suggestions during the paper preparation, ANR-13-BSV8-0020,Pentagate,Mécanismes d'activation et de désensibilisation d'un récepteur-canal pentamérique(2013), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
- Subjects
0301 basic medicine ,Gating ,Crystallography, X-Ray ,Divalent ,03 medical and health sciences ,chemistry.chemical_compound ,Allosteric Regulation ,Bacterial Proteins ,[CHIM.CRIS]Chemical Sciences/Cristallography ,structural biology ,Binding site ,crystallography ,Ion channel ,Tetramethylammonium ,chemistry.chemical_classification ,Multidisciplinary ,ligand-gated ion channel ,druggable cavity ,Hydrogen-Ion Concentration ,Ligand-Gated Ion Channels ,electrophysiology ,Transmembrane protein ,Quaternary Ammonium Compounds ,030104 developmental biology ,chemistry ,Structural biology ,PNAS Plus ,Biophysics ,Ligand-gated ion channel ,Gammaproteobacteria - Abstract
International audience; Pentameric ligand-gated ion channels (pLGICs) constitute a widespread class of ion channels, present in archaea, bacteria, and eukaryotes. Upon binding of their agonists in the extracellular domain, the transmembrane pore opens, allowing ions to go through, via a gating mechanism that can be modulated by a number of drugs. Even though high-resolution structural information on pLGICs has increased in a spectacular way in recent years, both in bacterial and in eukaryotic systems, the structure of the open channel conformation of some intensively studied receptors whose structures are known in a nonactive (closed) form, such as Erwinia chrysanthemi pLGIC (ELIC), is still lacking. Here we describe a gammaproteobacterial pLGIC from an endo-symbiont of Tevnia jerichonana (sTeLIC), whose sequence is closely related to the pLGIC from ELIC with 28% identity. We provide an X-ray crystallographic structure at 2.3 Å in an active conformation, where the pore is found to be more open than any current conformation found for pLGICs. In addition, two charged restriction rings are present in the vestibule. Functional characterization shows sTeLIC to be a cationic channel activated at alkaline pH. It is inhibited by divalent cations, but not by quaternary ammonium ions, such as tetramethylammonium. Additionally, we found that sTeLIC is allosterically potentiated by aromatic amino acids Phe and Trp, as well as their derivatives, such as 4-bromo-cinnamate, whose cocrystal structure reveals a vestibular binding site equivalent to, but more deeply buried than, the one already described for benzodiazepines in ELIC.
- Published
- 2018