title: Probing the conformation of a conserved glutamic acid within the Cl-pathway of a CLC H+/Cl-exchanger.
abstract: The CLC proteins form a broad family of anion-selective transport proteins that includes both channels and exchangers. Despite extensive structural, functional, and computational studies, the transport mechanism of the CLC exchangers remains poorly understood. Several transport models have been proposed but have failed to capture all the key features of these transporters. Multiple CLC crystal structures have suggested that a conserved glutamic acid, Gluex, can adopt three conformations and that the interconversion of its side chain between these states underlies H+/Cl-exchange. One of these states, in which Gluexoccupies the central binding site (Scen) while Cl-ions fill the internal and external sites (Sintand Sext), has only been observed in one homologue, the eukaryotic cmCLC. The existence of such a state in other CLCs has not been demonstrated. In this study, we find that during transport, the prototypical prokaryotic CLC exchanger, CLC-ec1, adopts a conformation with functional characteristics that match those predicted for a cmCLC-like state, with Gluextrapped in Scenbetween two Cl-ions. Transport by CLC-ec1 is reduced when [Cl-] is symmetrically increased on both sides of the membrane and mutations that disrupt the hydrogen bonds stabilizing Gluexin Scendestabilize this trapped state. Furthermore, inhibition of transport by high [Cl-] is abolished in the E148A mutant, in which the Gluexside chain is removed. Collectively, our results suggest that, during the CLC transport cycle, Gluexcan occupy Scenas well as the Sextposition in which it has been captured crystallographically and that hydrogen bonds with the side chains of residues that coordinate ion binding to Scenplay a role in determining the equilibrium between these two conformations.
authors.affiliation: Department of Anesthesiology, Weill Cornell Medical College, New York, NY 10021.
authors.forename: Malvin
authors.initials: M
authors.lastname: Vien
authors.affiliation: Department of Anesthesiology, Weill Cornell Medical College, New York, NY 10021.
authors.forename: Daniel
authors.identifier: http://orcid.org/0000-0002-5346-8921
authors.initials: D
authors.lastname: Basilio
authors.affiliation: Department of Anesthesiology, Weill Cornell Medical College, New York, NY 10021.
authors.forename: Lilia
authors.identifier: http://orcid.org/0000-0003-3987-4269
authors.initials: L
authors.lastname: Leisle
authors.affiliation: Department of Anesthesiology, Weill Cornell Medical College, New York, NY 10021 ala2022@med.cornell.edu.|Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY 10021.|Department of Biochemistry, Weill Cornell Medical College, New York, NY 10021.
authors.forename: Alessio
authors.identifier: http://orcid.org/0000-0002-6584-0102
authors.initials: A
authors.lastname: Accardi
mesh_terms: D019943:Amino Acid Substitution; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D017124:Conserved Sequence; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015640:Ion Channel Gating; D011522:Protons
chemical_list: D018118:Chloride Channels; D002712:Chlorides; C508370:ClC protein, E coli; D029968:Escherichia coli Proteins; D011522:Protons; D018698:Glutamic Acid