title: Revealing an outward-facing open conformational state in a CLC Cl(-)/H(+) exchange transporter.
abstract: CLC secondary active transporters exchange Cl(-) for H(+). Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its protonation. Using (19)F NMR, we show that as [H(+)] is increased to protonate Gluex and enrich the outward-facing state, a residue ~20 Å away from Gluex, near the subunit interface, moves from buried to solvent-exposed. Consistent with functional relevance of this motion, constriction via inter-subunit cross-linking reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gate-opening motions. In support of this model, mutations that decrease steric contact between Helix N (part of the extracellular gate) and Helix P (at the subunit interface) remove the inhibitory effect of the cross-link. Together, these results demonstrate the formation of a previously uncharacterized 'outward-facing open' state, and highlight the relevance of global structural changes in CLC function.
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States.
authors.forename: Chandra M
authors.initials: CM
authors.lastname: Khantwal
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States.
authors.forename: Sherwin J
authors.initials: SJ
authors.lastname: Abraham
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, United States.|College of Medicine, University of Illinois at Urbana-Champaign, Urbana, United States.|Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, United States.|Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States.
authors.forename: Wei
authors.initials: W
authors.lastname: Han
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, United States.|College of Medicine, University of Illinois at Urbana-Champaign, Urbana, United States.|Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, United States.|Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States.
authors.forename: Tao
authors.initials: T
authors.lastname: Jiang
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States.
authors.forename: Tanmay S
authors.initials: TS
authors.lastname: Chavan
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States.
authors.forename: Ricky C
authors.initials: RC
authors.lastname: Cheng
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States.
authors.forename: Shelley M
authors.initials: SM
authors.lastname: Elvington
authors.affiliation: Stanford Magnetic Resonance Laboratory, Stanford University School of Medicine, Stanford, United States.
authors.forename: Corey W
authors.initials: CW
authors.lastname: Liu
authors.affiliation: Stanford Synchrotron Radiation Lightsource, Stanford University, Menlo Park, United States.
authors.forename: Irimpan I
authors.initials: II
authors.lastname: Mathews
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, United States.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Stein
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, United States.
authors.forename: Hassane S
authors.initials: HS
authors.lastname: Mchaourab
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, United States.|College of Medicine, University of Illinois at Urbana-Champaign, Urbana, United States.|Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, United States.|Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States.
authors.forename: Emad
authors.identifier: http://orcid.org/0000-0001-8434-1010
authors.initials: E
authors.lastname: Tajkhorshid
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States.
authors.forename: Merritt
authors.initials: M
authors.lastname: Maduke
mesh_terms: D018118:Chloride Channels / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation
chemical_list: D018118:Chloride Channels; C508370:ClC protein, E coli; D029968:Escherichia coli Proteins
keywords: <i>e. coli</i>; antiporter; biophysics; crystallization; double electron-electron resonance spectroscopy; membrane exchanger; membrane protein; principal component analysis; structural biology