Revealing an outward-facing open conformational state in a CLC Cl-/H+ exchange transporter

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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 (Glu_ex) upon its protonation. Using ¹⁹F NMR, we show that as [H⁺] is increased to protonate Glu_ex and enrich the outward-facing state, a residue ~20 Å away from Glu_ex, 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.

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Chandra M Khantwal

Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States

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Sherwin J Abraham

Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States

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Wei Han

Department of Biochemistry, College of Medicine, Center for Biophysics and Computational Biology, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States

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Tao Jiang

Department of Biochemistry, College of Medicine, Center for Biophysics and Computational Biology, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States

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Tanmay S Chavan

Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States

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Ricky C Cheng

Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States

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Shelley M Elvington

Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States

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Corey W Liu

Stanford Magnetic Resonance Laboratory, Stanford University School of Medicine, Stanford, United States

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Irimpan I Mathews

Stanford Synchrotron Radiation Lightsource, Stanford University, Menlo Park, United States

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Richard A Stein

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, United States

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Hassane S Mchaourab

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, United States

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Emad Tajkhorshid

Department of Biochemistry, College of Medicine, Center for Biophysics and Computational Biology, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States

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Merritt Maduke

Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States

For correspondence
maduke@stanford.edu

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