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

  1. Chandra M Khantwal
  2. Sherwin J Abraham
  3. Wei Han
  4. Tao Jiang
  5. Tanmay S Chavan
  6. Ricky C Cheng
  7. Shelley M Elvington
  8. Corey W Liu
  9. Irimpan I Mathews
  10. Richard A Stein
  11. Hassane S Mchaourab
  12. Emad Tajkhorshid
  13. Merritt Maduke  Is a corresponding author
  1. Stanford University School of Medicine, United States
  2. University of Illinois at Urbana-Champaign, United States
  3. Stanford University, United States
  4. Vanderbilt University, United States

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 19F 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.

Article and author information

Author details

  1. Chandra M Khantwal

    Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Sherwin J Abraham

    Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. 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
    Competing interests
    The authors declare that no competing interests exist.
  4. 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
    Competing interests
    The authors declare that no competing interests exist.
  5. Tanmay S Chavan

    Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Ricky C Cheng

    Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Shelley M Elvington

    Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Corey W Liu

    Stanford Magnetic Resonance Laboratory, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Irimpan I Mathews

    Stanford Synchrotron Radiation Lightsource, Stanford University, Menlo Park, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Richard A Stein

    Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Hassane S Mchaourab

    Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. 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
    Competing interests
    The authors declare that no competing interests exist.
  13. Merritt Maduke

    Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States
    For correspondence
    maduke@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. Richard Aldrich, The University of Texas at Austin, United States

Publication history

  1. Received: August 27, 2015
  2. Accepted: January 14, 2016
  3. Accepted Manuscript published: January 22, 2016 (version 1)
  4. Version of Record published: February 18, 2016 (version 2)

Copyright

© 2016, Khantwal et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,264
    Page views
  • 642
    Downloads
  • 30
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Chandra M Khantwal
  2. Sherwin J Abraham
  3. Wei Han
  4. Tao Jiang
  5. Tanmay S Chavan
  6. Ricky C Cheng
  7. Shelley M Elvington
  8. Corey W Liu
  9. Irimpan I Mathews
  10. Richard A Stein
  11. Hassane S Mchaourab
  12. Emad Tajkhorshid
  13. Merritt Maduke
(2016)
Revealing an outward-facing open conformational state in a CLC Cl-/H+ exchange transporter
eLife 5:e11189.
https://doi.org/10.7554/eLife.11189

Further reading

    1. Neuroscience
    2. Structural Biology and Molecular Biophysics
    Tianzhi Li, Qiqi Cheng ... Cong Ma
    Research Article

    Exocytosis of secretory vesicles requires the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and small GTPase Rabs. As a Rab3/Rab27 effector protein on secretory vesicles, Rabphilin 3A was implicated to interact with SNAP-25 to regulate vesicle exocytosis in neurons and neuroendocrine cells, yet the underlying mechanism remains unclear. In this study, we have characterized the physiologically relevant binding sites between Rabphilin 3A and SNAP-25. We found that an intramolecular interplay between the N-terminal Rab-binding domain and C-terminal C2AB domain enables Rabphilin 3A to strongly bind the SNAP-25 N-peptide region via its C2B bottom α-helix. Disruption of this interaction significantly impaired docking and fusion of vesicles with the plasma membrane in rat PC12 cells. In addition, we found that this interaction allows Rabphilin 3A to accelerate SNARE complex assembly. Furthermore, we revealed that this interaction accelerates SNARE complex assembly via inducing a conformational switch from random coils to α-helical structure in the SNAP-25 SNARE motif. Altogether, our data suggest that the promotion of SNARE complex assembly by binding the C2B bottom α-helix of Rabphilin 3A to the N-peptide of SNAP-25 underlies a pre-fusion function of Rabphilin 3A in vesicle exocytosis.

    1. Chromosomes and Gene Expression
    2. Structural Biology and Molecular Biophysics
    Lena Maria Muckenfuss, Anabel Carmen Migenda Herranz ... Martin Jinek
    Research Article Updated

    3′ end formation of most eukaryotic mRNAs is dependent on the assembly of a ~1.5 MDa multiprotein complex, that catalyzes the coupled reaction of pre-mRNA cleavage and polyadenylation. In mammals, the cleavage and polyadenylation specificity factor (CPSF) constitutes the core of the 3′ end processing machinery onto which the remaining factors, including cleavage stimulation factor (CstF) and poly(A) polymerase (PAP), assemble. These interactions are mediated by Fip1, a CPSF subunit characterized by high degree of intrinsic disorder. Here, we report two crystal structures revealing the interactions of human Fip1 (hFip1) with CPSF30 and CstF77. We demonstrate that CPSF contains two copies of hFip1, each binding to the zinc finger (ZF) domains 4 and 5 of CPSF30. Using polyadenylation assays we show that the two hFip1 copies are functionally redundant in recruiting one copy of PAP, thereby increasing the processivity of RNA polyadenylation. We further show that the interaction between hFip1 and CstF77 is mediated via a short motif in the N-terminal ‘acidic’ region of hFip1. In turn, CstF77 competitively inhibits CPSF-dependent PAP recruitment and 3′ polyadenylation. Taken together, these results provide a structural basis for the multivalent scaffolding and regulatory functions of hFip1 in 3′ end processing.