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Unfolding of a ClC chloride transporter retains memory of its evolutionary history

Nature Chemical Biology volume 14, pages 489–496 (2018)Cite this article

Abstract

ClC chloride channels and transporters are important for chloride homeostasis in species from bacteria to human. Mutations in ClC proteins cause genetically inherited diseases, some of which are likely to involve folding defects. The ClC proteins present a challenging and unusual biological folding problem because they are large membrane proteins possessing a complex architecture, with many reentrant helices that go only partway through membrane and loop back out. Here we were able to examine the unfolding of the Escherichia coli ClC transporter, ClC-ec1, using single-molecule forced unfolding methods. We found that the protein could be separated into two stable halves that unfolded independently. The independence of the two domains is consistent with an evolutionary model in which the two halves arose from independently folding subunits that later fused together. Maintaining smaller folding domains of lesser complexity within large membrane proteins may be an advantageous strategy to avoid misfolding traps.

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Fig. 1: ClC-ec1 structure and single-molecule forced unfolding experiments.
Fig. 2: Separate unfolding of N- and C-terminal domains.
Fig. 3: A longer linker between domains increases the size of the pretransition.
Fig. 4: Characterization of the isolated N- and C-terminal domains.
Fig. 5: Molecular dynamics simulations and vesicle swelling assay.
Fig. 6: Stable off-pathway misfolded states.

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Acknowledgements

This work was supported by the National Institutes of Health (R01GM063919 to J.U.B. and U54GM087519 to W.I.), the National Science Foundation (MCB-1727508 to W.I.), and the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (NRF-2016R1A6A3A03007871 to D.M.). We thank J. L. Robertson at the University of Iowa for sending a plasmid template containing a monomeric ClC-ec1 gene and the members of our lab for comments on the manuscript. Anton 2 computer time was provided by the Pittsburgh Supercomputing Center (PSC) through grant R01GM116961 from the National Institutes of Health. The Anton 2 machine at PSC was generously made available by D. E. Shaw Research.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA, USA

    Duyoung Min, Robert E. Jefferson, Jing Yang Wang, Mark A. Arbing & James U. Bowie

  2. UCLA-DOE and Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Duyoung Min, Robert E. Jefferson, Jing Yang Wang, Mark A. Arbing & James U. Bowie

  3. School of Chemistry and Molecular Engineering, East China Normal University, Shanghai, China

    Yifei Qi

  4. Departments of Biological Sciences and Bioengineering, Lehigh University, Bethlehem, PA, USA

    Wonpil Im

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Contributions

D.M., R.E.J. and J.U.B. conceived and designed the experiments. Y.Q. and W.I. designed and performed molecular dynamics simulations. D.M., R.E.J., J.Y.W. and M.A.A. performed plasmid cloning and protein purification. D.M. performed DNA handle conjugation and single-molecule forced unfolding experiments. R.E.J. performed the domain isolation, SEC, CD and vesicle swelling experiments. D.M., R.E.J. and J.U.B. analyzed the experimental data. Y.Q., W.I., D.M. and J.U.B. analyzed the molecular dynamics simulations. D.M., R.E.J., Y.Q., W.I. and J.U.B. wrote the paper.

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Correspondence to James U. Bowie.

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Min, D., Jefferson, R.E., Qi, Y. et al. Unfolding of a ClC chloride transporter retains memory of its evolutionary history. Nat Chem Biol 14, 489–496 (2018). https://doi.org/10.1038/s41589-018-0025-4

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