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Conformational changes required for H+/Cl− exchange mediated by a CLC transporter

Nature Structural & Molecular Biology volume 21, pages 456–463 (2014)Cite this article

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Abstract

CLC-type exchangers mediate transmembrane Cl− transport. Mutations altering their gating properties cause numerous genetic disorders. However, their transport mechanism remains poorly understood. In conventional models, two gates alternatively expose substrates to the intra- or extracellular solutions. A glutamate was identified as the only gate in the CLCs, suggesting that CLCs function by a nonconventional mechanism. Here we show that transport in CLC-ec1, a prokaryotic homolog, is inhibited by cross-links constraining movement of helix O far from the transport pathway. Cross-linked CLC-ec1 adopts a wild-type–like structure, indicating stabilization of a native conformation. Movements of helix O are transduced to the ion pathway via a direct contact between its C terminus and a tyrosine that is a constitutive element of the second gate of CLC transporters. Therefore, the CLC exchangers have two gates that are coupled through conformational rearrangements outside the ion pathway.

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Figure 1: Structural arrangement of CLC-ec1.
Figure 2: Functional effects of cross-linking helices J, O and Q.
Figure 3: Structure of the A399C A432C cross-linked mutant.
Figure 4: Movement of helix O is coupled to the Cl− gates.
Figure 5: I402 couples helix O to Y445.
Figure 6: Effects of cross-linking helix O on the Cl−/H+ exchange stoichiometry.
Figure 7: Transport cycle for Cl−/H+ exchange in the CLC transporters.

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Acknowledgements

The authors wish to thank C. Miller (Brandeis University) for the generous gift of 36Cl and O. Boudker, C. Nimigean, O. Andersen and members of the Accardi laboratory for helpful discussions and comments on the manuscript. This work was supported by US National Institutes of Health grant GM085232 and an Irma T. Hirschl–Monique Weill-Caulier Scholar Award (to A.A.).

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Authors and Affiliations

  1. Department of Anesthesiology, Weill Cornell Medical College, New York, New York, USA

    Daniel Basilio, Kristin Noack, Alessandra Picollo & Alessio Accardi

  2. Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA

    Alessio Accardi

  3. Department of Biochemistry, Weill Cornell Medical College, New York, New York, USA

    Alessio Accardi

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  1. Daniel Basilio
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Contributions

D.B., K.N. and A.P. performed experiments; D.B. and A.A. analyzed the data; A.A. designed research and wrote the paper; and all authors contributed to the editing of the manuscript.

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Correspondence to Alessio Accardi.

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Integrated supplementary information

Supplementary Figure 1 Effect of single-cysteine mutants on Cl– transport.

Representative traces of Cl- efflux mediated by WT CLC-ec1 (black), G259C (red), A399C (green) and A432C (yellow) mutants of CLC-ec1. Averaged values of the Cl- transport rates are reported in Table 1.

Supplementary Figure 2 Gel shift assay to determine the number of free cysteines.

a-c) Time courses of reaction of the A399C and A432C single mutants (a), the A399C A432C double mutant before (b) and after (c) incubation with 30 μM Hg2+ for 60 min with 400 μM mPEG5K. Arrows denote the positions of the un-reacted and singly or doubly reacted proteins. Arrows indicate the positions of the protein when unreacted (black), singly (blue) or doubly reacted (red). d-e) Titration of Hg-induced protection from PEG-ylation of the strongly inhibited mutant A399C A432C (d) and of the non-inhibited mutant A392C T428C (e). f) Gel filtration profiles of the A399C A432C mutant before (black) and after (red) incubation with Hg2+.

Supplementary Figure 3 Hg2+ treatment completely protects the nine double mutants from PEGylation.

The proteins were incubated with 200 μM Hg2+ for 60 min prior to a 120 min incubation with 1 mM mPEG5K. Arrows denote the position of the unreacted proteins as in Supplementary Figure 2.

Supplementary Figure 4 Effect of cross-links at additional positions.

a-d) representative traces of Cl- efflux mediated by the A392C T428C (a), L252C P424C (b), A396C G429C (c) and G259C A399C (d) mutants of CLC-ec1 before (black) and after (red) crosslink formation. Average±s.e.m. of the Cl- transport rates are reported in Table 1.

Supplementary Figure 5 The A399C A432C cross-link acts on the Cl– pathway.

Averaged reduction of the transport rate induced by Hg-treatment for the indicated mutants. For reference the reduction of the WT Cys-less CLC-ec1 (black) and A399C A432CHg (red) are shown as dashed lines.

Supplementary Figure 6 Structural alignment of WT and A399C A432CHg CLC-ec1.

Alignment of the structures of WT (PDBID: 1OTS, blue) and A399C/A432CHg (PDBID: 4MQX, orange) in the ion binding region (a) and of helices O and Q around the crosslink region (b). Electron densities are contoured at 1σ and the Cl- ions are represented as blue (WT) and salmon (A399C A432CHg) colored spheres.

Supplementary Figure 7 Helix O moves during the transport cycle of CLC-ec1.

a, c) Location of the crosslink between helices J and Q (A432C G259C) (a) and of the intrahelix crosslink in helix O (A396C A399C) (c). b, d) Functional effect of crosslinks at G259C A432C (b) and A396C A399C (d) before (black) and after incubation with Hg2+ (red). Mean values of the transport rates are reported in Table 1.

Supplementary Figure 8 Helix O is kinked at Gly393.

a) Ribbon representation of helix O. For clarity the backbone atoms are also shown. Gly393 is colored in green. b) Alignment of the residues comprising the central portion of helix O from CLC-ec1, rCLC-7, hCLC-5, hCLC-1 and CLC-0. Gly393 is highlighted in green and Ala399 is shown in red.

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Supplementary Figures 1–8 and Supplementary Table 1 (PDF 13079 kb)

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Basilio, D., Noack, K., Picollo, A. et al. Conformational changes required for H+/Cl− exchange mediated by a CLC transporter. Nat Struct Mol Biol 21, 456–463 (2014). https://doi.org/10.1038/nsmb.2814

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