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Structural basis of pH-dependent activation in a CLC transporter

Nature Structural & Molecular Biology volume 31, pages 644–656 (2024)Cite this article

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An Author Correction to this article was published on 14 February 2024

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Abstract

CLCs are dimeric chloride channels and anion/proton exchangers that regulate processes such as muscle contraction and endo-lysosome acidification. Common gating controls their activity; its closure simultaneously silences both protomers, and its opening allows them to independently transport ions. Mutations affecting common gating in human CLCs cause dominant genetic disorders. The structural rearrangements underlying common gating are unknown. Here, using single-particle cryo-electron microscopy, we show that the prototypical Escherichia coli CLC-ec1 undergoes large-scale rearrangements in activating conditions. The slow, pH-dependent remodeling of the dimer interface leads to the concerted opening of the intracellular H+ pathways and is required for transport. The more frequent formation of short water wires in the open H+ pathway enables Cl− pore openings. Mutations at disease-causing sites favor CLC-ec1 activation and accelerate common gate opening in the human CLC-7 exchanger. We suggest that the pH activation mechanism of CLC-ec1 is related to the common gating of CLC-7.

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Fig. 1: pH- and Cl−-dependent conformational rearrangements of CLC-ec1.
Fig. 2: Opening of the H+ pathway is required for transport by CLC-ec1.
Fig. 3: smFRET-TIRF microscopy of interprotomer dynamics in a membrane environment.
Fig. 4: Substrate accessibilities of the H+ and Cl− pathways increase in Turn and Twist.
Fig. 5: Mechanism of pH-dependent activation of CLC-ec1.
Fig. 6: Activation mechanism of CLC-ec1.

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Data availability

All constructs are available on request to A.A. or O.B. All models and associated cryo-EM maps have been deposited into the Electron Microscopy Data Bank (EMDB) and the Protein Data Bank (PDB) under the following accession codes CLC-ec1 pH 7.5 0 mM Cl− Swap (PDB: 7RNX; EMD-24582); CLC-ec1 pH 7.5 100 mM Cl− Swap (PDB: 7RO0; EMD-24584); CLC-ec1 pH 4.5 0 mM Cl− Swap (PDB: 7RP6; EMD-24668); CLC-ec1 pH 4.5 0 mM Cl− Turn (PDB:7RSB; EMD-24583); CLC-ec1 pH 4.5 100 mM Cl− Swap (PDB: 7N8P; EMD-24241); CLC-ec1 pH 4.5 100 mM Cl− Turn (PDB: 7RP5; EMD-24612); CLC-ec1 pH 4.5 100 mM Cl− Twist (PDB: 7N0W;EMD-24263); R230C L249C pH 4.5 100 mM Cl− Swap (PDB: 8GA1; 29884); R230C L249C pH 4.5 100 mM Cl− Turn (PDB: 8GA3; EMD-29885); L25C A450C pH 4.5 100 mM Cl− Intermediate (PDB:8GA5; EMD-29890); L25C A450C pH 4.5 100 mM Cl− Twist (PDB: 8GAH; EMD-29899); E202Y pH 4.5 100 mM Cl− (PDB: 8GA0; EMD-29883), which are listed in Supplementary Table 7. The atomic coordinates of the free MD simulation at t = 0 and 1 µs of systems 1–8 listed in Supplementary Table 3 are available at https://doi.org/10.6084/m9.figshare.24777321.v1. Source data are provided with this paper.

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Acknowledgements

We thank members of the Accardi and Boudker labs, C. Nimigean and S. Scheuring for helpful discussions. We thank R. Hite for help with cryo-EM data analysis. We thank S. Blanchard for help with establishing smFRET resources. The work was supported by the National Institutes of Health (NIH) grants R01 GM128420 and R35 GM152012 (to A.A.) and R01GM120260 and R21GM126476 (to J.L.R.), and American Heart Association grant 19PRE34380215 (to D.C.). The electron microcopy data were collected on the Tiran Krios at the Cryo-EM Core Facility at NYU Langone Health, with the assistance of W. Rice and B. Wang (RRID: SCR_019202). Some of this work was performed at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center, supported by grants from the Simons Foundation (SF349247), NYSTAR and the NIH National Institute of General Medical Sciences (GM103310). The computational resource of this research was provided in part by TACC Stampede2, SDSC Expanse, PSC Bridges2 and NCSA Delta through allocation number MCB200216 from the Extreme Science and Engineering Discovery Environment (XSEDE), which was supported by National Science Foundation grant number no. 1548562, and in part by HPC resources supported by the Scientific Computing Unit at Weill Cornell Medicine.

Author information

Author notes

  1. Gerard Huysmans

    Present address: Erasmus University, Jette, Belgium

  2. These authors contributed equally: Eva Fortea, Sangyun Lee.

Authors and Affiliations

  1. Department of Physiology and Biophysics, Weill Cornell Medical School, New York, NY, USA

    Eva Fortea, Hatice Didar Ciftci, Gerard Huysmans, Olga Boudker & Alessio Accardi

  2. Department of Anesthesiology, Weill Cornell Medical School, New York, NY, USA

    Eva Fortea, Sangyun Lee, Yiorgos Argyros, Maria E. Falzone & Alessio Accardi

  3. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, USA

    Rahul Chadda, Robyn Mahoney-Kruszka & Janice L. Robertson

  4. Department of Biochemistry, Weill Cornell Medical School, New York, NY, USA

    Yiorgos Argyros, Maria E. Falzone & Alessio Accardi

  5. Department of Molecular Physiology and Biophysics, The University of Iowa, Iowa City, IA, USA

    Priyanka Sandal

  6. Tri-Institutional Training Program in Chemical Biology, New York, NY, USA

    Hatice Didar Ciftci

  7. Howard Hughes Medical Institute, Chevy Chase, MD, USA

    Olga Boudker

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Contributions

E.F. designed and performed cryo-EM experiments of WT CLC-ec1, collected and carried out initial processing of the CLC-ec1-E202Y dataset, smFRET measurements, patch–clamp recordings of CLC-7 and flux assays of WT and mutant CLC-ec1; S.L. designed and performed cryo-EM experiments and flux assays of Top and Bot crosslinked CLC-ec1, collected the CLC-ec1-E202Y dataset, completed processing and built the atomic model, carried out MD simulations and performed flux assays on mutant CLC-ec1; R.C., P.S., R.M.-K. and J.L.R. designed, carried out and analyzed dimerization experiments; Y.A. contributed to the electrophysiological experiments; M.E.F. contributed to the initial cryo-EM data processing; D.C. and G.H. contributed to the design of smFRET experiments, data acquisition and analysis; and O.B. and A.A. oversaw research and wrote the initial draft of the paper. All authors edited and approved the paper.

Corresponding authors

Correspondence to Olga Boudker or Alessio Accardi.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 CryoEM image processing scheme for the dataset at pH 4.5 in 100 mM Cl−.

(a) Representative cryoEM micrographs of CLC-ec1. (b) Representative 2D classes from CryoSPARC. (c) Summary of the image processing procedure. Red frames indicate classes that were selected for further processing. (d–f) Density maps (mesh) for αB, αH, αI, and the H-I loop with the refined models of Swap (D), Turn (E), and Twist (F). (g–j) EM density (mesh) and atomic models of E148 for Swap at pH 7.5 and 100 mM Cl− (G), Swap at pH 7.5 and 0 mM Cl− (H), Turn at pH 4.5 and 0 mM Cl− (I), and Twist and pH 4.5 and 100 mM Cl− (J). Data acquisition and refinement parameters are reported in Tables 1 and 2.

Extended Data Fig. 2 Validation of maps and models for WT CLC-ec1.

Fourier shell correlation (FSC) curves for the masked (tight mask from CryoSPARC) and unmasked density maps (left), FSC curves from model−map cross-validation (middle), and density maps colored by local resolution estimated in CryoSPARC (right) of maps obtained in the following conditions: pH 4.5 and 100 mM Cl− (A), pH 4.5 and 0 mM Cl− (B), pH 7.5 and 0 mM Cl− (C), pH 7.5 and 100 mM Cl− (D). Data acquisition and refinement parameters are reported in Table 1.

Source data

Extended Data Fig. 3 Structural comparisons between different CLC-ec1 conformations.

(a, b) Per residue Cα r.m.s.d. for CLC-ec1 in Swap at pH 7.5 and 100 mM Cl− relative to the crystal structure (PDB: 1OTS) (A) and for Turn at pH 4.5 and 100 mM Cl− relative to E113Q/E148Q/E203Q (QQQ, PDB: 6V2J) (B). (c–e) Close-up view of the rearrangements of the H-I loops in Swap (C), Turn (D), and Twist (E). The top panels are viewed from the plane of the membrane, and the bottom panels are viewed from the intracellular side. One protomer is colored gray, and the other is colored red (Swap), wheat (Turn), and Blue (Twist). Helices αH and αI are shown in cartoon representation, and residues Q207, F208, R209, Y210, T211, and L212 are shown in stick CPK representation. (f) Quantification of the surface area per protomer buried at the interface of the soluble (gray bars) and TM (black bars) domains in Swap, Turn, and Twist. (g, h) Cartoon representations of single protomers viewed from the dimer interface and colored by changes in per-residue contact areas in Turn (G) and Twist (H) relative to Swap. (i) Diameter of the Cl− permeation pathway visualized using HOLE 1 in cryoEM structures. The position of the external gate is defined as the midpoint of the backbone atoms of residues 147, 148, 356, and 357. Residues S107, E148, I356, and Y445 are shown as sticks. Blue indicates regions with R > 2.3 Å, green 2.3 Å>R > 1.15 Å, and red R < 1.15 Å.

Source data

Extended Data Fig. 4 Characterization of Top and Bot crosslinks.

(a, b) SDS PAGE showing the molecular mass of TCEP-treated (right) or untreated (left) CLC-ec1 WT, Top (R230C/L249C) and Bot (L25C/A450C) crosslinks, L25C and A450C single mutants, and the Top+Bot quadruple mutant. Arrows denoted by D and M indicate the position of the dimer and monomer, respectively. Note that L25C on helix αA spontaneously forms dimers, reflecting crosslinks between the two dynamic αA helices. This results in two dimer bands for the Bot and Top+Bot crosslinked constructs, where αA of one protomer is crosslinked either to αA or αR of the sister protomer. SDS PAGE results were consistent in three repeats from independently obtained proteo-liposome samples of WT and all mutants. (c) Time courses of Cl− efflux mediated by TCEP-treated (right) or untreated (left) proteoliposomes reconstituted with CLC-ec1 WT (black/blue), Bot (pink/dark green), L25C (light green/gray), Top (orange/brown), A450C (purple/yellow) and Top+Bot (red/cyan). Traces for WT and Bot ±TCEP are the same as those shown in Fig. 2. (d) Intracellular view of cryoEM density maps of CLC-ec1 Bot crosslink in Intermediate (left panel) and Twist (right panel) conformations imaged at pH 4.5 in 100 mM Cl−. One subunit is colored gray, and the other is pink (Intermediate) or yellow (Twist). (e) CryoEM density (mesh) contoured at 5.1 σ and atomic model of the crosslink between A450C and L25C in Bot Intermediate. (f) Intracellular view of cryoEM density maps of CLC-ec1 Top crosslink in Swap (left panel) and Turn (right panel) conformations imaged at pH 4.5 in 100 mM Cl−. One subunit is colored gray, and the other is green (Swap) or aquamarine (Turn). (g) CryoEM density (mesh) contoured at 6.0 σ and atomic model of the crosslink between A450C and L25C in Top Swap.

Source data

Extended Data Fig. 5 Validation of maps and models for CLC-ec1 mutants.

(a–c) FSC curves for the masked (tight mask from CryoSPARC) and unmasked density maps (left), FSC curves from model−map cross-validation (middle), and density maps colored by local resolution estimated in CryoSPARC (right) of maps obtained for CLC-ec1 (A) L25C/A450C Bot Intermediate and Twist, (B) R230C/L249C Top Swap and Turn, (C) E202Y. All data were collected at pH 4.5 and 100 mM Cl−. Data acquisition and refinement parameters are reported in Table 2.

Source data

Extended Data Fig. 6 Characterization of CLC-ec1 mutants used in smFRET experiments and the dynamics of the dimer interface.

(a) Representative normalized time course of chloride efflux from liposomes reconstituted at 0.2 μg protein/mg lipid with CLC-ec1 WT (black), Q24C (orange) and Q24C labelled with maleimide activated LD555p and LD650 fluorophores in the same molar ratio used in smFRET TIRF imaging (CLC-ec1:LD555p:LD650 = 1:2:2.5) (green). Traces were normalized to the total chloride content of the liposomes determined following the addition of detergent2. (b) The initial velocity of Cl− efflux from proteoliposomes containing WT CLC-ec1 (red), CLC-ec1 Q24C (orange), and Q24C-labelled (green) All values are means ± S.D. The number of repeats of independent experiments and preparations of proteo-liposome samples for all constructs are reported in Supplementary Table 1. (c) The experimental design to monitor dynamics of the TM dimer interface tracking the movement of E385C labelled with donor and acceptor fluorophores. CLC-ec1 dimer is shown as cartoon and colored as in Fig. 1. The labeling site is shown as a black sphere, and inter-dye distances are shown on the structures. Schematic representations of Swap, Turn, and Twist are on the left. (d-g) Population contour plots of labelled E385C at pH 7.5 in 0 mM Cl− (D), pH 7.5 in 100 mM Cl− (E), pH 4.5 in 0 mM Cl− (F), and pH 4.5 in 100 mM Cl− (G). N, the number of molecules used to construct the plots, is shown on each panel. Cumulative FRET state histograms are on the right of each panel plotted as means of three independent experiments ± SEM. Assignments of the FRET states to the protein conformations are on the right of panel G. (h–m) Dwell-time distributions of FRET states corresponding to: ‘full TM interface’ (high FRET for E385C, Swap or Turn) (H) or ‘reduced TM interface’ (low FRET for E385C, Twist) (I) in 100 mM Cl− or 0 Cl− (L and M, respectively); αA bound (high FRET for Q24C, Swap) (J) or αA loose (low FRET for Q24C, Turn and Twist) (K) in 0 mM Cl-. Dashed lines are the fluorophore survival probability, which refers to the fraction of molecules where both LD555 and D655 survived longer than the time indicated in the x-axis at pH 7.5 (blue) and pH 4.5 (red). Data are averages of three independent repeats, and error bars are SEM.

Source data

Extended Data Fig. 7 Examples of smFRET recordings of CLC-ec1.

(a-h) Representative smFRET traces of CLC-ec1 labelled at E385C at pH 7.5 and 0 mM Cl− (A), pH 7.5 and 100 mM Cl− (B), pH 4.5 and 0 mM Cl− (C), pH 4.5 and 100 mM Cl− (D), or CLC-ec1 labelled at Q24C at pH 7.5 and 0 mM Cl− (E), pH 7.5 and 100 mM Cl− (F), pH 4.5 and 0 mM Cl− (G) and pH 4.5 and 100 mM Cl− (H). Fluorescence emissions from LD555p (green) and LD655 (red) dyes are shown in the top panels, and FRET efficiency (blue) is in the bottom panels. The traces were selected to illustrate the heterogeneity of the dynamic behaviors observed in all collected datasets. The quantitative analysis of the FRET-efficiency and dwell time distributions is reported in Fig. 3 and Extended Data Fig. 6.

Source data

Extended Data Fig. 8 Water wire formation and Cl− pathway dynamics of CLC-ec1.

(a) The pKa values of the ionizable residues calculated using PropKa3,4. Black dotted lines correspond to pH 4.5 and 7.5. Data are not shown for residues with the calculated pKa values below 4.5 or above 7.5 in all conformations. (b-f) Spontaneous formation of water wires connecting Gluex and the intracellular bulk water within the H+ pathways of protomers A and B, observed in 10 independent 1 μs long simulations of CLC-ec1 WT in Swap (B), Turn (C), Twist (D), Bot Intermediate crosslinked (E), and E202Y (F). Each vertical line in the panels shows the occurrence of a water wire at that time point. The number of events of formation of water wires as a function of the lifetime of individual event. The lifetime of water wire is defined as the time duration for any hydrogen bond in the water wire to be disconnected. (H)Representative pore-radius profiles along the z-axis of the Cl− pathway, calculated using HOLE1 for conformations with the open (green) and closed (blue) external gate seen in the MD simulations of Twist. (i) The diameter of the Cl− permeation pathway, visualized using HOLE1 in MD snapshots of Twist with an open (right panel) and closed (left panel) extracellular gate. The external gate radius is calculated using the backbone atoms of residues 147, 148, 356, and 357. Residues S107, E148, I356, and Y445 are shown as sticks. Blue indicates regions with R>2.3 Å, green 2.3 Å>R>1.15 Å, and red R< 1.15 Å. (j) Representative snapshots of conformational rearrangements in the backbone of the Cl− pathway when the external gate is open (green) or closed (blue). * denotes the center of the Cl− permeation pathway.

Source data

Extended Data Fig. 9 Characterization of activating mutations in CLC-ec1 and CLC-7.

(a) Theoretical (top panels - dimer & monomer) and experimental (middle and bottom panels – WT & ΔN29 pH 7.5 & 4.5) photobleaching steps probability distributions (P1, P2, P3+) for CLC-ec1 in lipid bilayers as a function of the experimental subunit/lipid mole fraction, χobs. Data for WT and ΔN29 pH 7.5 & 4.5 are means ± SEM for n = 4 for WT pH 4.5 and n = 3 for all other systems, where n is the number of independently prepared samples. (b, d) Representative normalized time courses of chloride efflux from liposomes reconstituted at pH 4.5 (B) or 7.5 (D) with CLC-ec1 WT (black), Q24C (orange), I201W (purple), E202Q (red) and E202Y (grey) at 0.2 μg protein/mg lipid. Traces were normalized to the total chloride content of the liposomes determined following the addition of detergent2. (c-e) Initial velocity of Cl− efflux at pH 4.5 (C) and 7.5 (E) of proteoliposomes reconstituted with CLC-ec1 WT (white), Q24C (orange), I201W (purple), E202Q (red) and E202Y (grey). All values are shown as means ± S.D. The number of repeats of independent experiments and preparations of proteo-liposome samples for all constructs are reported in Supplementary Table 1. (f, g) smFRET population contour plots of CLC-ec1 E202Q (F) and E202Y (G) labelled at E385C at pH 7.5 (left) and 4.5 (right) in 100 mM Cl−. Data are average of 3 independent repeats, and errors are SEM. (h) Forward and reverse potential of mean force (PMF) profile as a function of the minimum distance between αA of monomer A and αR of monomer B with E202 protonated, E202(0), or deprotonated, E202(-), at pH 4.5 or pH 7.5 with no Cl− in the central binding site. Molecular systems are listed in Supplementary Table 4. Error bars (in SD) are estimated by calculating the PMF values in four consecutive blocks of the trajectories. (i) Free energy difference between local energy minima for αA ‘bound’ to and ‘loose’ from αR with E202 protonated (E2020) or deprotonated (E202−) at pH 4.5 or pH 7.5 with no Cl− in the central binding site. ΔG is taken from the free energy profiles in panel H. Error bars are estimated in the same fashion as in panel H. (j) Current density-voltage curves for CLC-7PM WT (black), E311Q (red), L310W (blue), G578W (pink), and L310W/G578W (WW, orange). Data are averages of 10 independent repeats, and error bars are SEM.

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Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2 and Supplementary Tables 1–7.

Reporting Summary

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Supplementary Data 1

Primer sequences used in mutagenesis

Supplementary Video 1

The pH and Cl− dependent rearrangements of the CLC-ec1 dimer interface are viewed from the intracellular side. Morph between Swap, Turn, Twist conformations of CLC-ec1 viewed from the intracellular solution. The transmembrane dimer interface helices αI, αH, αP and αQ are shown in light pink, and the other helices of each protomer are colored by white and yellow, respectively.

Supplementary Video 2

The pH and Cl− dependent rearrangements of the CLC-ec1 dimer are viewed from the plane of the membrane. The same morph as in Supplementary Video 1, but viewed from the plane of the membrane.

Source data

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Fortea, E., Lee, S., Chadda, R. et al. Structural basis of pH-dependent activation in a CLC transporter. Nat Struct Mol Biol 31, 644–656 (2024). https://doi.org/10.1038/s41594-023-01210-5

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