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A topological switch in CFTR modulates channel activity and sensitivity to unfolding

Nature Chemical Biology volume 17, pages 989–997 (2021)Cite this article

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Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs. Functional impairment of CFTR due to mutations in the cftr gene leads to cystic fibrosis. Here, we show that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternate conformation that departs from the canonical NBD fold previously observed. Crystallography reveals that this conformation involves a topological reorganization of NBD1. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by adenosine triphosphate binding. However, under destabilizing conditions, such as the disease-causing mutation F508del, this conformational flexibility enables unfolding of the β-subdomain. Our data indicate that, in wild-type CFTR, this conformational transition of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.

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Fig. 1: Single-molecule FRET analysis for the 426–519 reporter pair of NBD1.
Fig. 2: The G11a-bound structure reveals a new conformation of NBD1 with a different topology.
Fig. 3: The G11a nanobody binds to full-length CFTR in cells and increases channel activity.
Fig. 4: smFRET analysis for the 479–519 reporter pair of NBD1.
Fig. 5: The effects of F508del, temperature and S492P on the β-subdomain.
Fig. 6: Model of an unfolding pathway through the β-SS conformation.

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Data availability

Data supporting the findings of this manuscript are available from the corresponding author upon reasonable request. The atomic coordinates and structure factors reported in this paper have been deposited in the Protein Data Bank (PDB). The accession number for the structure reported in this paper is PDB 6ZE1. Source data are provided with this paper.

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Acknowledgements

We thank C. R. O’Riordan for C127 cells, J. Kappes for HEK293 CFTR cells and J. R. Riordan, T. Jensen and CF Foundation Therapeutics for anti-CFTR antibodies. C.G. acknowledges support by the Fonds Forton, Welbio (grant no. CR-2012S-04R), Vaincre la Mucoviscidose, Mukoviszidose e.V., the Association Luxembourgeoise de Lutte contre la Mucoviscidose, ABCF2, Chiesi Fondation, the Cystic Fibrosis Foundation, Fondation Air Liquide and Fondation ULB. D.N.S acknowledges support from the CF Trust and CF Foundation Therapeutics. J.S. acknowledges support from Instruct-ERIC, part of the European Strategy Forum on Research Infrastructures (ESFRI), Instruct-ULTRA (EU H2020 grant no. 731005) and the Research Foundation—Flanders (FWO) for support with nanobody discovery. C.G. is a senior Research Associate of the FRS-FNRS. D.S. was a fellow of the FRIA. We acknowledge Diamond Light Source for time on beamlines i02, i04 and i24 under proposals 12718 and 9426. We are grateful to H. Remaut and B. Kobilka for careful reading of the manuscript. We thank F. Sobott and J. Ault of the Biomolecular Mass Spectrometry department in the Astbury Centre for their support and assistance in this work, as well as the BBSRC (BB/M012573/1) for funding.

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Authors and Affiliations

  1. SFMB, Université Libre de Bruxelles, Brussels, Belgium

    Daniel Scholl, Maud Sigoillot, Marie Overtus, Rafael Colomer Martinez, Chloé Martens & Cédric Govaerts

  2. School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, UK

    Yiting Wang & David N. Sheppard

  3. VIB-VUB center for Structural Biology, VIB, Brussels, Belgium

    Els Pardon, Toon Laeremans & Jan Steyaert

  4. Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium

    Els Pardon, Toon Laeremans & Jan Steyaert

  5. Cellular and Molecular Microbiology, Université Libre de Bruxelles, Gosselies, Belgium

    Abel Garcia-Pino

  6. Dynamic Bioimaging Lab, Advanced Optical Microscopy Centre and Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium

    Jelle Hendrix

  7. Molecular Imaging and Photonics, Chemistry Department, KU Leuven, Leuven, Belgium

    Jelle Hendrix

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Contributions

D.S., D.N.S. and C.G. conceived and designed the experiments. D.S., M.S., M.O. and R.C.M. performed NBD1 and nanobody expression, purification and biochemical characterization. D.S., M.S. and M.O. performed nanobody discovery, cloning and purification, supervised by E.P. and T.L. M.S. designed and conducted crystallography experiments and collected the X-ray data. M.S., C.G., A.G.-P. and D.S. processed and refined the X-ray data. M.O. performed and analyzed flow cytometry and CFTR pulldown experiments. Y.W. performed patch-clamp electrophysiological experiments, supervised by D.N.S. C.M. conducted the HDX-MS data collection and analysis. D.S. performed modeling, construction and labeling of FRET reporters, as well as smFRET experiments. D.S., J.H. and R.C.M. recorded and analyzed the smFRET data. D.S., D.N.S., J.S., J.H. and C.G. co-wrote the manuscript. D.N.S., J.S., J.H. and C.G. directed the study. All authors approved the final version of the manuscript.

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Correspondence to Cédric Govaerts.

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Extended data

Extended Data Fig. 1 Setup of the two-color MFD-PIE microscope.

a, Representation of NBD1 containing two cysteines residues (yellow spheres) which are labeled by the fluorophores ATTO488 and Alexa647. b, FRET between both fluorophores attached to NBD1 is monitored while NBD1 diffuses through the confocal volume. c, Schematic illustration of the confocal microscope setup used here to study molecules freely diffusing in solution by multiparameter fluorescence detection with pulsed interleaved excitation (MFD-PIE)57,58. The lasers emit pulses with a fixed repetition rate and are synchronized by the TCSPC hardware (see below). An electronic delay leads to a lag time between green and red laser leading to interleaved excitation of the sample. Dichroic mirrors (DM) combine the lasers. They are then collimated and their beam profile is cleaned up by a single-mode optical fiber (SMF). Polychroic mirrors (PM) separate the excitation from the emission light both spectrally and spatially. The emission light is focused through a confocal pinhole and collimated by achromatic lenses (AL) to reduce chromatic aberrations. The emission light is then split up into two spectral ranges (green and red). Polarization beam splitters (PBS) further split the polarization of each color range into parallel (|) and antiparallel (⊥). Emission filters (EmF) finally transmit the appropriate spectral band and a lens focuses the emission light onto an avalanche photodiode (APD). The time-correlated single photon counting (TCSPC) hardware receives the signals from the APDs. Three parameters are registered per photon: the identity of the detector (red or green, parallel or antiparallel), the microtime (time since the last laser pulse) and the macrotime (time since the beginning of the experiment).

Extended Data Fig. 2 Acceptor fluorescence lifetime versus FRET efficiency plots for the 426–519 reporter pair.

a, Location of the smFRET reporters in the canonical conformation of NBD1 (based on the published structure of human NBD1, PDB: 2BBO). The positions of the labelled cysteines are shown as yellow spheres. The regulatory insertion (RI), which is not resolved in the structure, is depicted as a red dashed line. F508, located in the α-subdomain, and ATP are shown. b–d, Two-dimensional histograms of acceptor fluorescence lifetime (τA) versus FRET efficiency of the 426–519 reporter for the indicated conditions. The histograms provide a distinctive signature for each of the three observed FRET states. Small changes in acceptor lifetimes are due to an altered fluorescence quantum yield of the acceptor, likely due to sticking of the fluorophore to the protein. Correction of these effects did not substantially affect the observed FRET efficiency values (data not shown).

Source data

Extended Data Fig. 3 Characterization of the interactions between NBD1 variants and nanobody G11a.

a, Differential scanning fluorescence (DSF) of purified NBD1 variants with and without nanobody G11a. The samples were incubated with SYPRO Orange dye and fluorescence was measured as a function of temperature. The melting temperatures (Tm) were determined by the maxima of the first derivative of fluorescence. Curves depict mean of duplicates of one experiment representative of at least three independent experiments. b, Binding of nanobody G11a to NBD1 variants measured by ELISA. Biotinylated NBD1 variants were immobilized on neutravidincoated plates and incubated with increasing concentrations of nanobody. Binding of nanobody was followed by immunodetection of the His6-tag (see Methods). Representative curve of 3 independent experiments is shown. Error bars represent the standard deviation (SD) of duplicates. See Supplementary Table 1 for pEC50 and Tm values.

Source data

Extended Data Fig. 4 FRET-restrained positioning and screening simulation.

FRET-restrained positioning and screening (FPS30) simulations of the accessible volumes of the donor and acceptor dyes ATTO488 and Alexa647 when attached to cysteine residues in NBD1. a, Accessible dye positions when attached to residues 426 and 519 in the β-strand swapped conformation observed in the 2PT:G11a structure. b, Accessible dye positions in the 479–519 reporter in the canonical conformation, based on the canonical NBD1 structure (PDB: 2BBO). Simulation results for all used reporters in both β-strand swapped and canonical conformations are detailed in Supplementary Table 3). Figures were generated using PyMOL (DeLano Scientific LLC).

Extended Data Fig. 5 G11a binds wild-type human CFTR and alters its gating behaviour.

a–c, Summary of patch-clamp studies of wild‐type CFTR Cl− channel in an excised inside‐out membrane patch from a C127 cell heterologously expressing wild‐type human CFTR. The data were acquired at 37 °C in the presence of ATP (0.3 mM) and PKA (75 nM) in the intracellular solution. After the channel was fully activated, G11a (1 μM) was then directly added to the intracellular solution bathing the membrane patch. A large Cl− concentration gradient was imposed across the membrane patch ([Cl−]int, 147 mM; [Cl−]ext, 10 mM) and membrane voltage was clamped at –50 mV. Single‐channel current amplitude (i) mean burst duration (MBD) and interburst interval (IBI) of wild‐type CFTR in the absence and presence of G11a are quantified. Symbols represent individual values and columns are means ± SEM (a, n = 6; b, c, n = 3); *, P  = 0.03 vs control, Student’s t-test; two-sided. d–e, Open probability (Po) timecourses for an individual wild‐type CFTR Cl− channel in absence and presence of G11a (1 µM). Lowercase letters indicate the locations of the recordings shown in f and g during the Po timecourses. f-g, Representative recordings of an individual wild‐type CFTR Cl− channel in the absence and presence of G11a. The two recordings in the presence of G11a demonstrate the different gating patterns observed in its presence (b, steady‐state gating; c, high activity gating). Dotted lines indicate where the channel is closed and downward deflections correspond to channel openings.

Extended Data Fig. 6 Conformational dynamics of the 390–519 reporter pair.

a-b. Structures of the canonical conformation (PDB: 2BBO) and the β-strand swapped conformation reported here. Unresolved segments are depicted in dashed lines, the RI (404–436) in red and the N-terminal region (389–403) in magenta. c-d, Donor lifetime in presence of acceptor (τD(A)) vs FRET efficiency histograms of the 390–519 reporter pair with and without ATP. The red line is called the ‘static FRET line’ and characterizes the theoretical relationship between lifetime and FRET efficiency in absence of conformational dynamics on the timescale of the measurement (milliseconds). e, Overlay of FRET efficiency histograms of 2PT-390-519 without ATP and with 2 mM ATP. Raw data (fill) was fitted using the PDAFit software (thick lines, see Methods). f-g, Donor lifetime vs FRET efficiency plots of 2PT-390-519 without the RI and with the RI in presence of nanobody G11a. h, Reversibility of the conformational change was tested. At the beginning of the experiment no ATP was present and after 2500 s ATP was added to yield a final concentration of 2 mM. i, Deuterium build-up curves of the 392–399 peptide identified in mass spectral analyses of NBD1-2PT (blue) and ΔRI-NBD1 (black). Error bars represent the standard deviation of technical triplicates. Data shown are representative of three biological replicates.

Source data

Extended Data Fig. 7 Effects of the P492S substitution, temperature and F508del on the conformational equilibrium measured via the 479–519 reporter pair.

a–c, Overlay of FRET efficiency histograms of different 479–519 reporter pair variants. Raw data (fill) was fitted using the PDAFit software (thick lines, see Methods). The insets show the population of the canonical state. Error bars indicate 95% confidence interval.

Source data

Extended Data Fig. 8 Gating strategies used in flow cytometry (main Fig. 3a).

Cells that expressed CFTR and were successfully permeabilized were selected by gating for EGFP + (left, laser: 488 nm – detector: 525/50 nm) and DAPI + (right, laser: 405 nm – detector: 450/50 nm) as shown for each of the three nanobodies tested: the negative control, T2a and G11a (as indicated).

Supplementary information

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Scholl, D., Sigoillot, M., Overtus, M. et al. A topological switch in CFTR modulates channel activity and sensitivity to unfolding. Nat Chem Biol 17, 989–997 (2021). https://doi.org/10.1038/s41589-021-00844-0

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