Domain-interface dynamics of CFTR revealed by stabilizing nanobodies

Abstract

The leading cause of cystic fibrosis (CF) is the deletion of phenylalanine 508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR). The mutation affects the thermodynamic stability of the domain and the integrity of the interface between NBD1 and the transmembrane domain leading to its clearance by the quality control system. Here, we develop nanobodies targeting NBD1 of human CFTR and demonstrate their ability to stabilize both isolated NBD1 and full-length protein. Crystal structures of NBD1-nanobody complexes provide an atomic description of the epitopes and reveal the molecular basis for stabilization. Furthermore, our data uncover a conformation of CFTR, involving detachment of NBD1 from the transmembrane domain, which contrast with the compact assembly observed in cryo-EM structures. This unexpected interface rearrangement is likely to have major relevance for CF pathogenesis but also for the normal function of CFTR and other ABC proteins.

Fig. 1

Binding of NBD1-specific nanobodies to isolated 2PT-NBD1 and F508del-2PT-NBD1. a Nanobody binding to 2PT-NBD1 measured by ELISA. Biotinylated 2PT-NBD1 was immobilized on neutravidin-coated plates and incubated with increasing concentration of each 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. Data were normalized to maximum signal for each nanobody separately. b Nanobody binding to F508del-2PT-NBD1 measured by ELISA as described in (a). c Nanobody T4 binding to 2PT-NBD1 and F508del-2PT-NBD1. Data were normalized to maximum signal of T4 binding to 2PT-NBD1. d Nanobody T8 binding to 2PT-NBD1 and F508del-2PT-NBD1. Data were normalized to maximum signal of T8 binding to 2PT-NBD1. e Thermodynamic parameters of nanobody binding to 2PT-NBD1 determined using isothermal calorimetry (curves shown in Supplementary Fig. 2), and pEC₅₀ determined by ELISA (a). K_D values determined by ITC represent mean ± SD (n = 3). Source data are provided as a Source Data file

Fig. 2

Stabilization of isolated 2PT-NBD1 and F508del-2PT-NBD1 variants by nanobodies. a Differential scanning fluorescence (DSF) of purified 2PT-NBD1. The protein (alone or in complex with one or two different nanobodies) was 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 Summary of melting temperature differences (ΔTm) of 2PT-NBD1 in presence of different nanobodies determined using DSF as in (a). Data are mean ± SEM of duplicates from four independent experiments. c Summary of melting temperature differences (ΔTm) of F508del-2PT-NBD1 in presence of different nanobodies using DSF as in (a). Data are mean of duplicates ± SEM (n = 4). Source data are provided as a Source Data file

Fig. 3

Crystal structures of NBD1-nanobody complexes. a Structures of nanobodies D12, T2a and T27 bound to NBD1. Superimposition was performed on the NBD1 region. b Structure of NBD1-nanobody D12 complex highlighting the different structural elements of hNBD1 as well as the CDRs of the nanobody. c Details of the interface between nanobody D12 and NBD1. Polar interactions are highlighted by dashed lines (see text for description). Only side chains participating in the interface are explicitly shown. d Structures of nanobodies T4 and T8 bound to NBD1. Superimposition was performed on the NBD1 region. The view is rotated compared to (a). e Structure of NBD1-T4 nanobody complex highlighting the different structural elements of NBD1 as well as the CDRs of the nanobody f Details of the interface between nanobody T4 and NBD1. Polar interactions are highlighted by dashed lines (see text for description). Only side chains participating in the interface are explicitly shown. g Close-up of the interaction of F508 from NBD1 to residues in T4. Atoms are shown as space-filling model to highlight the contacts, occurring at van der Waals distances. h Structure of NBD1-G3a nanobody complex i Details of the interface between nanobody G3a and NBD1. Polar interactions are highlighted by dashed lines (see text for description). Only side chains participating in the interface are explicitly shown

Fig. 4

Binding of nanobodies to FL-CFTR. a Dose–response ELISA of interactions between wt-CFTR and nanobodies. Immobilized nanobodies (T2a, T8 and G3a) were incubated with different concentrations of purified CFTR. b Immobilized purified wt-CFTR was incubated with increasing concentrations of nanobodies. For both (a, b) Data were normalized to maximal response of T2a after subtraction of the signal from the negative control nanobody. Graph depicts one representative of at least three independent experiments. Error bars represent standard deviations of triplicates. c Average B_max of 3 independent experiments (±SEM) calculated for curves in (b). d Flow cytometry analysis of nanobodies T2a, T8 and G3a on parental BHK-21 cells shows no difference in labelling compared to a negative control nanobody while in (e) increased labelling is observed for the NBD1-specific nanobodies in BHK-21 cells overexpressing wt-CFTR. Data were normalized to the number of events acquired in each condition. Graph depicts one representative of at least three independent experiments. f Average median fluorescence (fold over negative control) for each of the three representative nanobodies as illustrated in (d, e). Quantification of at least 3 independent experiments (±SEM). g Immunoblot of CFTR from solubilized BHK-21 cells pulled-down with His₆-tagged nanobodies, including a non-CFTR nanobody as a control. Eluted nanobodies-CFTR complexes were separated by SDS-PAGE and presence of CFTR was detected with mAb 596 antibody after immunoblotting. Arrows indicate the mature (band C) and immature (band B) forms of CFTR. Representative of at least 3 independent experiments. h Flow cytometry analysis of nanobodies T2a, T8 and G3a on BHK-21 cells expressing 2PT-F508del showing increased labelling for T2a and G3a, but not T8. i Quantification of data illustrated in (h). Average of 3 independent experiments (±SEM). Source data are provided as a Source Data file

Fig. 5

Nanobodies reduce ATPase activity of CFTR but increase the temperature of thermal inactivation. a Influence of nanobody addition on ATPase activity of wt-CFTR. Conversion of α-³²P-ATP to ADP was measured after 1 h incubation of wt-CFTR with the different nanobodies. Data from replicate determinations are represented as mean ± SEM (n = 3, except for ATPase activity of wt-CFTR activity with nanobodies Neg and T4 for which n = 4). b Thermoprotection of wt-CFTR activity by nanobodies. Inactivation threshold temperatures were determined by measuring residual ATPase activity after 30 min heat challenge at various temperatures. Data from replicate determinations are represented as mean ± SEM (n = 3, except for wt-CFTR activity in absence of nanobody for which n = 4). c Thermostability of stab-CFTR measured by nanoDSF. First derivative of 350 nm fluorescence as a function of temperature showing the determination of Tm of purified stab-CFTR alone (black) or in complex with nanobody T2a (dark grey). Melting curve of nanobody T2a alone is depicted in light grey. One representative experiment shown. d Thermostability of stab-CFTR as in (c), in complex with nanobody T4 (dark grey). Melting curve of nanobody T4 alone is depicted in light grey. One representative experiment shown. e Summary of melting temperatures of stab-CFTR in complex with nanobodies T2a, T4 and T8 determined by nanoDSF. Data from triplicates are represented as mean ± SD (n = 2). Source data are provided as a Source Data file

Fig. 6

NBD1-nanobody complexes superimposed onto the structure of CFTR. a The previously reported cryo-EM structure of CFTR (PDB: 5UAK) was aligned with structure of ΔRI-NBD1-G3a complex showing that the epitope is located in the periphery of CFTR. b Same alignment as (a) with the structure 2PT-NBD1-T2a complex, showing a compatible binding of nanobody D12 in between the NBDs. c Same alignment as (a) with the ΔRI-NBD1-T8 complex where the nanobody overlaps with the TMDs, indicating that binding is not compatible with this conformational state of CFTR

Fig. 7

NBD1 must undock from the TMDs to allow binding of nanobodies T4 or T8. Current models indicate that CFTR alternates between a state where the two NBDs are in close contact (state a), leading to channel opening, and a state where the NBDs separate leading to channel closing (state b). State a would typically be induced by PKA phosphorylation. States a and b have been observed by cryo-EM and both bury F508 in the NBD1-TMD interface. Nanobodies T4 and T8 bind an epitope containing F508 (illustrated in red), thus requiring a transient undocking of NBD1 from the TMDs (state c). This transient state can be stabilized upon binding of these nanobodies (state d)