1. Consolidated peer review report (3 September 2021)

    GENERAL ASSESSMENT

    Cystic Fibrosis (CF) is a lethal genetic disease, caused by loss-of-function mutations in the cftr gene that encodes the epithelial ion channel CFTR (Cystic Fibrosis Transmembrane conductance Regulator), a mediator of chloride and bicarbonate transport across the cell membrane. In the past decade, we have witnessed the success of precision medicine strategies in CF clinics using small-molecule compounds targeting directly the CFTR protein. These drugs improve folding, maturation, stability and gating at the plasma membrane of mutant CFTR versions. The objective of this study is to improve our molecular understanding of how one type of these drugs, Class I correctors, specifically lumacaftor, VX-809, and tezacaftor, VX-661, work on CFTR.

    The manuscript describes three new cryo-electron microscopy structures of CFTR: both nucleotide-free (channel closed state) and ATP-bound (channel active state) lumacaftor-bound structures, and an ATP-bound tezacaftor-bound structure. All three structures show that type I correctors bind in the same small cavity at the cytoplasmic edge of the membrane of the first transmembrane domain, although the binding modes of the two drugs are slightly different within the site. The authors both confirm the location of binding site and find evidence for the distinct binding specificity of the two drugs by identifying mutations within this cavity that decrease the drugs' binding affinity and effectiveness of improving ΔF508-CFTR (the most common CF-causing mutation) folding in a cell-based assay. The results of these biochemical experiments are consistent with their structural observations, providing solid evidence that the binding pocket that is observed in is the one targeted by type I correctors to improve CFTR folding efficiency.

    The strength of the paper lies in the clarity and consistency of the structural data with maturation and binding assays, as well as with much of the existing literature. The three presented structures are consistent, making for strong evidence of the proposed mechanism for type I correctors, namely that they stabilize the transmembrane domain 1, enabling a larger proportion of the CFTR molecules to overcome the slow, defective folding of other domains, and of the whole CFTR protein overall. The manuscript includes a scholarly discussion of previous related literature that effectively adds support to the conclusions and illustrate how their new results are consistent with and help explain many previous observations. References to additional literature, as detailed in our recommendation, could further enhance the discussion.

    The manuscript provides evidence that other corrector types do not use the binding site they identify for type I correctors, but does not resolve their mechanism. It will therefore be exciting in future studies to use similarly elegant approaches to elucidate the mechanisms of action for other corrector types postulated to work through distinct mechanisms. Altogether, this work reflects a rigorous presentation of an investigation into the mechanism for type I correctors, and the current manuscript will be of great interest to many in the fields of cystic fibrosis, protein folding, and drug design. The reviewing team has the following minor recommendations to improve clarity of the presentation of these results.

    RECOMMENDATIONS

    Revisions essential for endorsement:

    None

    Additional suggestions for the authors to consider:

    1. Expand on background. Stating in the introduction, rather than later in the manuscript, that F508 is in NBD1 could help better convey the allostery argument made in the discussion that binding of a corrector in the TMD rescues the folding of a mutation in the NBD. At the end of the introduction, the phrase "support a specific mechanism of action" is ambiguous or vague. Do the authors mean that the mechanism is through specific interaction with CFTR (as opposed to a model where the drug acts indirectly by modifying membrane properties, as suggested earlier in that paragraph)? In general, it would be more effective summarize the actual mechanism. Page 11: WT CFTR may not traffic to the cell membrane efficiently in heterologous expression systems as reported previously, but they are processed far better in native epithelial cells.

    2. Expand on visual detail in the binding site. Enlarge the frame of Figure 2C and Figure 3B so that the side chain densities for K68 and R74 can be included. Some detailed description of the interaction between residues 371-375 and N66 and P67 may be warranted since lumacaftor can improve surface expression of P67L, a folding defective mutant. It would be helpful to include a figure panel that illustrates the interactions between TM1 and TM6 (and the molecules), perhaps in Figure 2E. Showing these interactions explicitly could help readers see the clasp between TM1 and TM6 that is later invoked when proposing the mechanism of action of type I correctors.

    3. Correctors also stabilise ΔF508-CFTR once fully folded and at the membrane. The authors state (page 3): "chaperones that increase the amount of folded CFTR are called correctors" and "some cause defects in channel function and others interfere with CFTR expression and folding". Most mutations, including ΔF508, interfere with both biogenesis and channel function. Also later "Patients with folding mutations..." Most folding mutations also cause gating impairment, justifying a combination therapy with both potentiator and corrector. Correctors are defined in a more pragmatic way as increasing the amount of CFTR at the plasma membrane - whether they act as chaperones (specifically helping with folding) or through stabilization of the protein at the membrane. Throughout the manuscript the authors only consider the action of class I correctors on CFTR folding. However, there is evidence that mature deltaF508-CFTR stability at the plasma membrane is also increased by VX-809 (Eckford et al. Chem Biol. 2014; Meng et al., J Biol Chem 2017). This should be mentioned and discussed.

    4. Cavity in which correctors bind: could this aspect of the data be used to give further insight? On page 11, the authors point out that there is a relatively large cavity into which the correctors can fit, without altering CFTR conformations. The empty cavity is predicted to destabilize CFTR. However, a similar-sized space is present in TMD2, lined by the elbow helix of TM7, TM9 and TM12. Does this TMD2 cavity also destabilize CFTR? Might correctors rationally designed to fill that space act in synergistic with class I correctors? The effect might be smaller, but even the more C-terminal TMD2 could impact on NBD2 folding, and on stability of the mature protein. Might it be interesting to perform more analysis, comparing size and hydrophobicity of the two cavities?

    5. Link between early stabilization of TMD1 and overall efficacy of maturation is supported by published biochemical data. On page 12, the authors state"As CFTR folding is a highly cooperative process, stabilizing TMD1 would ultimately increase the overall probability of forming a fully assembled structure and thereby allosterically rescue a large number of disease-causing mutants that reside in other parts of CFTR." The Braakman lab (Kleizen et al. JMB, 2021) show that the early effect of VX-809 on TMD1 stability correlates with an increase in total mature protein - consistent with reduced degradation at early stages, and cooperativity of conformational maturation (with an early stabilization of the TMD1/NBD1 interface positively affecting folding and assembly of downstream domains). In addition, mutations shown to render CFTR "hyper-responsive" to VX-809 are all linked to the VX-809 binding site presented here via secondary structure elements - P67L (via lasso helix), E92K (via TM1) and K166E and R170E (via TM2). It is plausible that these mutations worsen the intrinsically slow TMD1 packing, ER-membrane insertion and downstream biogenesis steps that correctors can accelerate. Readers may appreciate reading more details of how the structural data presented here is confirmed by these functional biochemical analyses.

    6. Synergy of class I correctors with suppressor mutations could be explained better. On page 12, the authors state: "This mechanism is also consistent with the synergy between lumacaftor and suppressor mutations (Farinha et al., 2013; Okiyoneda et al., 2013): lumacaftor extends the lifetime of TMD1 and the suppressor mutations stabilize different parts of CFTR or enhance inter-domain assembly, and thus together they achieve higher rescuing efficiency". This could be explained a bit better. Several papers demonstrate that the effect of VX-809 is less than additive with the effect of the second-site revertant R1070W mutation on ΔF508-CFTR (He et al., 2013 FASEB J; Okiyoneda et al., 2013 Nat Chem Biol). This seems to contradict the sentence above. However, the non-additivity is consistent with the mechanism the authors propose. According to the latter, correctors stabilize TMD1. This in turn stabilizes the TMD1/NBD1 interface, which provides a "nucleus" which helps folding of downstream domains, post-translational domain assembly and strengthens the functionally important TMD/NBD1 ball-and-socket joint. The R1070W revertant mutation provides molecular contacts at the NBD/TMD interface. Thus both rescue strategies affect similar steps in CFTR's maturation and improve interdomain interactions at, and stability of, the same interface. This overlap in mechanism likely explains the reduced effectiveness of the combination (corrector+revertant mutation). On the other hand, most other suppressor mutations stabilize NBD1. These have a very distinct mechanism and therefore a clear synergistic effect with VX-809.

    7. Interpretation of the conformational state of CFTR. Electrophysiological studies indicate that the phosphorylated, ATP bound CFTR is expected to have open probabilities close to 1, and yet, no transmembrane permeation pathway has been found in structures under these conditions (PDB ID: 6MSM, Zhang et al., 2017 Cell, and the structures in this paper). Previously, the authors speculated that this might be related to some distortion in the membrane-embedded TMDs in the cryo-EM structures. Since the drug-binding site described here is at the protein-lipid interface, how might these distortions impact the interpretation of your results? In addition, among all the structures of CFTR published so far from the authors' group, the apo states show lower resolution than the phosphorylated, ATP-bound states. It would be useful to understand how these inherent differences in structure determination might affect the overall interpretation.

    8. Expand on cryoEM methods. It would be helpful if the authors could provide some more explanations for Figure S5. For example, please explain the rationale for collecting three sets of 3D classes; What criteria did the authors use to pool the three green classes into one? How did the duplicated particles get removed? Please specify the condition for grid freezing or reference to previous publications.

    9. Expand on binding assays. In the methods, it would be helpful to specify whether the proteins used in the SPA assay were purified in the same way as the proteins used for the cryoEM (using both anti-GFP affinity chromatography in LMNG+CHS and SEC in digitonin), or whether a different protocol was used. In the first results section, the authors compare the Kd values that they obtained to previously published EC50 values. They refer to these previous experiments (by Van Goor et al 2016, Van Goor et al 2011, and Ren et al 2013) as "in vivo". But all these results were in fact in vitro, in cell-based experiments (and described as such in the original papers).

    10. Figure S6B is not discussed anywhere in the text, and it should be at least mentioned in the main text or the methods section. Is the extra density, attributed to a lipid acyl chain, present only in this specific structure? Also, the density seems rather large in diameter for an acyl chain. Are other acyl chains visible elsewhere in the structure and how do they compare?

    REVIEWING TEAM

    Reviewed by:

    Rachelle Gaudet, Professor, Harvard University, USA: structural biology of membrane proteins, ABC transporters and Nramp-family transporters, cadherin-family protein structure and assembly

    Tzyh-Chang Hwang, Professor, University of Missouri, USA: structure and function of CFTR, CFTR pharmacology, electrophysiology

    Paola Vergani, Associate Professor, UCL, UK: CFTR structure-function, CFTR pharmacology, epithelial physiology

    Curated by:

    Janice L. Robertson, Assistant Professor, Washington University in St. Louis, USA: membrane protein folding & stability, single-molecule microscopy, computational modeling

    (This consolidated report is a result of peer review conducted by Biophysics Colab on version 1 of this preprint. Minor corrections and presentational issues have been omitted for brevity.)

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