**5. Conclusions**

Overall, our data show that while the presence of the regulatory insertion (RI) seems to preclude full rescue of F508del-CFTR processing by VX-809, this region appears essential to rescue its function by VX-770, thus suggesting some contradictory role in rescue of F508del-CFTR by these two modulators (Figure 7). Nevertheless, this negative impact of removing RI on VX-770-stimulated currents on F508del-CFTR can be compensated by deletion of the regulatory extension which also leads to the stabilization of this mutant. We thus propose that, despite both these regions being conformationally dynamic, RI precludes F508del-CFTR processing while RE affects mostly its stability and channel opening.

**Figure 7.** Model for the modulation of F508del processing and gating by RI<sup>L</sup> and RES. Our data suggest that the RI destabilizes the F508del-CFTR structure. Therefore, deleting the RI (∆RIL) rescues F508del-CFTR processing and gating. However, the RI is essential for F508del-CFTR rescuing by VX-770. The RE is essential for gating and removal of its shorter version (∆RES) generates an F508del-CFTR variant which cannot be rescued pharmacologically. Simultaneous removal of both regions (∆RIL-∆RES) rescues F508del-CFTR processing and gating. The CFTR structure from Liu F et al. [22] depicts the overall NBD1 structure and the location of the RI and RE.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/12/ 4524/s1, **Figure S1.** ABC transporters alignment. Sequence alignment of NBD sequences from ABC transporters. Sequences correspond to NBD domains with experimentally determined 3D structures: human CFTR (hNBD1) and Tap1, mouse CFTR (mNBD1), HisP from *S. typhimurium*, MJ0796 and MJ1267 from *M. jannaschii* and Malk from *T. litoralis*. Only RI and RE regions are shown. Numbering is the one of human CFTR. The localization of the RI, RE and H9 are shown as arrows and shades. Blue and pink regions respectively highlight structured and unstructured stretches in human NBD1 crystal structures [19], **Figure S2.** Turnover and processing of wt-CFTR, alone or in *cis* with ∆RE<sup>S</sup> and ∆RIS. (A) BHK cells expressing wt, ∆RE<sup>S</sup> and ∆RI<sup>S</sup> were labelled with <sup>35</sup>S-methionine for 30 min and then chased for the indicated times (0, 0.5, 1, 2, 3h) before lysis. IP was performed with the anti-CFTR 596 antibody. After electrophoresis and fluorography, images were analysed by densitometry. (B) Turnover of immature (band B) CFTR for different CFTR variants is shown as the percentage of immature protein at a given time point of chase (P) relative to the amount at t = 0 (P<sup>0</sup> ). (C) Efficiency of processing of band B into band C is shown as the percentage of band C at a given time of chase relative to the amount of band B at t = 0. Data are mean <sup>±</sup> SEM at each point (n <sup>=</sup> 3), **Figure S3.** Functional characterization of the <sup>∆</sup>RE<sup>S</sup> and <sup>∆</sup>RI<sup>L</sup> variants of F508del-CFTR with and without VX-809 or VX-770 treatments. (A-L) Iodide efflux from BHK cells stably expressing ∆RES, ∆RIS- and ∆RIL-F508del-CFTR variants. Cells were incubated either with 3µM VX-809 (E-H) or equivalent concentration of DMSO (control) for 48h at 37◦C (A-D; I-L) and for the assay stimulated with Fsk/Gen (A-H) or Fsk/VX-770 (I-L), as indicated above the bar and for the period indicated by the bar, **Figure S4.** Functional characterization of the ∆RE<sup>S</sup> and ∆RI<sup>L</sup> variants of wt-CFTR. (A-C) Iodide efflux from BHK cells stably expressing ∆RES- or ∆RI-wt-CFTR alone (A) or jointly with revertants R1070W and G550E (B, C). Cells were stimulated during the assay with Fsk/Gen (A, B) or with Fsk/VX-770 (C), as indicated above the bar and for the period indicated by the bar. (D) Graph summarizing data from the iodide efflux peak magnitude generated by different BHK cells stably expressing the various CFTR variants. Data are shown as a percentage of wt-CFTR activity and as mean±SEM. (n) indicates number of independent experiments. "#" indicates significantly different from the

wt-CFTR stimulated with Fsk/Gen (*p* < 0.05). (E) Quantification of the iodide efflux data, showing peak response (time and value) and percentage of wt-CFTR activity, **Figure S5.** Relative localization of the RI and G550 in CFTR. The figure shows the structure of full length CFTR bound to ATP and VX-770 [34] viewed from the intracellular side, highlighting the NBDs and rendering secondary structure elements and protein surface. The RI is shown in shades of red: light red highlights the stretch which is resolved in the cryo-EM structure [34] (404Gly-Phe409) and dark red highlights the <sup>410</sup>Glu-Leu<sup>435</sup> stretch, which is not resolved. This stretch is shown as modelled by Serohijos et al. [4], after alignment to NBD1. Other elements are colored as in Figure 1A. (A) Overall structure. (B) Cut through the NBDs. Interaction between the RI and the G550 site is unlikely given their separation and the buried localization of G550.

**Author Contributions:** Conceptualization, I.U., A.C.d.P., and M.D.A.; data curation, I.U., A.C.d.P., H.M.B., and M.D.A.; formal analysis, I.U., H.M.B., and C.M.F.; funding acquisition, M.D.A.; Investigation, I.U., A.C.d.P., S.A., M.J.L., V.F., H.M.B., and C.M.F.; methodology, I.U., A.C.d.P., H.M.B., and M.D.A.; project administration, I.U.; Supervision, I.U. and M.D.A.; validation, I.U.; writing original draft, I.U., H.M.B., and M.D.A.; review and editing I.U., H.M.B. and M.D.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** Work supported by grants UIDB/04046/2020 and UIDP/04046/2020 centre grants (to BioISI) and research grant (to M.D.A.): "iDrugCF" (FCT/02/SAICT/2017/28800), both from FCT/MCTES Portugal '. I.U., A.C.P. and V.F. were recipients of SFRH/BD/69180/2010, SFRH/BD/17475/2004, SFRH/BD/87478/2012 PhD fellowships and H.M.B. of SFRH/BPD/93017/2013 post-doctoral fellowship (FCT, Portugal), respectively.

**Acknowledgments:** Authors acknowledge I Callebaut, B Hoffmann, and J-P Mornon for the revision of the manuscript and for the helpful comments.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
