**3. Discussion**

The main goal of this study was to understand how the removal of the regulatory extension (∆RE) alone or with the regulatory insertion (∆RI)—two highly conformationally dynamic regions—impact the rescue of F508del-CFTR processing and function by two compounds—VX-809 and VX-770—which in combination were recently approved to clinically treat F508del-homozygous patients. Indeed, CFTR is the sole ABC transporter that functions as a channel and thus, these highly dynamic RI and RE regions which are absent in other ABC transporters, may be of high relevance to understand how CFTR differs from other ABCs, namely in its function as a channel.

These regions RI and RE were originally suggested to be positioned to impede formation of the NBD1-NBD2 dimer required for channel gating [15,19]. Indeed, both RI and RE were shown to be mobile elements in solution that bind transiently to the core of NBD in the β-sheet and α/β subdomains of NBD1 [3]. Similarly to what demonstrated for RI [13], it is plausible to posit that the dynamic flexibility of the RE may also result in exposure of hydrophobic surfaces thus contributing to the dynamic instability of NBD1 and thus contribute to the low folding efficiency of F508del-CFTR. The RE is a ~30-residue segment at NBD1 C-terminus, so-called because it goes beyond canonical ABC NBDs [15]. Although this region was absent from the solved CFTR-NBD1 crystal structure [19], it was described as a helix packing against NBD1 at the NBD1:NBD2 interface and NMR data showed it has significant conformational flexibility [3,21]. Notwithstanding, there is some controversy regarding the RE boundaries. The RE (previously called H9c) was defined as <sup>655</sup>Ala-Ser<sup>670</sup> [15,19], <sup>639</sup>Asp-Ser670 20 , or <sup>654</sup>Ser-Gly<sup>673</sup> [3]. According to the crystal structure (PDB ID 2PZE), NBD1 extends only to <sup>646</sup>Gly, where RD begins, so RE could be proposed to start at <sup>647</sup>Cys [3,18], thus being considered as part of RD, the latter absent in other ABC transporters [1]. As for the RI (previously called S1-S2 loop) its limits are also variable, being firstly described as a ~35-residue segment (405Phe-436Leu) consisting of α-helices H1b and H1c [15] (Figure S1). Later, however, these limits were proposed to be <sup>404</sup>Gly-Leu<sup>435</sup> [13,20] or <sup>405</sup>Phe-Leu<sup>436</sup> [18].
