*3.1. Impact of RE and RI on CFTR Processing and Function*

Our data shown here on CFTR variants depleted of different versions of the RE dynamic region demonstrate that unlike RI deletion, removal of short RE—∆RE<sup>S</sup> (∆ <sup>654</sup>Ser-Gly673) did not per se rescue F508del-CFTR processing. Nevertheless, ∆RE<sup>S</sup> dramatically stabilized the immature form of F508del-CFTR (see Figure 6). In fact, our pulse-chase experiments show that the immature form of ∆RES-F508del-CFTR exhibits a turnover rate which is ~2-fold lower than that of wt-CFTR. Of note, when Aleksandrov et al. removed the dynamic region RI from F508del-CFTR they found a dramatic increase in the channel thermostability, even augmented for higher temperatures [13]. Interestingly however, while RE<sup>S</sup> removal from ∆RIL-F508del-CFTR did not significantly affect processing (71% vs. 78% for ∆RES-∆RIL-F508del-CFTR and ∆RIL-F508del-CFTR, respectively, in Table 1, it further reduced its function from 92 to 78% (∆RIL-F508del-CFTR vs. ∆RES-∆RIL-F508del-CFTR, respectively, Figure 5C).

The latter findings on function of ∆RES-wt-CFTR and ∆RES-∆RIL-F508del-CFTR were somewhat surprising since the RE was previously described to impede putative NBD1:NBD2 dimerization that is required for channel gating [20]. Accordingly, an increase in CFTR activity would be expected upon ∆RE<sup>S</sup> removal, which is actually the opposite of what we observe for wt-CFTR. Besides the fact that those authors studied a different RE version (639Asp-Ser670), this discrepancy could derive from structural constraints and lack of flexibility of the 'single polypeptide' protein that we used, vs. their 'split' CFTR channels (2 'halves' of 633 and 668 amino acid residues) for which channel function was indistinguishable from wt-CFTR [20]. Moreover, since RD phosphorylation is required for channel gating [24,25], the reduced function of ∆RES-wt-CFTR may also result from absence of <sup>660</sup>Ser and <sup>670</sup>Ser [17,21]. Although the RD contains more than ten PKA phospho-sites and no individual one is essential, phosphorylation of increasing numbers of sites enables progressively greater channel activity [26].

Removal of a longer RE version (∆REL: ∆ <sup>647</sup>Cys-Ser678) was without effect on F508del-CFTR processing and significantly reduced that of wt-CFTR to 88%. Such differential impact on wt- and F508del-CFTR is consistent with the conformational heterogeneity between these two proteins lacking both RI and RE [27].

Removal of RI short version, RI<sup>S</sup> (∆ <sup>412</sup>Ala-Leu428) significantly reduced wt-CFTR processing to less than half of its normal levels, while not rescuing F508del-CFTR processing, thus, being essential for CFTR proper folding. This is in contrast to removal of long RI (∆RIL) which led to 78% processing F508del-CFTR, as reported [13] but without impact on wt-CFTR. These data indicate that those 8/7 amino acid residues at the N-term/ C-term of RI<sup>S</sup> (and absent in RIL) impair the folding efficiency and processing of both wt- and F508del-CFTR. Despite the difficulty in speculating how those amino acid residues can specifically impact of F508del-CFTR folding and processing, the fact that structurally RI<sup>S</sup> is strictly the region described as destructured in the crystal structure [19] and RI<sup>L</sup> includes some flanking residues restricting its mobility may explain the observed difference.

Indeed, the recently published cryo-EM structures of human CFTR [22,28,29] indicate that RI<sup>S</sup> is structurally disordered. Consistently, the RI loop in NBD1 is described in the zebrafish CFTR cryo-structure [30,31] to contribute to the amorphous density that is observed between NBD1 and the elbow helix of TMD2 [32]. Our data for the RI are consistent with these findings.

From a structural point of view, models of the full-length CFTR protein suggest that the two regions (RI and RE) behave differently. The distances between the extremities of the deleted parts are indeed different (∆RI<sup>S</sup> = 18Å, ∆RIL= 10Å versus ∆RE<sup>S</sup> = 27Å, ∆RE<sup>L</sup> = 30Å). The longer distances observed for ∆RE imply a substantial reorganization of the C-terminal parts of NBD1 and NBD2, which precludes a clear understanding of what might happen upon these deletions. In contrast, the lower distances observed for ∆RI (near a possible minimum of 6Å) allows a better simulation of the possible structural behavior of these constructs. Indeed, preliminary molecular dynamics simulations of the ∆RIL-F508del-CFTR suggested that in this case, part of the RD is reorganized and partially takes space left by the ∆RI deletion, thereby substituting it for tight contacts with NBD2. Meanwhile, following a probable allosteric effect [13,18,32], contact of NBD1-F508 with ICL4 residue L1077 (a likely essential contact for channel opening), is nearly completely restored by I507. Such a feature is not observed in the F508del-CFTR model [33].

**Figure 6.** Summary of the most relevant results observed in the present study. CFTR structure used is the one by Liu F et al. [22] and putative bind sites of VX-809 and VX-770 shown are the NBD1:MSD2 (ICL4) interface and to the MSDs (although it is not defined the exact binding site), as described by Farinha et al. [5] and by Jih and Hwang [23], respectively. RI<sup>L</sup> and RE<sup>S</sup> are shown as red lines, the R region a dashed green line.
