*3.3. Impact of F508del-Revertants on CFTR Variants Lacking RE and RI*

Another goal of the present study was to assess how presence of the F508del-CFTR revertants G550E and R1070W [5,10,11] influence variants without RE and RI. Remarkably, our data show that the presence of either of these revertants did not affect ∆RES-F508del-CFTR processing, but both of them further increased processing (but not function) of ∆RIL-F508del-CFTR to almost levels of wt-CFTR: 92–96% (G550E) and 71–76% (R1070W).

In contrast, removal of RI<sup>S</sup> from either of these F508del-CFTR revertants completely abolished their processing, emphasizing how important the different residues between RI<sup>S</sup> and RI<sup>L</sup> are for F508del-CFTR conformers partially rescued by the revertants. We can speculate that RI<sup>S</sup> removal from F508del-CFTR has an effect on folding equivalent to that of either G550E or R1070W.

Interestingly, regarding wt-CFTR processing, G550E (at the NBD1:NBD2 dimer interface) partially recovered the negative effect caused by RI<sup>S</sup> removal, thus further suggesting that this revertant and the destructured region of RI may be allosterically coupled, since they do not plausibly interact (Figure S5). On the contrary, R1070W (at the NBD1:ICL4 interface), negatively affected processing of wt-CFTR (to 69%) and of ∆RIS-wt-CFTR (from 44% to 11%), while not affecting ∆RIL-wt-CFTR. R1070W rescues F508del-CFTR because <sup>1070</sup>Trp fills the gap created by deletion of residue <sup>508</sup>Phe [36]. It is not surprising that it perturbs CFTR folding due to clashing of <sup>508</sup>Phe and <sup>1070</sup>Trp residues. It is nevertheless, curious that R1070W affect more the processing ∆RIS-wt-CFTR than wt-CFTR, to levels of F508del-CFTR, suggesting that both changes affect the same region of the molecule. Rescue of both R1070W-wt-CFTR and R1070W-∆RIS-wt-CFTR by VX-809 further supports this concept.

The most striking effect of ∆RI<sup>L</sup> however, was the almost complete abolition of VX-770-stimulated current of G550E-F508del-CFTR to levels even lower than those observed for ∆RIL-F508del-CFTR (see above). It was suggested that VX-770 binds directly to CFTR to the MSDs (although it is not defined the exact binding site), in phosphorylation-dependent but ATP-independent manner and away from the canonical catalytic site [23,37]. Both RD and RI were suggested by Eckford and colleagues as putative binding regions for VX-770 [37]. Nevertheless, the pharmacological effect of VX-770 remains robust in the absence of the RD [23] and here we demonstrate that it does indeed require RI since VX-770 is unable to stimulate either ∆RIL-F508del-CFTR or ∆RIL-G550E- F508del-CFTR (see Figure 6). We can speculate that removal of RI<sup>L</sup> increases the chances of VX-770 blocking the pore. In contrast, this would not occur for genistein, which has been proposed to bind to the NBDs interface [38], thus probably explaining why it does not cause this effect.
