Trikafta rescues F508del-CFTR by tightening specific phosphorylation-dependent interdomain interactions.

Trikafta is well-known for correcting thermal and gating defects caused by the most common cystic fibrosis mutation F508del in the human cystic fibrosis transmembrane conductance regulator even at a physiological temperature. However, the exact correction pathway is still unclear. Here, noncovalent interactions among two transmembrane domains (TMD1 and TMD2), the regulatory (R) domain and two nucleotide binding domains (NBD1 and NBD2) were analyzed. The thermal stability of NBD1 was also evaluated through its tertiary constrained noncovalent interaction networks or thermoring structures. The results demonstrated that Trikafta binding to flexible TMD1 and TMD2 rearranged their interactions with the R domain upon phosphorylation, coupling tightened cytoplasmic TMD1-TMD2 interactions to tightened Mg/ATP-dependent NBD1-NBD2 dimerization, which stabilized NBD1 above human body temperature. Overall, although the deletion of F508 induces the primary thermal defect in NBD1 and then the gating defect at the TMD1-TMD2 interface, Trikafta rescued them in a reverse manner allosterically. These mechanistic insights into the precise correction pathway of this misfolded channel facilitate optimizing cystic fibrosis treatment.