CFTR
February 2024
CFTR
Cystic fibrosis transmembrane conductance regulator (CFTR) is a protein that belongs to the ATP-binding cassette (ABC) transporter family. It functions as an anion channel, regulating the transport of salt and water across epithelial cells. This process is essential for the proper functioning of cells, and its dysregulation induces progressive damage across multiple organs.
Mutations in CFTR are the cause of the genetic disease cystic fibrosis (CF), making this protein a critical CF pharmacological target. More than 300 mutations causing CF have been identified, but the most common one – found in around 90% of the patients – is the deletion of the phenylalanine at position 508 (Δ508). Δ508 mutation critically impacts CFTR folding and dimerization, leading to premature degradation and, thus, insufficient protein levels in the cells. Because of its intrinsic instability, the Δ508 CFTR mutant is also difficult to structurally characterize.
CFTR modulators have been recently developed to revert the effects of these mutations. So far, four small-molecule drugs have entered clinic treatment. They function either by enhancing CFTR activity (potentiators), increasing the amount of CFTR in cell membranes (correctors), or both (dual-function modulators). The most advanced available therapy is Trikafta, which is a combination of three of these drugs: ivacaftor (potentiator), tezacaftor (corrector), and elexacaftor (dual-function modulator).
To reveal the molecular mechanism of combined CFTR modulators' action in Trikafta, researchers solved the 3D structure of the Δ508 CFTR mutant in complex with these drugs. This study illustrates the structural basis for the synergistic rescue of Δ508 CFTR function by Trikafta.
The scientists used cryo-EM to determine the structure of the Trikafta: Δ508 CFTR complex (Image 1). The electron density map unambiguously showed the presence of all the ligands bound to the Δ508 CFTR, occupying distinct binding sites. Ivacaftor and tezacaftor interacted with the Δ508 CFTR mutant in the same way previously characterized in other studies (that used the non-mutated, wild-type CFTR). The elexacaftor binding site, instead, was elucidated for the first time in this work. This modulator binds a shallow pocket in a protein region critical for CFTR folding and function.
Image 1. 3D structure of Δ508 CFTR mutant (in white, PDB: 8EIQ) in complex with the modulators composing Trikafta: ivacaftor (in pink), elexacaftor (in yellow), and tezacaftor (in light blue). In the zoomed-in view are the binding sites for each modulator. The molecular surface of the protein is represented in white, and the electron densities around the ligands are shown as a green mesh. The pictures are produced with the 3decision® software.
By comparing the structure of the Trikafta-Δ508 CFTR mutant complex with the wild-type CFTR active structure, scientists observed that, overall, they were very similar (Image 2A). This indicates that the binding of these molecules with the Δ508 CFTR mutant restores the original structure of the CFTR protein, thus explaining the recovery of its function.
Image 2. Comparison of 3D structures of the Trikafta:Δ508 CFTR complex (in white, PDB: 8EIQ) and the wild-type CFTR (in blue, PDB: 6MSM). A) Overlay of the full structures. Superposition done with the 3decision® software. B) Zoomed-in view on the Phe508 position. TM11 is on the left side (as indicated), and NBD1 is on the right side (grey, dotted circle). The residues Arg1070 (from the mutant Δ508 in white, from the wild-type in blue) and Phe508 (only from the wild-type, since it is not present in the mutant Δ508) are labeled. The electron density of the Trikafta:Δ508 CFTR complex is represented as a green mesh. The pictures are produced with the 3decision® software.
Scientists analyzed the region around the Δ508 deletion (Image 2B), that is the Nucleotide Binding Domain 1 (NBD1). This domain is implicated in forming the functional, active dimer of CFTR. The phenylalanine 508 (Phe508) residue contributes to stabilizing the NBD1 by protruding the aromatic chain towards the transmembrane helix 11 (TM11), thus bridging these two regions of the protein. In the Δ508 CFTR mutant, NBD1 is usually very mobile and not stabilized. However, the Tikafta-corrected Δ508 CFTR structure shows that the large gap between the TM11 and NBD1 (naturally occupied by Phe508) gets filled by the Arginine 1070 side chain, which goes under a conformational change compared to the wild-type structure, and by a water molecule (or possibly an ion), indicated by the presence of an electron density blob. These allow the formation of interactions with the NBD1, compensating for the absence of the Phe508 interactions. In this way, the NBD1 gets stabilized, allowing the formation of the active dimer. Since NBD1 structural stability is crucial for the correct activity of the protein, this explains the restored function of the Tikafta-Δ508 CFTR complex.
The structural elucidation of the Δ508 CFTR mutant structure marks a significant advancement in the molecular understanding of cystic fibrosis and the mechanism underlying current therapies, providing a foundation for the structure-based design of future drugs.