Abstract
Patients with cystic fibrosis (CF) harboring the P67L variant in the cystic fibrosis transmembrane conductance regulator (CFTR) often exhibit a typical CF phenotype, including severe respiratory compromise. This rare mutation (reported in <300 patients worldwide) responds robustly to CFTR correctors, such as lumacaftor and tezacaftor, with rescue in model systems that far exceed what can be achieved for the archetypical CFTR mutant F508del. However, the specific molecular consequences of the P67L mutation are poorly characterized. In this study, we conducted biochemical measurements following low-temperature growth and/or intragenic suppression, which suggest a mechanism underlying P67L that (1) shares key pathogenic features with F508del, including off-pathway (non-native) folding intermediates, (2) is linked to folding stability of nucleotide-binding domains 1 and 2, and (3) demonstrates pharmacologic rescue that requires domains in the carboxyl half of the protein. We also investigated the “lasso” helices 1 and 2, which occur immediately upstream of P67. Based on limited proteolysis, pulse chase, and molecular dynamics analysis of full-length CFTR and a series of deletion constructs, we argue that P67L and other maturational processing (class 2) defects impair the integrity of the lasso motif and confer misfolding of downstream domains. Thus, amino-terminal missense variants elicit a conformational change throughout CFTR that abrogates maturation while providing a robust substrate for pharmacologic repair.
Highlights
The present studies suggest a model in which point mutations in the amino terminus (e.g., P67L) disrupt folding of a large transmembrane protein, in a manner reversed by cystic fibrosis transmembrane conductance regulator (CFTR) pharmacocorrection (Fig. 9)
Our data support a mechanism in which specific transmembrane alpha helices encoded by TMDs 1 and 2 are maintained in proper orientation by lasso helix 2 (Lh2), and that loss of this constraint leads to misfolding of downstream CFTR elements
Further studies will be necessary to determine precise ways in which Lh2 interacts with transmembrane alpha helices and thereby maintains proper conformational stability of CFTR
Summary
The primary CFTR sequence was aligned among multiple species (Fig. 1A). P67, which occurs between Lh2 and elbow helix 1 based on the recently published cryo-EM structure of CFTR [12] (Fig. 1, B and C), is invariant among all species we evaluated. Mutations in the lasso helical region, including those at positions less than 10 residues apart, can elicit nearly identical effects on overall CFTR maturational efficiency, yet exhibit pronounced differences with regard to pharmacocorrection (Fig. 3B). Unlike P67L (which exhibits partial rescue following either lumacaftor treatment or R555K suppression), W57G—a mutation found in a small number of patients worldwide (CFTR2 database [https://cftr2.org/])—exhibits negligible effect on either protein maturation or CFTR chloride transport following these maneuvers (Fig. S1). As an additional test of P67L CFTR response to lumacaftor, a “half molecule” construct truncated near the R-domain C terminus (CFTR 837X; residues 1–836) was coexpressed with the downstream half of the molecule (M837; residues 837–1480) (Fig. 4) [24]. These results indicate that correct N-terminal conformation enhances CFTR processing and lumacaftor response in a fashion that requires proper domain interactions between the two half molecules. The P67L trajectories were analyzed using the MDpocket algorithm [46] in search of putative lumacaftor-binding sites, and a distinct site immediately adjacent to Lh2 was observed (Fig. 8)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
