A new corrector rescues F508del-CFTR folding through stabilization of the TMD1-NBD1 linker
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- Reading List (BiophysicsColab)
Abstract
While Trikafta has turned many CF patients into people that lead relatively healthy lives, additional modulators are still needed. We developed an acylsulfonamide-type C2 corrector X307810 that is potent and shows a distinctive mode of action in rescuing F508del-CFTR from degradation at the endoplasmic reticulum. We tracked the de-novo folding and assembly of each CFTR domain with a radio-active pulse chase - limited proteolysis folding assay and determined when and where X307810 works on the newly synthesized protein. X307810 rescued the Golgi form of F508del-CFTR to 80% of wild-type values. To our knowledge, such a dramatic increase has never been attained with a single corrector. Zooming in on X307810 mode of action revealed that the corrector changed protease resistance of the linker between TMD1 and NBD1 during late stages of CFTR folding. X307810 is the first corrector that added a unique new fragment to the typical limited-proteolysis pattern. Other C2 correctors do not change fragments, but portray an increase in fragment quantity characteristic of domain assembly in wild-type and corrected F508del-CFTR. X307810 instead extended protection of TMD1 from intracellular loop 2 to the unstructured regulatory insertion of NBD1. These results provide new and important insights into the impact of acylsulfonamide correctors on CFTR structure and function.
SIGNIFICANCE STATEMENT
CFTR is an anion channel, mutations in which cause cystic fibrosis (CF). Corrector compounds allow misfolded missense CFTR mutants to escape from degradation in the endoplasmic reticulum, which is the basis for CF therapy, in combination with a potentiator. How correctors improve folding is incompletely understood. We here report on X307810 having a novel mode of action on post-translational domain assembly of newly synthesized CFTR. Our in-cell folding assay shows that X307810 stabilizes the linker between the first transmembrane domain (TMD1) and the first nucleotide binding domain (NBD1) into the Regulatory Insertion; it strengthens the TMD1-NBD1-ICL4 interface. The action of X307810 boosts egress of F508del-CFTR from the ER to an unprecedented 80% for a single modulator.
