No abstract

Conflict of interest: JLH is founder of and holds equity in Spectrum PhenomX LLC, which aims to commercialize quantitative high-throughput cell array phenotyping (Q-HTCP) technology.

Pharmacological intervention to treat the lethal genetic disease cystic fibrosis has become reality, even for the severe, most common folding mutant F508del CFTR. CFTR defects range from absence of the protein, misfolding that leads to degradation rather than cell-surface localization (such as F508del), to functional chloride-channel defects on the cell surface. Corrector and potentiator drugs improve cell-surface location and channel activity, respectively, and combination therapy of two correctors and a potentiator have shown synergy. Several combinations are in the drug-development pipeline and although the primary defect is not repaired, rescue levels are reaching those resembling a cure for CF. Combination therapy with correctors may also improve functional CFTR mutants and benefit patients on potentiator therapy.

The question how proteins fold is especially pointed for large multidomain, multispanning membrane proteins with complex topologies. We have uncovered the sequence of events that encompass proper folding of the ABC transporter CFTR in live cells, by combining kinetic radiolabeling with protease-susceptibility assays. We found that CFTR folds in two clearly distinct stages. The first, co-translational, stage involves folding of the 2 transmembrane domains TMD1 and TMD2, plus one nucleotide-binding domain, NBD1. The second stage is a simultaneous, post-translational increase in protease resistance for both TMDs and NBD2 caused by assembly of these domains onto NBD1. Our technology probes every 2-3 residues (on average) in CFTR. This in-depth analysis at amino-acid level allows detailed analysis of domain folding and importantly also the next level: the assembly of the domains to native, folded CFTR. Defects and changes brought about by medicines, chaperones or mutations also are amenable to analysis. We here show that the DXD motif in NBD1 that was identified to be required for export of CFTR from the ER turned out to be required for proper domain folding and assembly instead, upstream of transport. CFTR mutated in this motif phenocopies the misfolding and degradation of the well-known disease-causing mutant F508del that established cystic fibrosis as protein-folding disease. The highly modular process of domain folding and stepwise domain assembly explains the relatively high fidelity of folding and the importance of a step-wise folding process for such complex proteins.