Correction of Both NBD1 Energetics and Domain Interface Is Required to Restore ΔF508 CFTR Folding and Function

Abstract

The folding and misfolding mechanism of multi-domain proteins remains poorly understood. While thermodynamic instability of the first nucleotide binding domain (NBD1) of ΔF508-CFTR partly accounts for the mutant channel degradation in the endoplasmic reticulum and is considered as a drug target in cystic fibrosis, the link between NBD1 and CFTR misfolding remains unclear. Here we show that ΔF508 destabilizes NBD1 both thermodynamically and kinetically, but correction of either defect alone is insufficient to restore ΔF508-CFTR biogenesis. Instead, both ΔF508-NBD1 energetic and the NBD1-MSD2 (membrane spanning domain 2) interface stabilization are required for wild-type-like folding, processing and transport function, suggesting a synergistic role of NBD1 energetics and topology in CFTR coupled domain assembly. Identification of distinct structural deficiencies may explain the limited success of ΔF508-CFTR corrector molecules and suggests structure-based combination corrector therapies. These results may serve as a framework for understanding the mechanism of interface mutation in multi-domain membrane proteins.

Pharmacological Rescue of the Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Detected by Use of a Novel Fluorescence Platform

“…One possibility is that each corrector interacts with a different structural feature of F508del-CFTR to stabilize their folding or reduce nonproductive folding intermediates. Because the folding defect of F508del involves multiple domain and interdomain interactions, multiple interaction sites may be necessary for optimal correction of the misfolded protein (33)(34)(35)(36)(37). Alternatively, rescue might be achieved indirectly through the modulation of folding chaperones or by suppressing ER-associated degradation (ERAD) quality control pathways that promote F508del-CFTR ubiquitylation and degradation.…”

Cystic Fibrosis Transmembrane Conductance Regulator (ABCC7) Structure

“…The two papers supporting this inference both present systematic evaluations of different combinations of suppressor mutations in correcting the trafficking defect in F508del-CFTR in tissue culture cells. In these studies, none of the suppressor mutation sets confined to NBD1 restored maturation to wild-type levels, but full restoration or correction of the F508del defect was observed when combining suppressor mutations in NBD1 with mutations likely to strengthen the interdomain interface between NBD1 and the TMDs in F508del-CFTR (Mendoza et al 2012;Rabeh et al 2012). In contrast, the third recent paper showed essentially wild-type levels of maturation and stability in F508del-CFTR containing second-site suppressor mutations exclusively in NBD1 (i.e., the I539T mutation plus four proline substitutions found in chicken CFTR, which is naturally more thermostable than human CFTR) (Aleksandrov et al 2012).…”

“…However, the local structural perturbations caused by the F508del mutation in the folded conformation of NBD1 are likely to contribute at least indirectly to pathogenesis by disrupting structural interactions between NBD1 and the TMDs of CFTR (Lewis et al 2005Serohijos et al 2008;He et al 2010;Loo et al 2010;Thibodeau et al 2010). Recently published work suggests that developing effective drugs to correct the molecular defects caused by the F508del mutation in CFTR may require correction of these disrupted interdomain interactions (Mendoza et al 2012;Rabeh et al 2012).…”

“…Although the inability to date to purify the TMDs of CFTR in a native conformational state has prevented direct biophysical evaluation of this hypothesis, its validity is supported by the results from mutagenesis experiments conducted by several different laboratories Loo et al 2010;Thibodeau et al 2010;Mendoza et al 2012;Rabeh et al 2012). These experiments provide evidence that the defective trafficking of F508del-CFTR in tissue culture cells can be suppressed by strengthening the interaction between the perturbed surface on NBD1 and the cognate region of the TMD.…”

“…Two recent papers both concluded that strengthening the interface between NBD1 and the TMDs in F508del-CFTR would be required for effective pharmacological correction of its trafficking defect (Mendoza et al 2012;Rabeh et al 2012). However, another recent paper directly challenges this critical pharmacological inference (Aleksandrov et al 2012).…”