Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosis

Abstract

The lethal genetic disease cystic fibrosis is caused predominantly by in-frame deletion of phenylalanine 508 in the cystic fibrosis transmembrane conductance regulator (CFTR). F508 is located in the first nucleotide-binding domain (NBD1) of CFTR, which functions as an ATP-gated chloride channel on the cell surface. The F508del mutation blocks CFTR export to the surface due to aberrant retention in the endoplasmic reticulum. While it was assumed that F508del interferes with NBD1 folding, biophysical studies of purified NBD1 have given conflicting results concerning the mutation's influence on domain folding and stability. We have conducted isothermal (this paper) and thermal (accompanying paper) denaturation studies of human NBD1 using a variety of biophysical techniques, including simultaneous circular dichroism, intrinsic fluorescence, and static light-scattering measurements. These studies show that, in the absence of ATP, NBD1 unfolds via two sequential conformational transitions. The first, which is strongly influenced by F508del, involves partial unfolding and leads to aggregation accompanied by an increase in tryptophan fluorescence. The second, which is not significantly influenced by F508del, involves full unfolding of NBD1. Mg-ATP binding delays the first transition, thereby offsetting the effect of F508del on domain stability. Evidence suggests that the initial partial unfolding transition is partially responsible for the poor in vitro solubility of human NBD1. Second-site mutations that increase the solubility of isolated F508del-NBD1 in vitro and suppress the trafficking defect of intact F508del-CFTR in vivo also stabilize the protein against this transition, supporting the hypothesize that it is responsible for the pathological trafficking of F508del-CFTR.

Molecular Dynamics Flexible Fitting Simulations Identify New Models of the Closed State of the Cystic Fibrosis Transmembrane Conductance Regulator Protein

“…Yet these seemingly similar structures behave in very different ways in terms e.g., of their thermal stabilities and aggregation propensities. [56][57] The importance of this work is two-fold. First it provides new conformations of CFTR at the atomic level which were largely not discussed to date.…”

Dynamics Intrinsic to Cystic Fibrosis Transmembrane Conductance Regulator Function and Stability

“…Extensive chemical and thermal denaturation studies monitored by a variety of biophysical techniques have identified an aggregation-prone, partially unfolded intermediate state on the unfolding pathway that is enhanced by F508del (Richardson et al 2007;Protasevich et al 2010;Wang et al 2010a). F508del does not seem to affect unfolding of this partially unfolded intermediate, suggesting that the F508 region is already unfolded in the intermediate.…”

Intragenic Suppressing Mutations Correct the Folding and Intracellular Traffic of Misfolded Mutants of Yor1p, a Eukaryotic Drug Transporter

“…A recent characterization of these suppressing mutations revealed that their ability to compensate for folding lesions is specific for mutations that mapped within NBD1; mutations located in other domains, including ICL4, which forms an interface with the NBD1 surface, remain dysfunctional even in the presence of the suppressing mutations (34). Furthermore, introduction of these suppressing mutations into bacterially expressed NBD1 improved the soluble protein yield (39), likely through thermodynamic stabilization of the NBD1 fold (40) Together, these data suggest that the STE6-CFTR-⌬F suppressing mutations primarily correct the original lesion in NBD1 caused by ⌬F.…”