Allosteric Modulation Balances Thermodynamic Stability and Restores Function of ΔF508 CFTR

Abstract

Most cystic fibrosis is caused by a deletion of a single residue (F508) in CFTR that disrupts the folding and biosynthetic maturation of the ion channel protein. Progress towards understanding the underlying mechanisms and overcoming the defect remain incomplete. Here we show that the thermal instability of human ΔF508 CFTR channel activity evident in both cell-attached membrane patches and planar phospholipid bilayers is not observed in corresponding mutant CFTRs of several non-mammalian species. These more stable orthologs are distinguished from their mammalian counterparts by the substitution of proline residues at several key dynamic locations in the first nucleotide domain (NBD1), including the structurally diverse region (SDR), the gamma phosphate switch loop and the Regulatory Insertion (RI). Molecular Dynamic analyses revealed that addition of the prolines could reduce flexibility at these locations and increase the temperatures of unfolding transitions of ΔF508 NBD1 to that of the wild-type. Introduction of these prolines experimentally into full-length human ΔF508 CFTR together with the already recognized I539T suppressor mutation, also in the SDR, restored channel function and thermodynamic stability as well as its trafficking to and lifetime at the cell surface. Thus, while cellular manipulations that circumvent its culling by quality control systems leave ΔF508 CFTR dysfunctional at physiological temperature, restoration of the delicate balance between the dynamic protein’s inherent stability and channel activity returns a near-normal state.

Dynamics Intrinsic to Cystic Fibrosis Transmembrane Conductance Regulator Function and Stability

“…Recently, substitution of proline residues at key positions in the Q-loop, the SDR, and the RI in context of the I539T suppressor mutation has been shown to restore channel function and thermostability to fulllength F508del CFTR. MD studies support a role for these proline mutations in reducing flexibility and increasing the temperatures of unfolding transitions for F508del NBD1 (Aleksandrov et al 2012). Most convincingly, for a subset of suppressor mutants, a good correlation is observed between the improvement in NBD1 stability and CFTR processing efficiency Rabeh et al 2011).…”

Cystic Fibrosis Transmembrane Conductance Regulator (ABCC7) Structure

“…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). Based on this latter finding, small molecules that bind tightly to properly folded NBD1, and thereby significantly stabilize its native conformation, could be sufficient to correct the molecular pathologies caused by the F508del mutation and cure the disease in patients having this mutation.…”

“…However, another recent paper directly challenges this critical pharmacological inference (Aleksandrov et al 2012). 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.…”

“…Broader and deeper investigations are merited of this pharmacological approach focusing on thermodynamic correction of the folding defect in F508del-NBD1. It will be important to identify the location of compound binding sites in natively folded F508del-NBD1 and also to evaluate whether stabilization of NBD1 must be accompanied by stabilization of the NBD1-TMD interface to produce effective correction of the molecular defect caused by the F508del mutation, a claim supported by some studies cited in the last section (Mendoza et al 2012;Rabeh et al 2012) but questioned by others Aleksandrov et al 2012). …”

The N-terminal Domain Allosterically Regulates Cleavage and Activation of the Epithelial Sodium Channel

“…We used a graph theory-based computational approach to identify key residues involved in allosteric signal propagation from one region of a protein to another. 5 We have successfully used this approach to identify key residues involved in allosteric signal propagation in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator (34). 5 Recently, other groups have focused on extracellular events modulating gating of ENaC and related ion channels (35,36).…”