Restoration of NBD1 Thermal Stability Is Necessary and Sufficient to Correct ∆F508 CFTR Folding and Assembly

Abstract

CFTR (ABCC7), unique among ABC exporters as an ion channel, regulates ion and fluid transport in epithelial tissues. Loss of function due to mutations in the cftr gene causes cystic fibrosis (CF). The most common CF-causing mutation, the deletion of F508 (ΔF508) from the first nucleotide binding domain (NBD1) of CFTR, results in misfolding of the protein and clearance by cellular quality control systems. The ΔF508 mutation has two major impacts on CFTR: reduced thermal stability of NBD1 and disruption of its interface with membrane-spanning domains (MSDs). It is unknown if these two defects are independent and need to be targeted separately. To address this question we varied the extent of stabilization of NBD1 using different second site mutations and NBD1 binding small molecules with or without NBD1/MSD interface mutation. Combinations of different NBD1 changes had additive corrective effects on ΔF508 maturation that correlated with their ability to increase NBD1 thermostability. These effects were much larger than those caused by interface modification alone and accounted for most of the correction achieved by modifying both the domain and the interface. Thus, NBD1 stabilization plays a dominant role in overcoming the ΔF508 defect. Furthermore, the dual target approach resulted in a locked-open ion channel that was constitutively active in the absence of the normally obligatory dependence on phosphorylation by protein kinase A. Thus, simultaneous targeting of both the domain and the interface, as well as being non-essential for correction of biogenesis, may disrupt normal regulation of channel function.

Specific stabilization of CFTR by phosphatidylserine

“…Observation of somewhat greater CFTR stability in the presence of ATP than in its absence is not surprising, because differential scanning calorimetry studies have firmly established nucleotide stabilization of CFTR isolated NBDs [31]. Tm shifts achieved by addition of PS were larger than any stabilizations yet achieved through selective mutation of CFTR NBD1 [15, 32, 33] or the full length protein [12]. Further, we demonstrated proof of principle for combining thermal stabilization of CFTR by PS with stabilizing effects of introduced mutations (Figure 3).…”

ΔF508-CFTR Modulator Screen Based on Cell Surface Targeting of a Chimeric Nucleotide Binding Domain 1 Reporter

“…However, low temperature (27 °C) incubation strongly increased the HRP signal. BIA, a reported low-affinity ΔF508-NBD1 binder, 15 did not increase the HRP signal. Also, a panel of known CFTR modulators was tested, including correctors VX-809, VX-661, C3, and C18, which target the NBD1-MSD interface, 9 C4, which targets NBD2, 16 CoPo-22, a CFTR modulator with dual corrector and potentiator function, 12 and W1282X corr -A23, which corrects CFTR 1281 , the protein product generated by the W1282X-CFTR nonsense mutation.…”

Deletion of Phenylalanine 508 in the First Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Increases Conformational Exchange and Inhibits Dimerization

“…Because the mechanism of dimer inhibition involves an alternate Q-loop segment conformation and changes at the dimer interface, it seems likely that F508del will disrupt NBD1/NBD2 heterodimerization as well as the NBD1 homodimers observed here. Other published Q-loop segment suppressor mutations such as S492P and S495P (21,29) are also likely to modulate NBD dimerization. Besides these Q-loop effects, other influences on NBD1/ NBD2 heterodimerization include nucleotide binding, the RI and R region and their phosphorylation.…”

“…Evidence that NBD1 destabilization is problematic for proper processing was provided by NBD1-thermostabilizing mutations distant from the F508del site, including G550E, R553Q, R555K, and deletion of the RI. These NBD1-stabilizing mutations substantially improve processing efficiency of F508del CFTR without correcting structural perturbations near the F508del location (20,(27)(28)(29). Furthermore, the degree to which mutations stabilize or destabilize the isolated NBD1 domain is roughly correlated with full-length CFTR-processing efficiency (24,25), establishing F508del destabilization of NBD1 as a key mechanism for the deleterious effects of the mutation.…”