Abstract
Many disease-causing mutations impair protein stability. Here, we explore a thermodynamic strategy to correct the disease-causing F508del mutation in the human cystic fibrosis transmembrane conductance regulator (hCFTR). F508del destabilizes nucleotide-binding domain 1 (hNBD1) in hCFTR relative to an aggregation-prone intermediate. We developed a fluorescence self-quenching assay for compounds that prevent aggregation of hNBD1 by stabilizing its native conformation. Unexpectedly, we found that dTTP and nucleotide analogs with exocyclic methyl groups bind to hNBD1 more strongly than ATP and preserve electrophysiological function of full-length F508del-hCFTR channels at temperatures up to 37 °C. Furthermore, nucleotides that increase open-channel probability, which reflects stabilization of an interdomain interface to hNBD1, thermally protect full-length F508del-hCFTR even when they do not stabilize isolated hNBD1. Therefore, stabilization of hNBD1 itself or of one of its interdomain interfaces by a small molecule indirectly offsets the destabilizing effect of the F508del mutation on full-length hCFTR. These results indicate that high-affinity binding of a small molecule to a remote site can correct a disease-causing mutation. We propose that the strategies described here should be applicable to identifying small molecules to help manage other human diseases caused by mutations that destabilize native protein conformation.
Highlights
We use a novel thermal fluorescence selfquenching assay to identify nucleotide analogs that stabilize the first nucleotide-binding domain from human cystic fibrosis transmembrane conductance regulator (hCFTR) more strongly than the physiological nucleotide ligand ATP, and we use temperature-controlled single-channel electrophysiology assays to demonstrate that these analogs correct the thermodynamic defect in full-length hCFTR caused by the predominant disease-causing F508del mutation in human NBD1 (hNBD1)
Thermodynamic theory predicts that compounds with sufficient binding affinity for either the hNBD1 domain destabilized by the mutation or for the interdomain interfaces it forms in full-length hCFTR should be able to offset the thermal defect caused by the F508del mutation (Fig. 1)
Previous attempts to demonstrate direct correction of the thermodynamic defect caused by the F508del mutation focused on identification of small molecules that bind to hNBD1
Summary
The molecular pathology caused by the F508del mutation [42] in hCFTR is attributable at least in part to destabilization of the native conformation of hNBD1 relative to a molten globule folding intermediate [10, 11]. To gain insight into the structural basis of the thermodynamic stabilization of the domain by dTTP, we compared structures of F508-hNBD1⌬RI with dTTP versus dUTP (Fig. 5C), which gives a TSQ 3.3 °C lower than dTTP but differs exclusively by the absence of an exocyclic methyl group at the 5-position in the pyrimidine ring (Fig. 4B and Fig. S5B) This comparison, combined with the ITC data for the binding of these two nucleotides (Table S1), suggests that the enhanced binding affinity of dTTP compared with dUTP is due to displacement of hydrating water from interaction with the methyl group on residue Thr-465 in the Walker A motif. These observations are consistent with the prediction from the thermodynamic theory that compounds that stabilize either hNBD1 directly or the hNBD1– hNBD2 interface can correct the thermodynamic defect caused by the F508del mutation in hCFTR (Fig. 1)
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