ATP‐Dependent Thermo‐Ring Basis for the Heat Unfolding of the First Nucleotide‐Binding Domain Isolated From Human CFTR

ABSTRACT The creative concept of “thermo‐rings” has recently been developed to characterize the tightened noncovalent network of interactions that drive the tertiary folding of a protein. However, it is still challenging to accurately evaluate the melting temperature threshold for the initial heat unfolding of any domain that tethers with another domain in a protein. To this end, thermo‐ring structures were studied on the Mg/ATP‐dependent heat‐induced unfolding of the first nucleotide‐binding domain (hNBD1) isolated from the human cystic fibrosis transmembrane conductance regulator (hCFTR). The results showed that initial theoretical and experimental melting thresholds aligned well after three structural perturbations, including the F508del mutation, the most common cause of cystic fibrosis. This alignment further demonstrated that the heat‐induced unfolding process began with the disruption of the least‐stable noncovalent interaction within the biggest thermo‐ring along the single peptide chain. The release of the C‐terminal region, which was required for the normal Mg/ATP‐dependent dimerization of two nucleotide‐binding domains, emerged as a crucial prerequisite to stabilize hNBD1 and to rescue the F508del‐induced thermal and gating defects. Thus, the same thermo‐ring‐based methodology, once vigorously validated by an isolated domain, could be used to gauge the thermal stability of the same or similar disease‐related domain that links with another domain in CFTR.