Effects of Point Mutations on the Thermal Stability of the NBD1 Domain of hCFTR.

Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The first nucleotide-binding domain (NBD1) of the CFTR is considered to be a hotspot for CF-causing mutations, and some of these mutations compromise the domain's thermal stability as well as its interactions with other domains. The mechanisms by which such mutations exert their deleterious effects are important in the basic research of this complex disease as well as for the development of mutation-specific therapies. With this in mind, we studied two class-II, severe, CF-causing mutations, L467P and A559T, known to destabilize the domain by 19.3 and 10.7 °C, respectively, and to lead to a misfolded, nonfunctioning CFTR, by conducting microsecond-long molecular dynamics (MD) simulations at an elevated temperature of 410 K on L467P-NBD1 and A559T-NBD1 constructs. For comparison, similar simulations were also performed on the wild-type (WT) construct and on the 6SS-NBD1 and 2PT/M470V-NBD1 constructs, both bearing sets of stabilizing mutations that stabilize the domain by 17.5 and 8.2 °C, respectively. The resulting trajectories were analyzed using multiple metrics, leading to a good correlation between the experimental ΔTm values and the results of the simulations, as well as multiple experimental observations and results of previous modeling efforts. Specifically, our analyses point to specific regions within NBD1 that are substantially affected by the L467P and A559T mutations and, therefore, may play some role in their pathogenesis. Many of these regions are also known to be important for the proper folding and function of the full-length CFTR. Using time-dependent assignment of DSSP elements, we also found that the two mutants follow different disintegration pathways, that of L467P-NBD1 starting in region 464-471 which resides within the F1-like ATP-binding core subdomain and continues in regions 550-562 and 514-523 within the ABCα subdomain whereas that of A559T-NBD1 simultaneously starting at the 550-562 and 514-523 regions. We propose that the analyses presented in this work may pave the way toward the development of L467P and A559T-specific CF therapies and by extension to other mutation-specific therapies for CF and for other diseases involving mutations in NBDs of other proteins.