Nanomechanics combined with HDX reveal allosteric drug binding sites of CFTR NBD1

Cystic fibrosis is most frequently caused by the deletion of F508 (ΔF508) in CFTR9s nucleotide binding domain 1 (NBD1), thereby compromising CFTR folding, stability and domain assembly. Limitation to develop a successful therapy has been attributed to the lack of molecules that synergistically facilitate folding by targeting distinct structural defects of ΔF508-CFTR. To improve drug efficacy by targeting the ΔF508-NBD1 folding and stability, and to study potential ΔF508-NBD1 allosteric corrector binding sites at the atomic level, we combined molecular dynamics (MD) simulations, atomic force spectroscopy (AFM) and hydrogen-deuterium exchange (HDX) experiments to elucidate the mechanical and thermal stabilization mechanisms of ΔF508-NBD1 by 5-bromoindole-3-acetic acid (BIA). MD and AFM allowed us to describe unfolding intermediates and forces acting during NBD1 mechanical unfolding. Application of the low-potency corrector BIA increased the mechanical resistance of the ΔF508-NBD1 α-subdomain, which was confirmed as a binding site by computational modeling and HDX experiments. Our results underline the complementarity of MD and AFM despite their different pulling speeds and provide a possible strategy to improve folding correctors.