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

Abstract

AbstractCystic fibrosis is most frequently caused by the deletion of F508 (ΔF508) in CFTR’s nucleotide binding domain 1 (NBD1), compromising CFTR folding, stability and domain assembly. The limitation of developing a successful therapy is due 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. These methods allowed us to describe unfolding intermediates and forces acting during NBD1 mechanical unfolding and to elucidate the stabilization mechanism of ΔF508-NBD1 by 5-bromoindole-3-acetic acid (BIA). An NBD1 region, including the α-subdomain, was identified as a potentially important participant of the first folding steps, characterized by non-native interactions of F508, thus destabilized in the deletion mutant. The instability was counteracted by the low-potency corrector BIA, increasing 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 computational and experimental methods and provide a possible strategy to improve folding correctors.