Localization and suppression of a kinetic defect in cystic fibrosis transmembrane conductance regulator folding

A growing body of evidence indicates that the most common cystic fibrosis-causing mutation, Delta F508, alters the ability of the cystic fibrosis transmembrane conductance regulator (CFTR) protein to fold and transit to the plasma membrane, Here we present evidence that the Delta F508 mutation affects a step on the folding pathway prior to formation of the ATP binding site in the nucleotide binding domain (NBD), Notably, stabilization of the native state with 4 mM ATP does not alter the temperature-dependent folding yield of the mutant Delta F508 NBD1 in vitro. In contrast, glycerol, which promotes Delta F508-CFTR maturation in vivo, increases the folding yield of NBD1 Delta F and reduces the off pathway rate in vitro, although it does not significantly alter the free energy of stability, Likewise a second site mutation, R553M, which corrects the maturation defect in vivo, is a superfolder which counters the effects of Delta F508 on the temperature-dependent folding yield in vitro, but does not significantly alter the free energy of stability, A disease-causing mutation, G551D, which does not alter the maturation of CFTR in vivo but rather its function as a chloride channel, and the S549R maturation mutation have no discernible effect on the folding of the domain, These results demonstrate that Delta F508 is a kinetic folding mutation that affects a step early in the process, and that there is a significant energy barrier between the native state and the step affected by the mutation precluding the use of native state ligands to promote folding, The implications for protein folding in general are that the primary sequence may not necessarily simply define the most stable native structure, but rather a stable structure that is kinetically accessible.