This proposal focuses on the protein called CFTR, mutations in which cause Cystic Fibrosis (CF), a major disease frequently characterized by chronic lung infections. CFTR is an ATP hydrolysis-dependent Cl- channel consisting of 2 membrane domains, 2 nucleotide binding folds (NBF1 plus NBF2), and an R (regulatory) domain. Most cases of CF are caused by a single mutation (deltaF508) in NBF1 which prevents CFTR from trafficking from the E.R. to the plasma membrane where, in the lung, it aids in combating infections. Early work (Science, 1991; JBC, 1992, 93, 94), supported by this grant, demonstrated that NBF1 and NBF2 bind ATP and that the deltaF508 mutation results in a folding problem. Recent work has shown that NBF1 hydrolyzes ATP (JBC, 1995), interacts with the membrane (Biochem., 1997), participates in the formation of an NBF1 plus R/NBF2 complex (In Review), and maintains the critical F508 region as an alpha-helix (In Review). Despite these important advances, essential information is lacking about the catalytic mechanism of NBF1 and NBF2, their """"""""cross-talk"""""""" in the NBF1 plus R/NBF2 complex, and the location and role of the F508 region. For these reasons, the following 4 hypotheses will be tested: 1. ATP hydrolysis catalyzed by NBF1 or NBF2 of CFTR proceeds through a reaction pathway similar to that catalyzed by both the F1 moiety of ATP synthase/ATPase complexes and myosin. 2. The rate of ATP hydrolysis is enhanced when the 3 """"""""soluble"""""""" domains of CFTR form a NBF1 plus R/NBF2 complex in which both NBF1 NBF2 are catalytic but function in an alternating, cooperative manner. 3. The F508 region of NBF1, which contains a potential catalytic base (E504), is flexible, and contributes to the active """"""""ATPase"""""""" site in the NBF1 plus R/NBF2 complex but lies outside this site in NBF1 alone. 4. The deltaF508 mutation responsible for most cases of CF prevents NBF1 from undergoing an ATP-dependent conformational change while reducing the catalytic efficiency of this domain. The proposed studies are fundamental to understanding structure/function relationships within CFTR, to understanding the underlying basis of most cases of CF, and to developing new strategies to treat the disease.
