) The long range objective of this proposal is to obtain a molecular and chemical description of both the wild type CFTR protein and those mutant forms that cause cystic fibrosis. Work during the first 4 years of this project resulted in some novel, exciting and definitive progress. Both putative nucleotide domains (NBF1 and NBF2) of CFTR were shown by direct methods to bind ATP and ADP; the major disease causing mutation deltaF508 was shown to induce a marked instability and cause partial unfolding within NBF1; and microcrystals of an NBF1 fusion protein were obtained. In more recent work, NBF1 was shown to catalyze ATPase activity, be phosphorylated by protein kinase A, and associate with the membrane. In addition, 3-dimensional models of both NBF1 and NBF2 based on x-ray structures of known ATPases have been constructed for the first time. Future work will focus on relating the ATP binding and hydrolytic functions of NBF1 to a) phosphorylation capacity, b) structure, c) membrane binding, d) capacity to interact with and be modulated by NBF2, and e) mutant forms that cause cystic fibrosis.
Specific aims are 5-fold and will be to: 1. Define those conditions which are optimal for supporting the ATP binding and ATP hydrolytic functions of NBF1 by carefully examining thereon both the effect of covalent phosphorylation and the effect of physiological anions and cations. 2. Identify, using site directed mutagenesis and a 3-dimensional model as a guide, those amino acids most critical for the ATP binding and ATP hydrolytic functions of NBF1, and the effect on these functions of disease causing mutations residing within this domain. 3. Elucidate the nature of the membrane binding interaction of NBF1, recently discovered in this laboratory, when the peptide segment G404-N432 preceding the domain is present. 4. Establish whether NBF2 exhibits the capacity to hydrolyze or only bind ATP, and the extent to which this domain in the absence or presence of the R domain interacts with NBF1 in a functionally important manner. 5. Vigorously continue ongoing experiments to obtain preparations of NBF1 suitable for 3-dimensional structural analysis. These studies are fundamental to understanding those structure-function relationships within both wild type CFTR and in mutant forms thereof which cause most cases of cystic fibrosis.
