Expression and characterization of the NBD1-R domain region of CFTR: evidence for subunit-subunit interactions.

To study interactions between the contiguous NBD1 and R domains of CFTR, wild-type and DeltaF508 NBD1-R (amino acids 404-830, in fusion with His6 tag) were expressed as single proteins in Escherichia coli. NBD1-R (10-25 mg/L culture) was purified from inclusion bodies in 8 M urea by Ni-affinity chromatography, and renatured by rapid dilution at pH 5. In vitro phosphorylation by protein kinase A increased the apparent size of NBD1-R from approximately 52 to approximately 56 kDa by SDS-PAGE. The fluorescent ATP analogue TNP-ATP bound to renatured NBD1-R with of 0.81 +/- 0.1 microM (wild-type), 0.93 +/- 0.1 microM (wild-type, phosphorylated), 0.75 +/- 0.1 microM (DeltaF508 NBD1-R), and 0.72 +/- 0.1 microM (DeltaF508 NBD1-R, phosphorylated) with a stoichiometry of approximately 1 TNP-ATP site per NBD1-R molecule; TNP-ATP binding was reversed by ATP, AMP-PCP, and AMP-PNP with KIs of approximately 3.2, 4.2, and 4.6 mM, respectively. Secondary structure analysis by circular dichroism gave 19% alpha-helix, 43% beta-sheet and turn, and 38% "other" structure. To determine if nucleotide binding to NBD1 influenced R domain phosphorylation, NBD1-R was in vitro phosphorylated with protein kinase A and [gamma-32P]ATP in the presence of AMP-PCP, AMP-PNP, or TNP-ATP. Whereas the nucleotide analogues did not affect 32P-incorporation in control proteins (Kemptide, GST-R domain), phosphorylation of NBD1-R was reduced >75% by AMP-PNP or AMP-PCP (0.25 mM) and >50% by TNP-ATP (0.25 microM). Analysis of phosphorylation sites indicated that inhibition involved multiple sites in NBD1-R, including serines 660, 712, 737, 795, and 813. These results establish the conditions for NBD1-R expression, purification, and renaturation. The inhibition of R domain phosphorylation by nucleotide binding to the NBD1 domain indicates significant domain-domain interactions and suggests a novel mechanism for regulation of CFTR phosphorylation.