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 ΔF508 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 ~ 52 to ~ 56 kDa by SDS-PAGE. The fluorescent ATP analogue TNP-ATP bound to renatured NBD1-R with K(app) of 0.81 ± 0.1 μM (wild-type), 0.93 ± 0.1 μM (wild-type, phosphorylated), 0.75 ± 0.1 μM (AF508 NBD1-R), and 0.72 ± 0.1 μM (AF508 NBD1-R, phosphorylated) with a stoichiometry of ~ 1 TNP- ATP site per NBD1-R molecule; TNP-ATP binding was reversed by ATP, AMP-PCP, and AMP-PNP with K(IS) of ~ 3.2, 4.2, and 4.6 mM, respectively. Secondary structure analysis by circular dichroism gave 19% α-helix, 43% β-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 [γ-³²P]ATP in the presence of AMPPCP, 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 AMPPNP or AMP-PCP (0.25 mM) and ≤50% by TNP-ATP (0.25 μM). 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 NBD 1-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.