Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter family. CFTR consists of two transmembrane domains, two nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain. Previous biochemical reports suggest NBD1 is a site of stable nucleotide interaction with low ATPase activity, whereas NBD2 is the site of active ATP hydrolysis. It has also been reported that NBD2 additionally possessed adenylate kinase (AK) activity. Knowledge about the intrinsic biochemical activities of the NBDs is essential to understanding the Cl(-) ion gating mechanism. We find that purified mouse NBD1, human NBD1, and human NBD2 function as adenylate kinases but not as ATPases. AK activity is strictly dependent on the addition of the adenosine monophosphate (AMP) substrate. No liberation of [(33)P]phosphate is observed from the gamma-(33)P-labeled ATP substrate in the presence or absence of AMP. AK activity is intrinsic to both human NBDs, as the Walker A box lysine mutations abolish this activity. At low protein concentration, the NBDs display an initial slower nonlinear phase in AK activity, suggesting that the activity results from homodimerization. Interestingly, the G551D gating mutation has an exaggerated nonlinear phase compared with the wild type and may indicate this mutation affects the ability of NBD1 to dimerize. hNBD1 and hNBD2 mixing experiments resulted in an 8-57-fold synergistic enhancement in AK activity suggesting heterodimer formation, which supports a common theme in ABC transporter models. A CFTR gating mechanism model based on adenylate kinase activity is proposed.
Highlights
We found that the mouse NBD1 (mNBD1) adenylate kinase activity was inhibited by Ap5A with IC50 ϭ 70 nMϮ 4 nM (ATP and adenosine monophosphate (AMP) substrates were at Km) (Fig. 2C)
Reversibility of the CFTR nucleotide-binding domains (NBDs) Adenylate Kinase Activity—We have demonstrated that mNBD1, hNBD1, and hNBD2 proteins have the ability to catalyze adenylate kinase (AK) activity
NBD1-R C-terminal Domain Has an Inhibitory Role on the Adenylate Kinase Activity—We believe that our experiments have clearly demonstrated that the NBDs catalyze AK activity, but the possibility remains that larger CFTR protein fragments may gain extra functionality (e.g. ATPase) from additional contiguous residues
Summary
Cloning of the Mouse and Human CFTR NBDs—The NBDs were amplified by PCR from mouse and human cDNA libraries using Pfu/ Taq polymerases (Stratagene). The packed column was washed with buffer 3 (25 mM Tris-HCl, pH 7.5, 8 M urea, and 10 mM 2-mercaptoethanol) containing sequentially increasing amounts of imidazole. The reactions (30 l) contained 50 mM Tris-HCl, pH 7.5, 2.5 mM MgCl2, 50 mM NaCl, 1 mM dithiothreitol, 500 M [␥-33P]ATP, 400 M AMP, and varying amounts of NBD were incubated at 37 °C for 60 min. The reactions contained 50 mM Tris-HCl, pH 7.5, 2.5 mM MgCl2, 50 mM NaCl, 1 mM dithiothreitol, 400 M [8-14C]ADP, and varying amounts of NBD were incubated at 37 °C for 60 min. Determination of Kinetic Parameters and Data Analysis—The apparent Km and Vmax values for ATP, AMP, and ADP were determined by fitting the rectangular hyperbolic plot of linear rates of product (M/ min) versus increasing substrate concentration (M) to the Michaelis-.
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