title: Nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator, an ABC transporter, catalyze adenylate kinase activity but not ATP hydrolysis.
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.
authors.affiliation: Vertex Pharmaceuticals Inc., Cambridge, Massachusetts 02139, USA.
authors.forename: Christian H
authors.initials: CH
authors.lastname: Gross
authors.forename: Norzehan
authors.initials: N
authors.lastname: Abdul-Manan
authors.forename: John
authors.initials: J
authors.lastname: Fulghum
authors.forename: Judith
authors.initials: J
authors.lastname: Lippke
authors.forename: Xun
authors.initials: X
authors.lastname: Liu
authors.forename: Prakash
authors.initials: P
authors.lastname: Prabhakar
authors.forename: Debra
authors.initials: D
authors.lastname: Brennan
authors.forename: Melissa Swope
authors.initials: MS
authors.lastname: Willis
authors.forename: Carlos
authors.initials: C
authors.lastname: Faerman
authors.forename: Patrick
authors.initials: P
authors.lastname: Connelly
authors.forename: Scott
authors.initials: S
authors.lastname: Raybuck
authors.forename: Jonathan
authors.initials: J
authors.lastname: Moore
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000263:Adenylate Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002712:Chlorides / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000502:physiology; D019281:Dimerization; D006801:Humans; D006868:Hydrolysis; D017136:Ion Transport; D051379:Mice; D009711:Nucleotides / Q000378:metabolism*
chemical_list: D002712:Chlorides; D009711:Nucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase; D000251:Adenosine Triphosphatases