ATP and AMP mutually influence their interaction with the ATP-binding cassette (ABC) adenylate kinase cystic fibrosis transmembrane conductance regulator (CFTR) at separate binding sites.
Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP &lrarr2; 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5'-triphosphate (8-N3-ATP) and 8-azidoadenosine 5'-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P(1),P(5)-di(adenosine-5') pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2.
Departments of Pediatrics. Electronic address: christoph-randak@uiowa.edu.
Christoph O
CO
Randak
Departments of Pediatrics.
Qian
Q
Dong
Departments of Pediatrics.
Amanda R
AR
Ver Heul
Departments of Biochemistry.
Adrian H
AH
Elcock
Departments of Internal Medicine; Molecular Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242; Howard Hughes Medical Institute, Iowa City, Iowa 52242.
Michael J
MJ
Welsh
D000249:Adenosine Monophosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000263:Adenylate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D017434:Protein Structure, Tertiary
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase
ABC Transporter; AMP; ATP; CFTR; Cystic fibrosis; Genetic Diseases