title: Mg2+ -dependent ATP occlusion at the first nucleotide-binding domain (NBD1) of CFTR does not require the second (NBD2).
abstract: ATP binding to the first and second NBDs (nucleotide-binding domains) of CFTR (cystic fibrosis transmembrane conductance regulator) are bivalent-cation-independent and -dependent steps respectively [Aleksandrov, Aleksandrov, Chang and Riordan (2002) J. Biol. Chem. 277, 15419-15425]. Subsequent to the initial binding, Mg(2+) drives rapid hydrolysis at the second site, while promoting non-exchangeable trapping of the nucleotide at the first site. This occlusion at the first site of functional wild-type CFTR is somewhat similar to that which occurs when the catalytic glutamate residues in both of the hydrolytic sites of P-glycoprotein are mutated, which has been proposed to be the result of dimerization of the two NBDs and represents a transient intermediate formed during ATP hydrolysis [Tombline and Senior (2005) J. Bioenerg. Biomembr. 37, 497-500]. To test the possible relevance of this interpretation to CFTR, we have now characterized the process by which NBD1 occludes [(32)P]N(3)ATP (8-azido-ATP) and [(32)P]N(3)ADP (8-azido-ADP). Only N(3)ATP, but not N(3)ADP, can be bound initially at NBD1 in the absence of Mg(2+). Despite the lack of a requirement for Mg(2+) for ATP binding, retention of the NTP at 37 degrees C was dependent on the cation. However, at reduced temperature (4 degrees C), N(3)ATP remains locked in the binding pocket with virtually no reduction over a 1 h period, even in the absence of Mg(2+). Occlusion occurred identically in a DeltaNBD2 construct, but not in purified recombinant NBD1, indicating that the process is dependent on the influence of regions of CFTR in addition to NBD1, but not NBD2.
authors.affiliation: Department of Biochemistry/Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: Luba
authors.initials: L
authors.lastname: Aleksandrov
authors.forename: Andrei
authors.initials: A
authors.lastname: Aleksandrov
authors.forename: John R
authors.initials: JR
authors.lastname: Riordan
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism; D002478:Cells, Cultured; D003080:Cold Temperature; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D019281:Dimerization; D008274:Magnesium / Q000494:pharmacology*; D017434:Protein Structure, Tertiary
chemical_list: D001386:Azides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D008274:Magnesium