title: The First Nucleotide Binding Domain of Cystic Fibrosis Transmembrane Conductance Regulator Is a Site of Stable Nucleotide Interaction, whereas the Second Is a Site of Rapid Turnover.
abstract: As in other adenine nucleotide binding cassette (ABC) proteins the nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator (CFTR) bind and hydrolyze ATP and in some manner regulate CFTR ion channel gating. Unlike some other ABC proteins, however, there are preliminary indications that the two domains of CFTR are nonequivalent in their nucleotide interactions (Szabo, K., Szakacs, G., Hegeds, T., and Sarkadi, B. (1999) J. Biol. Chem. 274, 12209-12212; Aleksandrov, L., Mengos, A., Chang, X., Aleksandrov, A., and Riordan, J. R. (2001) J. Biol. Chem. 276, 12918-12923). We have now characterized the interactions of the 8-azido-photoactive analogues of ATP, ADP, and 5'-adenyl-beta,gamma-imidodiphosphate (AMP-PNP) with the two domains of functional membrane-bound CFTR. The results show that the two domains appear to act independently in the binding and hydrolysis of 8-azido-ATP. At NBD1 binding does not require a divalent cation. This binding is followed by minimal Mg(2+)-dependent hydrolysis and retention of the hydrolysis product, 8-azido-ADP, but not as a vanadate stabilized post-hydrolysis transition state complex. In contrast, at NBD2, MgN(3)ATP is hydrolyzed as rapidly as it is bound and the nucleoside diphosphate hydrolysis product dissociates immediately. Confirming this characterization of NBD1 as a site of more stable nucleotide interaction and NBD2 as a site of fast turnover, the non-hydrolyzable N(3)AMP-PNP bound preferentially to NBD1. This demonstration of NBD2 as the rapid nucleotide turnover site is consistent with the strong effect on channel gating kinetics of inactivation of this domain by mutagenesis.
authors.affiliation: Mayo Foundation and Mayo Clinic Scottsdale, S. C. Johnson Medical Research Center, Scottsdale, Arizona 85259, USA.
authors.forename: Luba
authors.initials: L
authors.lastname: Aleksandrov
authors.forename: Andrei A
authors.initials: AA
authors.lastname: Aleksandrov
authors.forename: Xiu-Bao
authors.initials: XB
authors.lastname: Chang
authors.forename: John R
authors.initials: JR
authors.lastname: Riordan
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels; D019943:Amino Acid Substitution; D000818:Animals; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000378:metabolism*; D015640:Ion Channel Gating; D007473:Ion Channels / Q000502:physiology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; D001386:Azides; D007473:Ion Channels; D009711:Nucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C017040:8-azidoadenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C021622:8-azidoadenosine diphosphate