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, Mg2+ 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 [32P]N3ATP (8-azido-ATP) and [32P]N3ADP (8-azido-ADP). Only N3ATP, but not N3ADP, can be bound initially at NBD1 in the absence of Mg2+. Despite the lack of a requirement for Mg2+ for ATP binding, retention of the NTP at 37 °C was dependent on the cation. However, at reduced temperature (4 °C), N3ATP remains locked in the binding pocket with virtually no reduction over a 1 h period, even in the absence of Mg2+. Occlusion occurred identically in a ΔNBD2 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.
Cysteine Residues in the Nucleotide Binding Domains Regulate the Conductance State of CFTR Channels