Mg2+ dependent ATP occlusion at the first nucleotide binding domain (NBD1) of CFTR does not require the second (NBD2).

ATP binding to the first (NBD1) and second (NBD2) nucleotide binding sites of CFTR are divalent cation independent and dependent steps, respectively (Aleksandrov et al, J Biol Chem 277, 15419-25, 2002). 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 glutamates 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 represent a transient intermediate formed during ATP hydrolysis (Tombline and Senior, J Bioenerg Biomembr 37, 497-500, 2005). To test the possible relevance of this interpretation to CFTR, we have now characterized the process by which NBD1 occludes [32P]-8N3ATP and [32P]-8N3ADP. 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 nucleoside triphosphate at 37 degrees C was dependent on the cation. However, at reduced temperature (4 degrees C), N3ATP remains locked in the binding pocket with virtually no reduction over a 1 hour period even in the absence of Mg2+. 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.

PMID: 18605986 [PubMed - as supplied by publisher]