Biochemical characterization and NMR studies of the nucleotide-binding domain 1 of multidrug-resistance-associated protein 1: evidence for interaction between ATP and Trp(653)

摘要

Multidrug-resistance-associated protein 1 (MRP1/ABCC1) is a human ATP-binding cassette transporter that confers cell resistance to antitumour drugs. Its NBDs (nucleotide-binding domains) bind/hydrolyse ATP, a key step in the activation of MRP1 function. To relate its intrinsic functional features to the mechanism of action of the full-size transporter, we expressed the N-terminal NBD1 domain (Asn(642) to Ser(871)) in Escherichia coli. NBD1 was highly purified under native conditions and was characterized as a soluble monomer. N-15-labelling allowed recording of the first two-dimensional NMR spectra of this domain. The NMR study showed that NBD1 was folded, and that Trp(653) was a key residue in the NBD1-ATP interaction. Thus, interaction of NBD1 with ATP/ADP was studied by intrinsic tryptophan fluorescence. The affinity for ATP and ADP were in the same range (K-d(ATP) = 118 muM and K-d(ADP) = 139 muM). Binding of nucleotides did not influence the monomeric state of NBD1. The ATPase activity of NBD1 was magnesium-dependent and very low [V.. and Km values of 5 x 10(-5) pmol of ATP . (pmol NBD1)(-1). s(-1) and 833 muM ATP respectively]. The present study suggests that NBDI has a low contribution to the ATPase activity of full-length MRP1 and/or that this activity requires NBD1-NBD2 heterodimer formation.