title: Biochemical characterization and NMR studies of the nucleotide-binding domain 1 of multidrug-resistance-associated protein 1: evidence for interaction between ATP and Trp653.
abstract: 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. (15)N-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 microM and K (d(ADP))=139 microM). Binding of nucleotides did not influence the monomeric state of NBD1. The ATPase activity of NBD1 was magnesium-dependent and very low [V (max) and K (m) values of 5x10(-5) pmol of ATP x (pmol NBD1)(-1) x s(-1) and 833 microM ATP respectively]. The present study suggests that NBD1 has a low contribution to the ATPase activity of full-length MRP1 and/or that this activity requires NBD1-NBD2 heterodimer formation.
authors.affiliation: Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique (CNRS) Unité Propre de Recherche 2301, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
authors.forename: Odile
authors.initials: O
authors.lastname: Ramaen
authors.forename: Sandrine
authors.initials: S
authors.lastname: Masscheleyn
authors.forename: Francis
authors.initials: F
authors.lastname: Duffieux
authors.forename: Olivier
authors.initials: O
authors.lastname: Pamlard
authors.forename: Marine
authors.initials: M
authors.lastname: Oberkampf
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Lallemand
authors.forename: Véronique
authors.initials: V
authors.lastname: Stoven
authors.forename: Eric
authors.initials: E
authors.lastname: Jacquet
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D018922:HL-60 Cells; D006801:Humans; D008274:Magnesium / Q000502:physiology; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism* / Q000502:physiology
chemical_list: D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D008274:Magnesium; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011994:Recombinant Proteins; D014364:Tryptophan; C421109:multidrug resistance-associated protein 1