title: Nucleotide binding to the human multidrug resistance protein 3, MRP3.
abstract: We used a spin-labeled ATP analog, SL-ATP, to study nucleotide binding to highly purified human multidrug resistance protein 3, MRP3, which had been expressed in the yeast Pichia pastoris. SL-ATP was shown to be a good substrate analog and is hydrolyzed by MRP3 at about 10% of the Vmax for normal ATP. ESR titrations showed that 2 mol of SL-ATP readily bound per mole of MRP3 with a dissociation constant of about 100 microM in the presence of Mg(2+) ions. The binding curve was easily fitted for a hyperbolic binding relationship. SL-ATP also bound readily to MRP3 in the absence of divalent ions and presence of EDTA. The resulting binding curve, however, could not be satisfactorily fitted using the equation for hyperbola. Analysis showed that a good fit was only obtained with the Hill equation using a Hill coefficient of 4 or close to 4. Lower Hill coefficients resulted in lower goodness of the fit. Such cooperative binding may be explained by a dimerization event triggered in the absence of divalent ions and a close communication of nucleotide binding sites of the interacting dimers. These findings may be of great importance for the overall mechanism and regulation of multidrug resistance proteins.
authors.affiliation: Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275, USA.
authors.forename: Andrea D
authors.initials: AD
authors.lastname: Hoffman
authors.forename: Ina L
authors.initials: IL
authors.lastname: Urbatsch
authors.forename: Pia D
authors.initials: PD
authors.lastname: Vogel
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*; D010843:Pichia; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism*; D013113:Spin Labels
chemical_list: D000255:Adenosine Triphosphate; D008274:Magnesium; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011994:Recombinant Proteins; D013113:Spin Labels; C114462:multidrug resistance-associated protein 3; C026652:3'-O-(1-oxyl-2,2,5,5-tetramethyl-3-carbonyl pyrrolidine)-adenosine 5'-triphosphate