title: Nucleotide binding to the multidrug resistance P-glycoprotein as studied by ESR spectroscopy.
abstract: Electron spin resonance (ESR) spectroscopy using spin-labeled ATP was used to study nucleotide binding to and structural transitions within the multidrug resistance P-glycoprotein, P-gp. Spin-labeled ATP (SL-ATP) with the spin label attached to the ribose, was observed to be an excellent substrate analogue for P-gp. SL-ATP was hydrolyzed in a drug-stimulated fashion at about 14% of the rate for normal ATP and allowed reversible trapping of the enzyme in transition and ground states. Equilibrium binding of a total of two nucleotides per P-gp was observed with a binding affinity of 366 microM in the presence of Mg2+ but in the absence of transport substrates such as verapamil. Binding of SL-ATP to wild-type P-gp in the presence of verapamil resulted in reduction of the protein-bound spin-label moiety, most likely due to a conformational transition within P-gp that positioned cysteines in close proximity to the spin label to allow chemical reduction of the radical. We circumvented this problem by using a mutant of P-gp in which all naturally occurring cysteines were substituted for alanines. Equilibrium binding of SL-ATP to this mutant P-gp resulted in maximum binding of two nucleotides; the binding affinity was 223 microM in the absence and 180 microM in the presence of verapamil. The corresponding ESR spectra of wild-type and Cys-less P-gp in the presence of SL-ATP indicate that a cysteine side chain of P-gp is located close to the ribose of the bound nucleotide. Trapping SL-ATP as an AlF(x)-adduct resulted in ESR spectra that showed strong immobilization of the radical, supporting the formation of a closed conformation of P-gp in its transition state. This study is the first to employ ESR spectroscopy with the use of spin-labeled nucleotide analogues to study P-glycoprotein. The study shows that SL-ATP is an excellent substrate analogue that will allow further exploration of structure and dynamics within the nucleotide binding domains of this important enzyme.
authors.affiliation: Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275, USA.
authors.forename: Sabine
authors.initials: S
authors.lastname: Delannoy
authors.forename: Ina L
authors.initials: IL
authors.lastname: Urbatsch
authors.forename: Gregory
authors.initials: G
authors.lastname: Tombline
authors.forename: Alan E
authors.initials: AE
authors.lastname: Senior
authors.forename: Pia D
authors.initials: PD
authors.lastname: Vogel
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D005800:Genes, Fungal; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics; D011485:Protein Binding; D014700:Verapamil / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002121:Calcium Channel Blockers; D000255:Adenosine Triphosphate; D014700:Verapamil; D000251:Adenosine Triphosphatases