title: Asymmetric ATP hydrolysis cycle of the heterodimeric multidrug ABC transport complex TmrAB from Thermus thermophilus.
abstract: ATP-binding cassette (ABC) systems translocate a wide range of solutes across cellular membranes. The thermophilic gram-negative eubacterium Thermus thermophilus, a model organism for structural genomics and systems biology, discloses ∼46 ABC proteins, which are largely uncharacterized. Here, we functionally analyzed the first two and only ABC half-transporters of the hyperthermophilic bacterium, TmrA and TmrB. The ABC system mediates uptake of the drug Hoechst 33342 in inside-out oriented vesicles that is inhibited by verapamil. TmrA and TmrB form a stable heterodimeric complex hydrolyzing ATP with a K(m) of 0.9 mm and k(cat) of 9 s(-1) at 68 °C. Two nucleotides can be trapped in the heterodimeric ABC complex either by vanadate or by mutation inhibiting ATP hydrolysis. Nucleotide trapping requires permissive temperatures, at which a conformational ATP switch is possible. We further demonstrate that the canonic glutamate 523 of TmrA is essential for rapid conversion of the ATP/ATP-bound complex into its ADP/ATP state, whereas the corresponding aspartate in TmrB (Asp-500) has only a regulatory role. Notably, exchange of this single noncanonic residue into a catalytic glutamate cannot rescue the function of the E523Q/D500E complex, implicating a built-in asymmetry of the complex. However, slow ATP hydrolysis in the newly generated canonic site (D500E) strictly depends on the formation of a posthydrolysis state in the consensus site, indicating an allosteric coupling of both active sites.
authors.affiliation: Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, D-60438 Frankfurt, Germany.
authors.forename: Ariane
authors.initials: A
authors.lastname: Zutz
authors.forename: Jan
authors.initials: J
authors.lastname: Hoffmann
authors.forename: Ute A
authors.initials: UA
authors.lastname: Hellmich
authors.forename: Clemens
authors.initials: C
authors.lastname: Glaubitz
authors.forename: Bernd
authors.initials: B
authors.lastname: Ludwig
authors.forename: Bernd
authors.initials: B
authors.lastname: Brutschy
authors.forename: Robert
authors.initials: R
authors.lastname: Tampé
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000494:Allosteric Regulation / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D018432:Drug Resistance, Multiple / Q000502:physiology; D006868:Hydrolysis; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases