TY  - JOUR
A1  - Zutz, Ariane
A1  - Hoffmann, Jan
A1  - Hellmich, Ute
A1  - Glaubitz, Clemens
A1  - Ludwig, Bernd
A1  - Brutschy, Bernd
A1  - Tampé, Robert
T1  - Asymmetric ATP hydrolysis cycle of the heterodimeric multidrug ABC transport complex TmrAB from Thermus thermophilus
T2  - Journal of biological chemistry
N2  - 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 Km of 0.9 mm and kcat 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.
KW  - ABC Transporter
KW  - ATPases
KW  - Membrane Proteins
KW  - Multidrug Transporters
KW  - Transport Drugs
Y1  - 2021
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/76565
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-765653
SN  - 0021-9258
VL  - 286
IS  - 9
SP  - 7104
EP  - 7115
PB  - American Society for Biochemistry and Molecular Biology Publications
CY  - Bethesda, Md
ER  -