Asymmetric ATP hydrolysis cycle of the heterodimeric multidrug ABC transport complex TmrAB from Thermus thermophilus
- 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.
Author: | Ariane ZutzGND, Jan HoffmannORCiDGND, Ute HellmichORCiDGND, Clemens GlaubitzORCiDGND, Bernd LudwigGND, Bernd BrutschyGND, Robert TampéORCiDGND |
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URN: | urn:nbn:de:hebis:30:3-765653 |
DOI: | https://doi.org/10.1074/jbc.M110.201178 |
ISSN: | 0021-9258 |
Pubmed Id: | https://pubmed.ncbi.nlm.nih.gov/21190941 |
Parent Title (English): | Journal of biological chemistry |
Publisher: | American Society for Biochemistry and Molecular Biology Publications |
Place of publication: | Bethesda, Md |
Document Type: | Article |
Language: | English |
Date of Publication (online): | 2021/01/04 |
Year of first Publication: | 2011 |
Publishing Institution: | Universitätsbibliothek Johann Christian Senckenberg |
Release Date: | 2023/11/09 |
Tag: | ABC Transporter; ATPases; Membrane Proteins; Multidrug Transporters; Transport Drugs |
Volume: | 286 |
Issue: | 9 |
Page Number: | 12 |
First Page: | 7104 |
Last Page: | 7115 |
HeBIS-PPN: | 516508326 |
Institutes: | Biochemie, Chemie und Pharmazie |
Exzellenzcluster / Exzellenzcluster Makromolekulare Komplexe | |
Dewey Decimal Classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Sammlungen: | Universitätspublikationen |
Licence (German): | Creative Commons - CC BY - Namensnennung 4.0 International |