PMID:22451937
| Citation |
Eicher, T, Cha, HJ, Seeger, MA, Brandstätter, L, El-Delik, J, Bohnert, JA, Kern, WV, Verrey, F, Grütter, MG, Diederichs, K and Pos, KM (2012) Transport of drugs by the multidrug transporter AcrB involves an access and a deep binding pocket that are separated by a switch-loop. Proc. Natl. Acad. Sci. U.S.A. 109:5687-92 |
|---|---|
| Abstract |
AcrAB-TolC is the major efflux protein complex in Escherichia coli extruding a vast variety of antimicrobial agents from the cell. The inner membrane component AcrB is a homotrimer, and it has been postulated that the monomers cycle consecutively through three conformational stages designated loose (L), tight (T), and open (O) in a concerted fashion. Binding of drugs has been shown at a periplasmic deep binding pocket in the T conformation. The initial drug-binding step and transport toward this drug-binding site has been elusive thus far. Here we report high resolution structures (1.9-2.25 Å) of AcrB/designed ankyrin repeat protein (DARPin) complexes with bound minocycline or doxorubicin. In the AcrB/doxorubicin cocrystal structure, binding of three doxorubicin molecules is apparent, with one doxorubicin molecule bound in the deep binding pocket of the T monomer and two doxorubicin molecules in a stacked sandwich arrangement in an access pocket at the lateral periplasmic cleft of the L monomer. This access pocket is separated from the deep binding pocket apparent in the T monomer by a switch-loop. The localization and conformational flexibility of this loop seems to be important for large substrates, because a G616N AcrB variant deficient in macrolide transport exhibits an altered conformation within this loop region. Transport seems to be a stepwise process of initial drug uptake in the access pocket of the L monomer and subsequent accommodation of the drug in the deep binding pocket during the L to T transition to the internal deep binding pocket of the T monomer. |
| Links |
PubMed PMC3326505 Online version:10.1073/pnas.1114944109 |
| Keywords |
Binding Sites; Biocatalysis; Biological Transport; Doxorubicin/chemistry; Doxorubicin/metabolism; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/metabolism; Minocycline/chemistry; Minocycline/metabolism; Models, Molecular; Multidrug Resistance-Associated Proteins/chemistry; Multidrug Resistance-Associated Proteins/metabolism; Pharmaceutical Preparations/metabolism; Protein Binding; Protein Structure, Secondary |
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