Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 109, Issue 15, Pages 5687-5692Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1114944109
Keywords
antibiotics; multidrug resistance; drug efflux; RND transporter
Categories
Funding
- Swiss National Foundation
- German Research Foundation [SFB 807]
- Swiss National Center of Competence in Research (NCCR) Structural Biology
- Forschungskredit der Universitat Zurich
- DFG (Cluster of Excellence Macromolecular Complexes at the Goethe-University Frankfurt) [EXC115]
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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 angstrom) 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.
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