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. 2012 Apr 10;109(15):5687-92. doi: 10.1073/pnas.1114944109. Epub 2012 Mar 26.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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

Substances

  • AcrB protein, E coli
  • Escherichia coli Proteins
  • Multidrug Resistance-Associated Proteins
  • Pharmaceutical Preparations
  • Doxorubicin
  • Minocycline

Associated data

  • PDB/4DX5
  • PDB/4DX6
  • PDB/4DX7