The assembled structure of a complete tripartite bacterial multidrug efflux pump

Proc Natl Acad Sci U S A. 2009 Apr 28;106(17):7173-8. doi: 10.1073/pnas.0900693106. Epub 2009 Apr 2.

Abstract

Bacteria like Escherichia coli and Pseudomonas aeruginosa expel drugs via tripartite multidrug efflux pumps spanning both inner and outer membranes and the intervening periplasm. In these pumps a periplasmic adaptor protein connects a substrate-binding inner membrane transporter to an outer membrane-anchored TolC-type exit duct. High-resolution structures of all 3 components are available, but a pump model has been precluded by the incomplete adaptor structure, because of the apparent disorder of its N and C termini. We reveal that the adaptor termini assemble a beta-roll structure forming the final domain adjacent to the inner membrane. The completed structure enabled in vivo cross-linking to map intermolecular contacts between the adaptor AcrA and the transporter AcrB, defining a periplasmic interface between several transporter subdomains and the contiguous beta-roll, beta-barrel, and lipoyl domains of the adaptor. With short and long cross-links expressed as distance restraints, the flexible linear topology of the adaptor allowed a multidomain docking approach to model the transporter-adaptor complex, revealing that the adaptor docks to a transporter region of comparative stability distinct from those key to the proposed rotatory pump mechanism, putative drug-binding pockets, and the binding site of inhibitory DARPins. Finally, we combined this docking with our previous resolution of the AcrA hairpin-TolC interaction to develop a model of the assembled tripartite complex, satisfying all of the experimentally-derived distance constraints. This AcrA(3)-AcrB(3)-TolC(3) model presents a 610,000-Da, 270-A-long efflux pump crossing the entire bacterial cell envelope.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Escherichia coli / chemistry
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Models, Molecular
  • Multidrug Resistance-Associated Proteins / genetics
  • Multidrug Resistance-Associated Proteins / metabolism*
  • Protein Multimerization*
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • Pseudomonas aeruginosa / chemistry
  • Pseudomonas aeruginosa / genetics
  • Pseudomonas aeruginosa / metabolism

Substances

  • Multidrug Resistance-Associated Proteins

Associated data

  • PDB/2V4D