title: Transport of drugs by the multidrug transporter AcrB involves an access and a deep binding pocket that are separated by a switch-loop.
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.
authors.affiliation: Institute of Biochemistry and Cluster of Excellence Frankfurt-Macromolecular Complexes,  Goethe-University Frankfurt, D-60438 Frankfurt am Main,  Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Eicher
authors.forename: Hi-jea
authors.initials: HJ
authors.lastname: Cha
authors.forename: Markus A
authors.initials: MA
authors.lastname: Seeger
authors.forename: Lorenz
authors.initials: L
authors.lastname: Brandstätter
authors.forename: Jasmin
authors.initials: J
authors.lastname: El-Delik
authors.forename: Jürgen A
authors.initials: JA
authors.lastname: Bohnert
authors.forename: Winfried V
authors.initials: WV
authors.lastname: Kern
authors.forename: François
authors.initials: F
authors.lastname: Verrey
authors.forename: Markus G
authors.initials: MG
authors.lastname: Grütter
authors.forename: Kay
authors.initials: K
authors.lastname: Diederichs
authors.forename: Klaas M
authors.initials: KM
authors.lastname: Pos
mesh_terms: D001665:Binding Sites; D055162:Biocatalysis; D001692:Biological Transport; D004317:Doxorubicin / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D008911:Minocycline / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary
chemical_list: D004317:Doxorubicin; D029968:Escherichia coli Proteins; D008911:Minocycline; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D004364:Pharmaceutical Preparations; C083496:AcrB protein, E coli