title: AcrB-AcrA Fusion Proteins That Act as Multidrug Efflux Transporters.
abstract: The AcrAB-TolC system in Escherichia coli is an intrinsic RND-type multidrug efflux transporter that functions as a tripartite complex of the inner membrane transporter AcrB, the outer membrane channel TolC, and the adaptor protein AcrA. Although the crystal structures of each component of this system have been elucidated, the crystal structure of the whole complex has not been solved. The available crystal structures have shown that AcrB and TolC function as trimers, but the number of AcrA molecules in the complex is now under debate. Disulfide chemical cross-linking experiments have indicated that the stoichiometry of AcrB-AcrA-TolC is 1:1:1; on the other hand, recent cryo-electron microscopy images of AcrAB-TolC suggested a 1:2:1 stoichiometry. In this study, we constructed 1:1-fixed AcrB-AcrA fusion proteins using various linkers. Surprisingly, all the 1:1-fixed linker proteins showed drug export activity under both acrAB-deficient conditions and acrAB acrEF double-pump-knockout conditions regardless of the lengths of the linkers. Finally, we optimized a shorter linker lacking the conformational freedom imparted by the AcrB C terminus. These results suggest that a complex with equal amounts of AcrA and AcrB is sufficient for drug export function.
  
  
  OBJECTIVE
  The structure and stoichiometry of the RND-type multidrug exporter AcrB-AcrA-TolC complex are still under debate. Recently, electron microscopic images of the AcrB-AcrA-TolC complex have been reported, suggesting a 1:2:1 stoichiometry. However, we report here that the AcrB-AcrA 1:1 fusion protein is active for drug export under acrAB-deficient conditions and also under acrAB acrEF double-deficient conditions, which eliminate the aid of free AcrA and its close homolog AcrE, indicating that the AcrB-AcrA 1:1 stoichiometry is enough for drug export function. In addition, the AcrB-AcrA fusion protein can function without the aid of free AcrA. We believe that these results are very important for considering the structure and mechanism of AcrAB-TolC-mediated multidrug export.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan The Institute of Scientific and Industrial Research (ISIR), Osaka University, Ibaraki, Osaka, Japan Japan Society for the Promotion of Science, Tokyo, Japan.
authors.forename: Katsuhiko
authors.identifier: http://orcid.org/0000-0001-7359-5939
authors.initials: K
authors.lastname: Hayashi
authors.affiliation: The Institute of Scientific and Industrial Research (ISIR), Osaka University, Ibaraki, Osaka, Japan CREST, Japan Science and Technology Agency, Saitama, Japan.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Nakashima
authors.affiliation: The Institute of Scientific and Industrial Research (ISIR), Osaka University, Ibaraki, Osaka, Japan CREST, Japan Science and Technology Agency, Saitama, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Sakurai
authors.affiliation: The Institute of Scientific and Industrial Research (ISIR), Osaka University, Ibaraki, Osaka, Japan.
authors.forename: Kimie
authors.initials: K
authors.lastname: Kitagawa
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan The Institute of Scientific and Industrial Research (ISIR), Osaka University, Ibaraki, Osaka, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Yamasaki
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan The Institute of Scientific and Industrial Research (ISIR), Osaka University, Ibaraki, Osaka, Japan.
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Nishino
authors.affiliation: The Institute of Scientific and Industrial Research (ISIR), Osaka University, Ibaraki, Osaka, Japan CREST, Japan Science and Technology Agency, Saitama, Japan akihito@sanken.osaka-u.ac.jp.
authors.forename: Akihito
authors.initials: A
authors.lastname: Yamaguchi
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D019295:Computational Biology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D004996:Ethidium / Q000378:metabolism*; D017353:Gene Deletion; D019450:Genes, MDR / Q000502:physiology*; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D004996:Ethidium; D008074:Lipoproteins; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011993:Recombinant Fusion Proteins; C083497:AcrA protein, E coli; C083496:AcrB protein, E coli; C073126:tolC protein, E coli