title: Different functions of MdtB and MdtC subunits in the heterotrimeric efflux transporter MdtB(2)C complex of Escherichia coli.
abstract: In contrast to homotrimeric transporters of the RND (resistance-nodulation-division) superfamily, which often conduct efflux transport of a wide range of substrates by the functionally rotating mechanism, the mechanism utilized by the heterotrimeric members of this family, which also perform multidrug efflux, is unclear. We examined one heterotrimeric transporter, the MdtB(2)C complex of Escherichia coli, by an extensive cysteine scanning mutagenesis of residues likely involved in ligand transport. Many such mutations in MdtC strongly decreased the level of cloxacillin transport, whereas mutations of corresponding residues in MdtB did not affect transport. Furthermore, many such residues in MdtC were covalently modified by fluorescein maleimide, which acted as a substrate and presumably produced labeling of the residues in the substrate path. In contrast, few residues in MdtB were labeled. Together with the previous data showing that the inactivation of proton translocation channel in MdtC has an only modest effect on transport yet in MdtB totally inactivated the activity, these results suggest that the two subunits, MdtB and MdtC, play very different roles, MdtC likely involved in substrate binding and transport and MdtB presumably inducing the conformational change needed for transport through proton translocation. Three-dimensional models of MdtB and MdtC, based on sequence homology with the AcrB transporter, also support this interpretation.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, USA.
authors.forename: Hong-Suk
authors.initials: HS
authors.lastname: Kim
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nikaido
mesh_terms: D000595:Amino Acid Sequence; D001692:Biological Transport / Q000235:genetics; D003023:Cloxacillin / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D008301:Maleimides / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D055503:Protein Multimerization
chemical_list: D003023:Cloxacillin; D003545:Cysteine; D029968:Escherichia coli Proteins; D008301:Maleimides; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C547958:MdtB protein, E coli; C547959:MdtC protein, E coli; C043592:maleimide