Multidrug efflux pump MdtBC of Escherichia coli is active only as a B2C heterotrimer.
RND (resistance-nodulation-division) family transporters in Gram-negative bacteria frequently pump out a wide range of inhibitors and often contribute to multidrug resistance to antibiotics and biocides. An archetypal RND pump of Escherichia coli, AcrB, is known to exist as a homotrimer, and this construction is essential for drug pumping through the functionally rotating mechanism. MdtBC, however, appears different because two pump genes coexist within a single operon, and genetic deletion data suggest that both pumps must be expressed in order for the drug efflux to occur. We have expressed the corresponding genes, with one of them in a His-tagged form. Copurification of MdtB and MdtC under these conditions showed that they form a complex, with an average stoichiometry of 2:1. Unequivocal evidence that only the trimer containing two B protomers and one C protomer is active was obtained by expressing all possible combinations of B and C in covalently linked forms. Finally, conversion into alanine of the residues, known to form a proton translocation pathway in AcrB, inactivated transport only when made in MdtB, not when made in MdtC, a result suggesting that MdtC plays a different role not directly involved in drug binding and extrusion.
Kim
Hong-Suk
HS
Department of Molecular and Cell Biology, 426 Barker Hall, University of California, Berkeley, CA 94720-3202, USA.
Nagore
Daniel
D
Nikaido
Hiroshi
H
D018432:Drug Resistance, Multiple; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D055503:Protein Multimerization
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C547958:MdtB protein, E coli; C547959:MdtC protein, E coli