Extramembrane Central Pore of Multidrug Exporter AcrB in Escherichia coli Plays an Important Role in Drug Transport

Abstract

We previously reported the crystal structure of the major multidrug exporter AcrB in Escherichia coli (Murakami, S., Nakashima, R., Yamashita, E., and Yamaguchi, A. (2002) Nature 419, 587-593). The extramembrane headpiece of the AcrB trimer contains a central pore composed of three ␣-helices. Each pore helix belongs to a different monomer. In this study, we constructed cysteine-scanning mutants as to the residues comprising the pore helix. Of the 21 mutants (D99C to P119C), 5 (D101C, V105C, N109C, Q112C, and P116C) showed significantly reduced drug resistance and drug-exporting activity. These residues are localized on one side of the pore helix, i.e. on the wall of the pore. These observations strongly indicate the important role of this pore in the drug transport process. A N-ethylmaleimide binding experiment revealed that the pore is in the closed state, and the thickness of the permeability barrier in the middle of the pore corresponds to 2.5 ␣-helical turns. Two mutants (V105C and Q112C), which showed the greatest loss of activity of all of the pore mutants, were detected in the form of disulfide cross-linking dimers under normal conditions, suggesting that a conformational change of the pore is indispensable during the transport process.

Vacuuming the Periplasm

“…This conclusion is in good agreement with results from several laboratories that indicate that residues in two large periplasmic loops of RND transporters determine their substrate specificity (8,11,12,22,24,27,35).…”

“…The proximity of these pockets to the upper portal might allow their connection to the portal upon a conformational change associated with proton transport. It has previously been postulated (27,28) that substrates that accumulate in the central cavity are actively transported into the upper portal space via the channel that should open along the central axis of the structure (solid blue arrows). However, a very significant conformational change associated with channel opening would have to be coupled with proton transport (turquoise arrow) via the transmembrane domain in order to accommodate the passage of substrates.…”

AcrB-AcrA Fusion Proteins That Act as Multidrug Efflux Transporters

“…The antibody reaction was performed with an iBind Western system (Life Technologies). An anti-His tag mouse monoclonal antibody was purchased from Sigma-Aldrich (catalog number H1029-0.2ML), and the anti-AcrA and anti-AcrB rabbit polyclonal antibodies that were used were described previously (51). For His tag, AcrA, and AcrB detection, we used 15 g, 2 g, and 15 g membrane protein, respectively.…”

The Role of the Transcription Factors MtrR and MtrA in the Fitness of the Pathogen Neisseria gonorrhoeae

“…shown that the inner membrane portion of the RND efflux pumps can recognize and efflux substrates in both the cytoplasm and periplasm of the bacterium (128). Mutation of the periplasmic loops of the P. aeruginosa inner membrane pump component MexB, has shown that substrates are recognized and effluxed from the,periplasmic space and/or the inner membrane (188).…”