, 2006; Nakashima et al., 2011). Consequently, RND-type drug transporters have been termed ‘cis transporters’. This is in contrast to drug transporters from the major facilitator superfamily and ATP-binding cassette families where drug transport occurs through an alternating access mechanism across the cytoplasmic membrane, the so-called trans-transporters (Eicher et al., 2009; Nikaido & Takatsuka, 2009; Doshi et al., 2011). However, studies on purified and reconstituted AcrD from E. coli revealed that this RND
transporter could efflux the SGI-1776 solubility dmso membrane-impermeable aminoglycoside gentamycin from either side of the liposomes, consistent with the existence of both periplasmic and cytoplasmic drug efflux pathways (Aires & Nikaido, 2005). It is therefore highly likely that, in addition to the periplasmic drug efflux pathway, a pathway exists for removal of drugs directly from the cytosol or inner membrane leaflet. Das and co-workers
(Das et al., 2007) predicted that conserved Phe residues on the small N-terminal helix of AcrB which line a 15 Å opening on the cytoplasmic side of AcrB could play a role in the discrimination, uptake and transport of drug molecules from the cytoplasm through the central cavity formed by the membrane domains of the AcrB trimer. Phenylalanine residues are hugely important in EPZ015666 mouse substrate recognition and binding in the RND-type drug transporters. The binding protomer in the asymmetric AcrB structure forms a hydrophobic pocket lined by phenylalanines 136, 178, 610, 615, 617 and 628 (Murakami et al., 2006; Seeger et al., 2006; Sennhauser et al., 2009) while minocycline, rifampicin and erythromycin are in direct contact with Phe residues (Murakami et al., 2006; Nakashima et al., 2011). Replacement of these phenylalanines with alanines reduced the ability of AcrB to confer resistance to a wide range of compounds (Bohnert et al., 2008; Vargiu et al.,
2011), with the F610A mutation having the greatest effect. Similarly, site-directed mutagenesis of F386, F388, F458 and F459 in AcrB resulted in a decrease in the minimum inhibitory concentration (MIC) for tetracycline, erythromycin, dequalinium and acriflavine (Yu et al., 2005). In this study, we aimed to provide more insight on the role of the conserved phenylalanine residues on the small L-NAME HCl N-terminal helix of MexB in drug efflux. These Phe residues were mutated to Ala residues to generate FAFA MexB. The ability of the mutant protein to confer resistance to toxic compounds and to efflux dyes was compared to that of the wild-type protein. For cytotoxicity and transport assays, plasmids were propagated in E. coli strain BW25113 lacking either AcrB or AcrA and AcrB (a kind gift from Professor Martin Pos, Institute of Biochemistry, Goethe-University, Frankfurt am Main, Germany). The plasmids used were pET41a (+), pUC18 (Novagen) and derivatives expressing MexB with a C-terminal His tag (pMexBH; Barrera et al.