AcrB Multidrug Efflux Pump of Escherichia coli : Composite Substrate-Binding Cavity of Exceptional Flexibility Generates Its Extremely Wide Substrate Specificity

Gram-negative bacteria have, in general, much higher intrinsic levels of resistance to various antibiotics, antiseptics, dyes, and detergents than do gram-positive bacteria.This is, in part, due to the effectiveness of the outer membrane as a barrier.The porin channels exclude large compounds (for example, vancomycin) and drastically slow down the influx of most antibiotics, which are usually much larger than common nutrients.Most antibiotics and chemotherapeutic agents that act on targets in the cytosol must cross the inner, cytoplasmic membrane, usually by spontaneous diffusion, and this necessitates their being at least moderately lipophilic.These compounds can in principle diffuse across the lipid bi-layer domain of the outer membrane.Transmembrane diffusion rates across this domain, however, are about two orders of magnitude slower than through the conventional phospholipid bi-layers (29), because the outer leaflet, composed exclusively of lipopolysaccharides (11), acts as an effective barrier.The outer membrane barrier alone, however, only slows down the influx of most of the noxious agents, and the gramnegative bacteria need the additional contribution of multidrug efflux pumps in order to achieve their characteristic levels of intrinsic resistance (24).The pumps belonging to the resistance-nodulation-division (RND) family are especially effective in generating resistance, as they form a tripartite complex together with the periplasmic proteins belonging to the membrane-fusion-protein (MFP) family and the outer membrane channels (Fig. 1), so that drugs are pumped out directly into the external medium.The RND pumps often have a very wide substrate specificity ( 23).An extreme case is the AcrB pump of Escherichia coli, which by forming a complex with an MFP, AcrA, and an outer membrane channel TolC pumps out tetracycline, chloramphenicol, ␤-lactams, novobiocin, fusidic acid, nalidixic acid, and fluoroquinolones among antibiotics and chemotherapeutic agents, SDS, Triton X-100, and bile salts among detergents, various cationic dyes and disinfectants, and even solvents (23,40,42).Examination of the structures of these substrates and the finding that carbenicillin and ceftriaxone, which cannot penetrate into the cytoplasm, were good substrates for an AcrB homolog, MexB (15), suggested in 1994 that a major pathway for the capture of substrates consists of