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Resistance to therapeutic drugs encompasses a diverse range of biological systems, which all have a human impact. From the relative simplicity of bacterial cells, fungi and protozoa to the complexity of human cancer cells, resistance has become problematic. Stated in its simplest terms, drug resistance decreases the chance of providing successful treatment against a plethora of diseases. Worryingly, it is a problem that is increasing, and consequently there is a pressing need to develop new and effective classes of drugs. This has provided a powerful stimulus in promoting research on drug resistance and, ultimately, it is hoped that this research will provide novel approaches that will allow the deliberate circumvention of well understood resistance mechanisms. A major mechanism of resistance in both microbes and cancer cells is the membrane protein-catalysed extrusion of drugs from the cell. Resistant cells exploit proton-driven antiporters and/or ATP-driven ABC (ATP-binding cassette) transporters to extrude cytotoxic drugs that usually enter the cell by passive diffusion. Although some of these drug efflux pumps transport specific substrates, many are transporters of multiple substrates. These multidrug pumps can often transport a variety of structurally unrelated hydrophobic compounds, ranging from dyes to lipids. If we are to nullify the effects of efflux-mediated drug resistance, we must first of all understand how these efflux pumps can accommodate a diverse range of compounds and, secondly, how conformational changes in these proteins are coupled to substrate translocation. These are key questions that must be addressed. In this review we report on the advances that have been made in understanding the structure and function of drug efflux pumps.

Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP.

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Multidrug resistance in Gram-negative bacteria arises in part from the activities of tripartite drug efflux pumps. In the pathogen Vibrio cholerae, one such pump comprises the inner membrane proton antiporter VceB, the periplasmic adaptor VceA, and the outer membrane channel VceC. Here, we report the crystal structure of VceC at 1.8 Å resolution. The trimeric VceC is organized in the crystal lattice within laminar arrays that resemble membranes. A well resolved detergent molecule within this array interacts with the transmembrane ␤-barrel domain in a fashion that may mimic proteinlipopolysaccharide contacts. Our analyses of the external surfaces of VceC and other channel proteins suggest that different classes of efflux pumps have distinct architectures. We discuss the implications of these findings for mechanisms of drug and protein export.To expel drugs and other toxic compounds, Gram-negative bacteria use specialized machinery that guides the compounds across two membranes and over the separating interstitial space, known as the periplasm. One type of such transport machinery is the complex formed by an inner membrane proton antiporter, an outer membrane channel, and a periplasmic protein that consolidates the assembly (1, 2). These energy-dependent tripartite pumps extrude actively a variety of noxious compounds from the cytoplasm or inner membrane to the extracellular medium. A number of such pumps have been implicated in multidrug resistance of Gram-negative species, and representative examples that have been well characterized include the Escherichia coli AcrAB-TolC and the Pseudomonas aeruginosa MexAB-OprM assemblies (3, 4).Advances are being made in understanding the mechanism of multidrug efflux in Gram-negative bacteria at the level of stereochemistry. To date, high resolution crystal structures have become available for the outer membrane components TolC (5) and OprM (6), the inner membrane proton antiporter AcrB (7,8), and the periplasmic adaptor MexA (9, 10). Based on these structures, several models have been proposed for the organization of the AcrAB-TolC assembly as a representative efflux pump (9 -11). However, the detailed interactions of the components have not been established nor is it clear how the transport of drugs is coupled to proton translocation.Tripartite pumps are likely to contribute to drug resistance of the Gram-negative pathogen Vibrio cholerae, which is the causative agent of the disease cholera. Isolates of V. cholerae have been described that are resistant to chemically diverse antibiotics, such as ampicillin, penicillin, streptomycin, nitrofurantoin, and erythromycin, as well as to toxic metals like Pb 2ϩ and Zn 2ϩ (12). A putative tripartite pump has been identified from V. cholerae comprising the inner membrane antiporter VceB, the periplasmic adaptor protein VceA, and the outer membrane channel VceC (13). Although the function of this pump in V. cholerea has not been demonstrated, its components were found to collectively complement the multidrug resistance phenotype in ...