Multidrug Recognition 151 The Ostensible Paradox of Multidrug Recognition

The ability of multidrug-efflux transporters to recognize scores of dissimilar organic compounds has always been considered paradoxical because of its apparent contradiction to some of the basic dogmas of biochemistry. In order to understand, at least in principle, how a protein can recognize multiple compounds, we analysed the transcriptional regulator of the Bacillus subtilis multidrug transporter Bmr. This regulator, BmrR, binds multiple dissimilar hydrophobic cations and, by activating expression of the Bmr transporter, causes their expulsion from the cell. Crystallographic analysis of the complexes of the inducer-binding domain of BmrR with some of its inducers revealed that ligands penetrate the hydrophobic core of the protein, where they form multiple van der Waals and stacking interactions with hydrophobic amino acids and an electrostatic bond with the buried glutamate. Mutational analysis of the binding site suggests that each ligand forms a unique set of atomic contacts with the protein: each tested mutation exerted disparate effects on the binding of different ligands. The example of BmrR demonstrates that a protein can bind multiple hydrophobic compounds with micromolar affinities by using only electrostatic and hydrophobic interactions. Its ligand specificity can be further broadened by the flexibility of the binding site. It appears, therefore, that the commonly expressed fascination with the relaxed substrate specificity of multidrug transporters is misdirected and originates from an almost exclusive familiarity with the more sophisticated processes of specific molecular recognition that predominate among proteins analyzed to date. The Problem of Multidrug Recognition All living cells express multidrug-efflux transporters. Each of these membrane proteins actively extrudes structurally dissimilar organic chemicals thus protecting cells from their toxic effects. The first such transporter, mammalian Pglycoprotein, has been discovered in the early seventies. To date, due to the efforts of many research groups, the list of multidrug transporters has grown to at least a hundred and includes transporters of mammalian cells, lower eukaryotes, archaebacteria, and both Gram-positive and Gram-negative eubacteria (Ambudkar et al., 1999; Kolaczkowski and Goffeau, 1997; Loo and Clarke, 1999, Nikaido, 1998; van Veen and Konings, 1997). Since many antitumor, antibacterial and antifungal agents used in clinics are substrates of multidrug transporters, these proteins create serious obstacles for chemotherapy of cancer and infectious diseases and thus are of immense clinical importance. However, it is not the clinical importance alone which makes the phenomenon of multidrug efflux interesting. The problem that has fascinated researchers since the discovery of Pglycoprotein is the apparent ability of multidrug transporters to interact functionally with moderately hydrophobic organic compounds having no obvious structural consensus. Many of these proteins have been demonstrated to promote efflux of dozens of dissimilar drugs, even hundreds in the case of the most thoroughly analyzed P-glycoprotein. It should be noted that multidrug transporters are not nonspecific but rather polyspecific: although they effect efflux of many hydrophobic compounds, not every hydrophobic drug can be effluxed by them. Furthermore, different substrates of a multidrug transporter can significantly differ in transport affinities while different multidrug transporters can differ in the spectra of transported drugs. Nevertheless, the level of substrate promiscuity of these transporters goes far beyond that of any other known transporter or enzyme. The traditional model of membrane transport postulates that a transporter first binds the transported substrate and then undergoes a conformational change forcing the bound substrate to dissociate on the other side of the membrane. However, from what we know about the binding of substrates to enzymes, such binding must involve the establishment of a specific set of atomic interactions between the enzyme residues and the substrate molecule. Not surprisingly, the very thought that a binding site of a transporter would be able to establish such interactions with scores of structurally dissimilar molecules has been perceived by many as a “violation of fundamental laws of biology and chemistry” (Roepe, 2000). In order to circumvent the seemingly intractable multidrug recognition problem, a number of researchers entertained an idea that multidrug efflux occurs indirectly, without physical contact between the transporter and its structurally diverse substrates. One of such hypotheses, most frequently expressed in the literature (see, for example, Wadkins and Roepe, 1997), postulates that multidrug transporters transport not drugs but mineral ions, thus changing the pH gradient across the membrane and dissipating membrane negative electric potential. As a result, drugs, most of which are positively charged or can be protonated to become positively charged, redistribute from cells into the surrounding medium according to the Nernst equation. In spite of the ingenuity of this and other indirect efflux ideas, they contradict a number of experimental facts. 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