Crystal structure of MtaN, a global multidrug transporter gene activator.

MtaN (Multidrug Transporter Activation, N terminus) is a constitutive, transcriptionally active 109-residue truncation mutant, which contains only the N-terminal DNA-binding and dimerization domains of MerR family member Mta. The 2.75 A resolution crystal structure of apo-MtaN reveals a winged helix-turn-helix protein with a protruding 8-turn helix (alpha5) that is involved in dimerization by the formation of an antiparallel coiled-coil. The hydrophobic core and helices alpha1 through alpha4 are structurally homologous to MerR family member BmrR bound to DNA, whereas one wing (Wing 1) is shifted. Differences between the orientation of alpha5 with respect to the core and the revolution of the antiparallel coiled-coil lead to significantly altered conformations of MtaN and BmrR dimers. These shifts result in a conformation of MtaN that appears to be incompatible with the transcription activation mechanism of BmrR and suggest that additional DNA-induced structural changes are necessary.

Efflux-mediated drug resistance in Gram-positive bacteria.

Gram-positive bacteria express numerous membrane transporters that promote the efflux of various drugs, including many antibiotics, from the cell to the outer medium. Drug transporters can be specific to a particular drug, or can have broad specificity, as in so-called multidrug transporters. This broad specificity can be a consequence of the hydrophobic nature of transported molecules, as suggested by recent structural studies of soluble multidrug-binding proteins. Although the functions of drug transporters may involve both the protection of bacteria from outside toxins and the transport of natural metabolites, their clinical importance lies largely in providing Gram-positive pathogens with resistance to macrolides, tetracyclines and fluoroquinolones. A number of agents, discovered in recent years, that inhibit drug transporters can potentially be used to overcome efflux-associated antibiotic resistance.

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.

Molecular sieve mechanism of selective release of cytoplasmic proteins by osmotically shocked Escherichia coli.

Escherichia coli cells, the outer membrane of which is permeabilized with EDTA, release a specific subset of cytoplasmic proteins upon a sudden drop in osmolarity in the surrounding medium. This subset includes EF-Tu, thioredoxin, and DnaK among other proteins, and comprises approximately 10% of the total bacterial protein content. As we demonstrate here, the same proteins are released from electroporated E. coli cells pretreated with EDTA. Although known for several decades, the phenomenon of selective release of proteins has received no satisfactory explanation. Here we show that the subset of released proteins is almost identical to the subset of proteins that are able to pass through a 100-kDa-cutoff cellulose membrane upon molecular filtration of an E. coli homogenate. This finding indicates that in osmotically shocked or electroporated bacteria, proteins are strained through a molecular sieve formed by the transiently damaged bacterial envelope. As a result, proteins of small native sizes are selectively released, whereas large proteins and large protein complexes are retained by bacterial cells.

Crystallization and preliminary X-ray diffraction studies on the DNA-binding domain of the multidrug transporter activation protein (MtaN) from Bacillus subtilis.

The N-terminal DNA-binding domain of the multidrug transporter activation protein (MtaN) was crystallized by the hanging-drop vapour-diffusion method using lithium chloride as a precipitant. The crystals are orthorhombic and belong to the space group I2(1)2(1)2(1), with unit-cell parameters a = 49.4, b = 67.8, c = 115. 0 A. Diffraction data have been collected at 100 K to 2.75 A resolution at a synchrotron-radiation source.

Recognition of multiple drugs by a single protein: a trivial solution of an old paradox.

Multidrug-efflux transporters recognize scores of structurally dissimilar toxic compounds and expel them from cells. The broad chemical specificity of these transporters challenges some of the basic dogmas of biochemistry and remains unexplained. 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 the 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 cause disordering of the surface alpha-helix and 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 glutamic residue. 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 compounds with micromolar affinities by using only electrostatic and hydrophobic interactions. Its ligand specificity can be broadened by the flexibility of the binding site. It therefore seems that the commonly expressed fascination with the broad 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 existing proteins.

A structure-based mechanism for drug binding by multidrug transporters.

Multidrug transporters bind chemically dissimilar, potentially cytotoxic compounds and remove them from the cell. How these transporters carry out either of these functions is unknown. On the basis of crystal structures of the multidrug-binding domain of the transcription activator BmrR and mutagenesis studies on the bacterial multidrug transporter MdfA, we propose a possible mechanism for the binding of cationic lipophilic drugs by multidrug transporters. The key element of this mechanism includes a conformational change in the transporter that exposes a buried charged residue in the substrate-binding pocket and allows access to this site by only those drugs that are its steric and electrostatic complements.

Mechanism of ligand recognition by BmrR, the multidrug-responding transcriptional regulator: mutational analysis of the ligand-binding site.

The Bacillus subtilis transcriptional regulator BmrR recognizes dissimilar hydrophobic cations and, in response, activates the expression of a multidrug transporter which expels them out of the cell. The structure of the inducer-binding domain of BmrR, both free and in complex with one of the inducers, tetraphenylphosphonium (TPP), revealed an unusual internal binding site, covered by an amphipathic alpha-helix. Upon unfolding of this helix, the TPP molecule penetrates into the core of the protein, where it contacts six hydrophobic residues and forms an electrostatic bond with a buried glutamate, E134 [Zheleznova et al. (1999) Cell 96, 353-362]. Here, a structure-based mutational analysis was used to understand how BmrR interacts with a wide variety of ligands. We determined the effects of alanine substitutions of each of the seven residues interacting with TPP, and mutations within the amphipathic alpha-helix, on the binding affinities of six different BmrR inducers. The E134A substitution abolished the binding of all but one inducer. Mutations of the hydrophobic residues contacting the ligand, and of the alpha-helix, had more moderate effects, often with the affinity for some inducers increasing and others decreasing as a result of the same substitution. These results indicate that each inducer forms a unique set of contacts within the binding site. The flexible geometry of this site and the lack of involvement of hydrogen bonds in ligand binding are the likely reasons for the extremely broad inducer specificity of BmrR. The similarly broad substrate specificity of multidrug transporters can be governed by the same structural principles.

Multiple novel inhibitors of the NorA multidrug transporter of Staphylococcus aureus.

The multidrug transporter NorA contributes to the resistance of Staphylococcus aureus to fluoroquinolone antibiotics by promoting their active extrusion from the cell. Previous studies with the alkaloid reserpine, the first identified inhibitor of NorA, indicate that the combination of a chemical NorA inhibitor with a fluoroquinolone could improve the efficacy of this class of antibiotics. Since reserpine is toxic to humans at the concentrations required to inhibit NorA, we sought to identify new inhibitors of NorA that may be used in a clinical setting. Screening of a chemical library yielded a number of structurally diverse inhibitors of NorA that were more potent than reserpine. The new inhibitors act in a synergistic manner with the most widely used fluoroquinolone, ciprofloxacin, by substantially increasing its activity against both NorA-overexpressing and wild-type S. aureus isolates. Furthermore, the inhibitors dramatically suppress the emergence of ciprofloxacin-resistant S. aureus upon in vitro selection with this drug. Some of these new inhibitors, or their derivatives, may prove useful for augmentation of the antibacterial activities of fluoroquinolones in the clinical setting.

Mta, a global MerR-type regulator of the Bacillus subtilis multidrug-efflux transporters.

Little is known about the natural functions of multidrug-efflux transporters expressed by bacteria. Although identified as membrane proteins actively extruding exogenous toxins from the cell, they may actually be involved in the transport of as yet unidentified specific natural substrates. The expression of two highly similar multidrug transporters of Bacillus subtilis, Bmr and Blt, is regulated by specific transcriptional activators, BmrR and BltR, respectively, which respond to different inducer molecules, thus suggesting distinct functions for the two transporters. Here, we describe an alternative mechanism of regulation, which involves a global transcriptional activator, Mta, a member of the MerR family of bacterial regulatory proteins. The individually expressed N-terminal DNA-binding domain of Mta interacts directly with the promoters of bmr and blt and induces transcription of these genes. Additionally, this domain stimulates the expression of the mta gene itself and at least one more gene, ydfK, which encodes a hypothetical membrane protein. These results and the similarity of Mta to the thiostrepton-induced protein TipA of Streptomyces lividans strongly suggest that Mta is an autogenously controlled global transcriptional regulator, whose activity is stimulated by an as yet unidentified inducer. This stimulation is mimicked by the removal of the C-terminal inducer-binding domain. The fact that both Bmr and Blt are controlled by this regulator demonstrates that some of their functions are either identical or, at least, related. Further analysis of Mta-mediated regulation may reveal the natural function of the system of multidrug transporters in B. subtilis and serve as a paradigm for similar systems in other bacteria.

Structural basis of multidrug recognition by BmrR, a transcription activator of a multidrug transporter.

Multidrug-efflux transporters demonstrate an unusual ability to recognize multiple structurally dissimilar toxins. A comparable ability to bind diverse hydrophobic cationic drugs is characteristic of the Bacillus subtilis transcription regulator BmrR, which upon drug binding activates expression of the multidrug transporter Bmr. Crystal structures of the multidrug-binding domain of BmrR (2.7 A resolution) and of its complex with the drug tetraphenylphosphonium (2.8 A resolution) revealed a drug-induced unfolding and relocation of an alpha helix, which exposes an internal drug-binding pocket. Tetraphenylphosphonium binding is mediated by stacking and van der Waals contacts with multiple hydrophobic residues of the pocket and by an electrostatic interaction between the positively charged drug and a buried glutamate residue, which is the key to cation selectivity. Similar binding principles may be used by other multidrug-binding proteins.

Paradoxical enhancement of the activity of a bacterial multidrug transporter caused by substitutions of a conserved residue.

Substitution of threonine or serine for the evolutionary conserved intramembrane proline P347 of the Bacillus subtilis multidrug transporter Bmr significantly increases the toxin-effluxing activity of Bmr without affecting its abundance in the cell. In cocultivation experiments, we demonstrate that although the mutant T347 Bmr is advantageous to cells growing in the presence of a toxin, the wild-type P347 Bmr is advantageous under the conditions of nutritional limitation. This may explain why Bmr has evolved the way it did, that is, with proline at position 347. These observations provide a basis for speculating that the evolution of Bmr has been determined by its presently unidentified natural function rather than by its ability to expel diverse toxins from the cell.

Broad ligand specificity of the transcriptional regulator of the Bacillus subtilis multidrug transporter Bmr.

The expression of the Bacillus subtilus multidrug-efflux transporter Bmr can be induced by two of its structurally dissimilar substrates, rhodamine 6G and tetraphenylphosphonium, through their direct interaction with the transcriptional regulator BmrR (Ahmed et al., J. Biol. Chem. 269, 28506). Here, by screening a chemical library, we identified four additional ligands of BmrR inducing Bmr expression at micromolar concentrations. BmrR ligands, although sharing a positive charge and moderate hydrophobicity, are structurally very diverse. At the same time, not all hydrophobic positively charged compounds, including many structural analogs of the inducers, induce Bmr expression, thus suggesting that local chemical interactions and not merely physical properties of the ligands are important for their recognition by BmrR. These results confirm that this soluble protein, like the membrane transporter it regulates, has a uniquely broad substrate specificity.

Natural functions of bacterial multidrug transporters.

Bacteria express several multidrug transporters that recognize structurally dissimilar toxic molecules and expel them from cells. These transporters may have evolved to protect bacteria from diverse environmental toxins or to transport specific physiological compounds with the ability to expel drugs being only a fortuitous side effect.

Apparent involvement of a multidrug transporter in the fluoroquinolone resistance of Streptococcus pneumoniae.

A Streptococcus pneumoniae strain selected for resistance to ethidium bromide demonstrated enhanced energy-dependent efflux of this toxic dye. Both the ethidium resistance and the ethidium efflux could be inhibited by the plant alkaloid reserpine. The ethidium-selected cells demonstrated cross-resistance to the fluoroquinolones norfloxacin and ciprofloxacin; this resistance could also be completely reversed by reserpine. Furthermore, reserpine potentiated the susceptibility of wild-type S. pneumoniae to fluoroquinolones and ethidium. The most plausible explanation for these results is that S. pneumoniae, like some other gram-positive bacteria, expresses a reserpine-sensitive multidrug transporter, which may play an important role in both intrinsic and acquired resistances of this pathogen to fluoroquinolone therapy.

Mutations affecting substrate specificity of the Bacillus subtilis multidrug transporter Bmr.

The Bacillus subtilis multidrug transporter Bmr, a member of the major facilitator superfamily of transporters, causes the efflux of a number of structurally unrelated toxic compounds from cells. We have shown previously that the activity of Bmr can be inhibited by the plant alkaloid reserpine. Here we demonstrate that various substitutions of residues Phe143 and Phe306 of Bmr not only reduce its sensitivity to reserpine inhibition but also significantly change its substrate specificity. Cross-resistance profiles of bacteria expressing mutant forms of the transporter differ from each other and from the cross-resistance profile of cells expressing wild-type Bmr. This result strongly suggests that Bmr interacts with its transported drugs directly, with residues Phe143 and Phe306 likely to be involved in substrate recognition.

Efflux of the natural polyamine spermidine facilitated by the Bacillus subtilis multidrug transporter Blt.

Multidrug transporters pump structurally dissimilar toxic molecules out of cells. It is not known, however, if detoxification is the primary physiological function of these transporters. The chromosomal organization of the gene encoding the Bacillus subtilis multidrug transporter Blt suggests a specific function for this protein; it forms a single operon with another gene, bltD, whose protein product is identified here as a spermine/spermidine acetyltransferase, an enzyme catalyzing a key step in spermidine degradation. Overexpression of the Blt transporter in B. subtilis leads not only to the multidrug-resistance phenotype but also to the efflux of large amounts of spermidine into the medium; this efflux is supressed by an inhibitor of Blt, reserpine. Taken together, these results strongly suggest that the natural function of the Blt transporter is the efflux of spermidine, whereas multiple drugs may be recognized by Blt merely opportunistically.

Preliminary structural studies on the multi-ligand-binding domain of the transcription activator, BmrR, from Bacillus subtilis.

In the bacterium Bacillus subtilis, the DNA-binding regulatory protein, BmrR, activates transcription from the multidrug transporter gene, bmr, after binding either rhodamine or tetraphenylphosphonium. These two compounds, which have no structural similarity, are also substrates for the bacterial multidrug transporter. BmrR belongs to the MerR family of transcription activators but differs from the other family members in its ability to bind unrelated small molecule activators. As an initial step in the elucidation of the mechanism by which BmrR recognizes rhodamine and tetraphenylphosphonium and activates transcription, we have crystallized the 144-amino acid-residue carboxy terminal dimerization/ligand-binding domain of the BmrR, named the BRC (BmrR C-terminus). Tetragonal crystals of ligand-free BRC take the space group P4(1)2(1)2, or its enantiomorph P4(3)2(1)2, with unit cell dimensions a = b = 76.3 A, c = 96.0 A, alpha = beta = gamma = 90 degrees. Diffraction is observed to at least 2.7 A resolution at room temperature. In addition, we determined the secondary structure content of ligand-free and rhodamine-bound BRC by circular dichroism. In the ligand-free form, BRC has considerable beta-sheet content (41%) and little alpha-helix structure (13%). After BRC binds rhodamine, its beta-sheet content increases to 47% while the alpha-helix structure decreases to 11%. The structure of BRC will provide insight not only into its multidrug recognition mechanism but could as well aid in the elucidation of the recognition and efflux mechanisms of Bmr and other bacterial multidrug transporters.

The drug-binding activity of the multidrug-responding transcriptional regulator BmrR resides in its C-terminal domain.

Rhodamine and tetraphenylphosphonium, the substrates of the Bacillus subtilis multidrug efflux transporter Bmr, induce the expression of Bmr through direct interaction with its transcriptional activator BmrR. Here we show that the C-terminal domain of BmrR, expressed individually, binds both these compounds and therefore can be used as a model for molecular analysis of the phenomenon of multidrug recognition.