A Single Acidic Residue Can Guide Binding Site Selection but Does Not Govern QacR Cationic-Drug Affinity

Structures of the multidrug-binding repressor protein QacR with monovalent and bivalent cationic drugs revealed that the carboxylate side-chains of E90 and E120 were proximal to the positively charged nitrogens of the ligands ethidium, malachite green and rhodamine 6G, and therefore may contribute to drug neutralization and binding affinity. Here, we report structural, biochemical and in vivo effects of substituting these glutamate residues. Unexpectedly, substitutions had little impact on ligand affinity or in vivo induction capabilities. Structures of QacR(E90Q) and QacR(E120Q) with ethidium or malachite green took similar global conformations that differed significantly from all previously described QacR-drug complexes but still prohibited binding to cognate DNA. Strikingly, the QacR(E90Q)-rhodamine 6G complex revealed two mutually exclusive rhodamine 6G binding sites. Despite multiple structural changes, all drug binding was essentially isoenergetic. Thus, these data strongly suggest that rather than contributing significantly to ligand binding affinity, the role of acidic residues lining the QacR multidrug-binding pocket is primarily to attract and guide cationic drugs to the “best available” positions within the pocket that elicit QacR induction

Regulation of Bacterial Drug Export Systems

The active transport of toxic compounds by membrane-bound efflux proteins is becoming an increasingly frequent mechanism by which cells exhibit resistance to therapeutic drugs. This review examines the regulation of bacterial drug efflux systems, which occurs primarily at the level of transcription. Investigations into these regulatory networks have yielded a substantial volume of information that has either not been forthcoming from or complements that obtained by analysis of the transport proteins themselves. Several local regulatory proteins, including the activator BmrR from Bacillus subtilis and the repressors QacR from Staphylococcus aureus and TetR and EmrR from Escherichia coli, have been shown to mediate increases in the expression of drug efflux genes by directly sensing the presence of the toxic substrates exported by their cognate pump. This ability to bind transporter substrates has permitted detailed structural information to be gathered on protein-antimicrobial agent-ligand interactions. In addition, bacterial multidrug efflux determinants are frequently controlled at a global level and may belong to stress response regulons such as E. coli mar, expression of which is controlled by the MarA and MarR proteins. However, many regulatory systems are ill-adapted for detecting the presence of toxic pump substrates and instead are likely to respond to alternative signals related to unidentified physiological roles of the transporter. Hence, in a number of important pathogens, regulatory mutations that result in drug transporter overexpression and concomitant elevated antimicrobial resistance are often observed

Transmembrane Helix 12 of the Staphylococcus aureus Multidrug Transporter QacA Lines the Bivalent Cationic Drug Binding Pocket▿

An acidic residue in transmembrane segment (TMS) 10 is important for recognition of bivalent cationic substrates by the QacA multidrug transporter. Remarkably, an acidic residue in TMS 12 compensated for the absence of such a residue in TMS 10, suggesting that TMS 12 is a component of the bivalent cation-binding region

Replication Control of Staphylococcal Multiresistance Plasmid pSK41: an Antisense RNA Mediates Dual-Level Regulation of Rep Expression

Replication of staphylococcal multiresistance plasmid pSK41 is negatively regulated by the antisense transcript RNAI. pSK41 minireplicons bearing rnaI promoter (P(rnaI)) mutations exhibited dramatic increases in copy number, approximately 40-fold higher than the copy number for the wild-type replicon. The effects of RNAI mutations on expression of the replication initiator protein (Rep) were evaluated using transcriptional and translational fusions between the rep control region and the cat reporter gene. The results suggested that when P(rnaI) is disrupted, the amount of rep mRNA increases and it becomes derepressed for translation. These effects were reversed when RNAI was provided in trans, demonstrating that it is responsible for significant negative regulation at two levels, with the greatest repression exerted on rep translation initiation. Mutagenesis provided no evidence for RNAI-mediated transcriptional attenuation as a basis for the observed reduction in rep message associated with expression of RNAI. However, RNA secondary-structure predictions and supporting mutagenesis data suggest a novel mechanism for RNAI-mediated repression of rep translation initiation, where RNAI binding promotes a steric transition in the rep mRNA leader to an alternative thermodynamically stable stem-loop structure that sequesters the rep translation initiation region, thereby preventing translation

Analysis of Tryptophan Residues in the Staphylococcal Multidrug Transporter QacA Reveals Long-Distance Functional Associations of Residues on Opposite Sides of the Membrane▿

Tryptophan residues can possess a multitude of functions within a multidrug transport protein, e.g., mediating interactions with substrates or distal parts of the protein, or fulfilling a structural requirement, such as guiding the depth of membrane insertion. In this study, the nine tryptophan residues of the staphylococcal QacA multidrug efflux protein were individually mutated to alanine and phenylalanine, and the functional consequences of these changes were determined. Phenylalanine substitutions for each tryptophan residue were functionally tolerated. However, alanine modifications revealed an important functional role for three tryptophan residues, W58, W149, and W173, each of which is well conserved among QacA-related transport proteins in the major facilitator superfamily. The most functionally compromising mutation, an alanine substitution for W58, likely to be located at the extracellular interface of transmembrane segment 2, abolished all detectable QacA-mediated resistance and transport function. Second-site suppressor analyses identified several mutations that rescued the function of the W58A QacA mutant. Remarkably, all of these suppressor mutations were shown to be located in cytoplasmic loops between transmembrane helices 2 and 3 or 12 and 13, demonstrating novel functional associations between amino acid positions on opposite sides of the membrane and in distal N- and C-terminal regions of the QacA protein