Structural basis of tetracycline resistance by efflux pump TetL

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DESCRIPTION (provided by applicant): Antibiotic resistance is a major threat to public health and resistance to tetracyclines in particular has severely limited the use of this once efficacious, broad-spectrum family of antibiotics. A major mechanism of resistance to antibiotics like tetracyclines is mediated by membrane transporter proteins that catalyze efflux of tetracyclines in the form of tetracycline-magnesium complex. The structural basis for substrate recognition by such efflux proteins is poorly understood. The efflux pump TetL from Bacillus subtilis, with 14 transmembrane 1-helices, is a member of the family of antibiotic resistance efflux proteins (Tet) in Gram-positive bacterial pathogens, including Bacillus anthracis, Bacillus cereus, Streptococcus pneumoniae, and Staphylococcus aureus. Clostridium spp., Enterococcus spp. and Listeria spp. All Tet transporters belong to the major facilitator superfamily (MFS). The substrate binding sites of these Tet proteins are expected to be similar to homologous regions of the Gram-negative Tet proteins which have 12 transmembrane 1-helices. The proposed studies build upon recent preliminary structural work on TetL and on earlier structure-function studies using site- directed mutagenesis and in vitro assays. Specifically, our aims are: (I). To understand the molecular basis of efflux-mediated tetracycline resistance, we propose to determine the crystal structure of TetL. (II). To understand the structural basis of TetL's substrate specificity, we will characterize key tetracycline-binding residues using a combination of structural, computational, mutagenesis and biochemical approaches. (III). To investigate the role of TetL dimerization in substrate transport. PUBLIC HEALTH RELEVANCE: Antibiotic resistance is a major threat to public health. The major mechanism of resistance to antibiotics like tetracyclines is efflux mediated by membrane transporter proteins. The structural basis for substrate recognition by such efflux proteins is lacking. The efflux pump TetL from Bacillus subtilis exports tetracycline in the form of tetracycline-magnesium complex, and is responsible for this bacterium's resistance to the once widely efficacious antibiotic. A crystal structure of TetL, in combination with biochemical and biophysical studies, not only will greatly advance our understanding of the molecular mechanism of antibiotic resistance, it will also suggest new ways to modify tetracycline to reverse resistance.