Mechanisms of substrate transport by the major facilitator superfamily multidrug transporter LmrP

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Drug resistance has become a global threat to health care. Concerns about the lack of effective antimicrobial drugs are communicated in the media with increased frequency. Pathogenic microorganisms have evolved several ways by which they can resist the toxic effects of antibiotics and disinfectants. One very powerful mechanism is based on the transport (efflux) of antimicrobial agents from the cell's interior by membrane transport proteins, also referred to as drug efflux pumps. Due to the activity of these efflux pumps, the drugs only reach subtoxic concentrations inside the cell, making the antimicrobial agents ineffective in the treatment of infectious diseases. There are many medically relevant and academically interesting questions that need to be answered about drug efflux pumps. One question concerns the ability of multidrug efflux pumps to recognize a very wide range of antimicrobial agents. As efflux pumps need to perform osmotic work during drug efflux,another question concerns the ways by which the cell can provide metabolic energy for this efflux activity. One form of metabolic energy is based on the existence of ion gradients across the plasma membrane where transport proteins reside. The drug efflux pumps contain a pathway along which these ions can move down their gradients into the cell, but this movement can only occur if it is coupled to the movement of drugs in the opposite direction (= efflux). Thus, these drug efflux pumps can couple to metabolic energy to drug efflux by mediating a drug-proton antiport reaction. This project will focus on a well-studied drug-proton antiporter termed LmrP from the Gram-positive bacterium Lactococcus lactis. LmrP is known to contain pockets in which substrates and protons can bind. In this project, we will test the hypothesis that the surface properties in these pockets change from attracting water to repelling water at different stages of the transport reaction so thatsubstrates can bind while protons are released, and vice versa. Several biochemical and biophysical approaches will be used to measure surface properties of the pockets while LmrP is kept in a state where the binding pockets are exposed to the interior of the cell (inward-facing state) or exterior of the cell (outward-facing state). This research will increase our understanding of the mechanisms by which LmrP and other drug efflux pumps work. The results will allow us in the future to generate new drugs that block or bypass the efflux activity of membrane transporters