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

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 that
substrates 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

The Major Facilitator Superfamily multidrug transporter LmrP can mediate the efflux of 22 clinically-used antibiotics and other cytotoxic agents from bacterial cells. The protein contains 3 catalytic carboxylates on the surface of an internal substrate binding chamber. These carboxylates contribute to proton/substrate antiport. Although residues in the catalytic machinery in membrane transporters usually have very precise relative locations to enable optimal interactions, we found in our very recent research on LmrP that the location of the catalytic carboxylates in the internal binding chamber is relatively flexible. In this proposal, we will test the hypothesis that the catalytic activity of these carboxylates does not require interactions with specific neighbouring residues, but that the interactions of LmrP with protons and substrates during transport are dependent on changes in the hydrophobicity of the cavity surface.

Who might benefit from this research?

Academia: The outcomes of this project have the potential to produce long-term impact in our understanding of the mechanisms of membrane transporters and drug efflux, mechanisms of drug resistance, and alternative physiological roles of drug efflux pumps.

Business/Industry: In the longer term, this project may lead to the generation of novel drugs in infectious diseases and diseases in mammals, and of novel inhibitors that can rejuvenate existing antibiotics for which drug resistance has developed. The results of our studies might offer new directions to pharmaceutical and biotech companies in their drug development programmes.

General Public: Infectious diseases are re-emerging due to the development of drug resistance and the lack of development of new antibiotics at a significant rate. This type of research might ultimately enable us to control this crisis.

Schools: The development of antibiotic resistance is partly related to the extensive and sometimes improper use of antibiotics in the veterinary and human medicine. Lay talks in primary and secondary schools are an important way to increase public awareness of the problems and solutions.

How might they benefit from this research?

The PI and postdoctoral worker will engage in academic dissemination through peer reviewed publications and talks at conferences and workshops, whilst wider public dissemination is more likely to be undertaken by the PI. Our project web site will be mounted by IT staff at Cambridge University and thereafter populated by material provided by the staff on the project. With so many pharmaceutical companies in the vicinity, Cambridge offers an excellent environment to communicate science and to investigate the translational opportunities it creates. This is especially facilitated by Cambridge Enterprise, who assist staff to commercialise their expertise and ideas, and by new initiatives such as the Milner Therapeutics Institute and Consortium, Cambridge New Therapeutics Forum, and Cambridge Academy of Therapeutic Sciences. Dissemination to society in general will be effected via community events and publicly accessible websites with lay summaries of our findings, via social media (Twitter), and via school outreach activities. We will engage the print and broadcast media in collaboration with the Office of Communications of Cambridge University and BBSRC Media Office with articles written by them and ourselves.