Transport mechanism of a multidrug transporter from Vibrio cholerae

We live in a time in which drug resistance has become a global threat to health care and in which concerns about the lack of effective antimicrobial drugs are communicated with increasing frequency. One very powerful mechanism of drug resistance in microorganisms is based on the transport of the drugs out of the cell by pump proteins that can recognise an extraordinarily broad range of antibiotics and disinfectants. In this project, we ask a few simple questions about these proteins: how do they bind substrates and how do they use cell's energy to pump? Answers to these questions will allow us to generate inhibitors of these pump proteins and new antibiotics that can bypass drug pumping.

In the past decade, crystal structures have been determined for multidrug transporters belonging to four families. However, the structure of members of the final family, that of multidrug and toxic compound extrusion (MATE) proteins, has only recently been determined. MATE transporters may be last in the list of crystallised multidrug transporters, but they are certainly not the least. They are present in all kingdoms of life and, in addition to their role in microbial drug resistance, they are essential for the detoxification of metabolic products in plants and for the excretion of toxins by the liver and kidney in mammals. Over the past decade, crystallographic evidence has been obtained supporting the general concept of alternating access for a variety of membrane transporters, demonstrating that this mechanism has been evolutionarily conserved. However, the structural details and biochemical reactions underlying the necessary conformational changes in multidrug transporters are only partly understood. This is particularly true for MATE transporters, for which very few studies on structure-function relationships have been published to date. In this project we will study the mechanism by which the MATE transporter NorM from Vibrio cholerae mediates antiport between coupling ions and substrates. The involvement of Na(+) in the transport reaction of NorM offers a unique opportunity compared to 'conventional' H(+)/drug antiporters to study the antiport reaction from the perspective of the transported substrate AND coupling ion, as Na(+) can be detected by using radioactive 22Na+. We will identify catalytic residues in NorM that are important for interactions with Na(+) and substrates, and we will challenge NorM's mechanism by relocating these residues to various positions in binding pockets. In this way, we will unravel details of the mechanism of transport and identify possible routes by which multidrug transporters can be inhibited.

The development and spread of multidrug resistance in microorganisms impairs the chemotherapeutic treatment of microbial infections in humans and animals. Our aim is to study the fundamental mechanisms of one member of the MATE (the multidrug and toxic compound extrusion) family that can confer drug resistance on the cell by mediating the transport of multiple drugs away from their intracellular targets. The activities and mechanisms established will be relevant for other bacterial, plant and mammalian members in this family as well as multidrug transporters from other protein families in eubacterial and eukaryotic cells.

Our research has clear social and economic impact as increased knowledge of the biochemical mechanism of multidrug transporters will generate new avenues for modulation of their activity in a clinical setting. This will offer considerable potential for modulator development programs in pharmaceutical industries and commercial institutes. These modulators can rejuvenate existing antimicrobials when target cells are resistant. This research might also lead to the development of new antibiotics that can bypass drug pumping.