At a hospital, a breast cancer patient is fighting a battle against cancer because the tenaciously dividing cancer cells in her body are spitting out anticancer drugs back into her blood stream, preventing them from attacking her tumour. In another setting, a pathogenic bacterium in our gut is collecting nutrients required for maintenance and growth of the cell. But within the cocktail trapped in the gut lies a mixture of chemical toxins, some natural (such bile acids which can permeabilize and dissolve the microbial cells membrane) and some man-made (such as antibiotics). Yet as soon as the poisons find their way into the interior of the bug, they are jettisoned out of the cell by the same molecular gadgetry as those present in the tumour cells of the cancer patient. Nature’s common currency is a protein called a ‘multidrug’ transporter. Embedded within a cell’s plasma membrane, this protein protects a cell by ejecting a variety of molecules - in many cases, toxins - on contact. The cell might be a bacterium, in which case the toxins are antibiotics. With cancer cells, the toxins are chemotherapeutic drugs.
We live in an era when drug resistance has spread at an alarming rate. The World Health Organization states that antibiotic resistance is one the three greatest threats to human health. There are so many interesting questions about multidrug transporters: How do they recognize so many different drugs? Can they be inhibited so that existing drugs can still be used in treatment of diseases? Can drugs be developed that bypass multidrug transporters?
In this laboratory, we work on the answers to these questions. Our methods are based on biochemistry, cell biology and biophysics, and include techniques such as DNA cloning, gene expression, molecular modelling, site-directed mutagenesis, protein purification and reconstitution, and drug binding and transport using radiolabelled and fluorescent substrates. We study multidrug transporters that are members of different families of membrane proteins, that originate from various pathogenic microorganisms and human tumour cells, and that are structurally different but have a conserved function. It is an exciting research group to be part of.
The following sections give an overview of some of our ongoing projects.
ABC (ATP-binding cassette) transporters comprise one of the largest families of structurally related membrane proteins, and are thought to mediate substrate transport in a reaction driven by ATP-binding and hydrolysis. One of the human transporter that we study is the Breast Cancer Resistance Protein (ABCG2). A variety of techniques is used in this laboratory to investigate the basic mechanisms by which the Breast Cancer Resistance Protein is able to recognize and transport multiple drugs. Our recent work on ABCG2 revealed for the first time the presence of a steroid hormone-binding element at the external face of the transporter (Fig. 1). As this element modulates ABCG2 activity, its further characterization might provide an opportunity for the development of new therapeutic ligands for ABCG2 in a clinical setting.
Certain human ABC transporters are well conserved in bacteria. The ABC multidrug transporter LmrA from Lactococcus lactis is a homologue of the human multidrug resistance P-glycoprotein (ABCB1), another major drug pump involved in drug resistance of human cancer cells. LmrA and ABCB1 have very similar specificities for chemotherapeutic drugs and modulators. LmrA can even functionally substitute for ABCB1 in human lung fibroblast cells. Although ABC transporters are generally thought to be unidirectional extrusion systems, we discovered that LmrA contains a reversible pathway for drugs. Moreover, we demonstrated that if the membrane-embedded, drug-binding part of the transporter is decoupled from the cytosolic part that breaks down ATP to provide metabolic energy (the 'engine' of the transporter) then LmrA acts to import drugs rather than export them. Interestingly, the membrane-embedded part of LmrA was shown to mediate a protonmotive force-dependent transport reaction, which might suggest a functional link between this ABC transporter and ion-coupled multidrug transporters (see below). Our findings open up the possibility to use LmrA-like transporters for drug delivery in cells.
This laboratory studies the molecular properties of the LmrA and ABCB1 homologue Sav1866 from the Gram-positive pathogen Staphylococcus aureus, which is responsible for outbreaks of MRSA (methicilline-resistant staphylococcus aureus) in hospitals and clinics. As the structure of the staphylococcal ABC transporter has recently been resolved by X-ray crystallography (Fig. 2), we can examine detailed structure-function relationships underlying drug binding and translocation using structural biology as a basis.
- Figure 2. Ribbon representation of the crystallized homodimeric ABC transporter Sav1866 from Staphylococcus aureus (side view) showing 2 x 6 transmembrane helices and 2 intracellular nucleotide-binding domains. Out and In refer to the outside and inside of the plasma membrane, respectively.
One of the interesting issues regarding multidrug transporters is their role in cell physiology. Multidrug transporters might have a purely protective function, but they could also be involved in the transport of substrates (e.g. lipids and lipid soluble metabolites) that share physico-chemical properties with drugs. The ABC transporter MsbA is expressed in the plasma membrane of many Gram-negative bacteria including pathogens such as Salmonella typhimurium and Vibrio cholera. MsbA is thought to interact with lipid A, which is the lipid anchor of lipopolysaccharides in the outer membrane, and which is a potent activator of innate immunity in mammals via Toll-like receptors. In a recent study on the kinetics of drug and lipid-A binding/transport by MsbA from Escherichia coli, we found that both types of substrates can interact with MsbA with a comparable affinity, and that lipid-A binding can inhibit drug binding. Hence, the lipid environment of a multidrug transporter can be an important factor its drug selectivity.
This laboratory also studies the molecular properties of ion-coupled multidrug transporters, which mediate drug efflux in a reaction driven by the transmembrane electrochemical gradient of protons and/or sodium ions. Many resistance plasmids in pathogenic bacteria contain genes encoding this type of transporter. Ion-coupled multidrug transporters can be classified into various families including the Major Facilitator Super (MFS) family and the Resistance-Nodulation-cell Division (RND) family.
LmrP is a MFS multidrug transporter from L. lactis. Previous work has indicated that ethidium, a monovalent cationic substrate, is exported by LmrP by electrogenic antiport with two (or more) protons. This observation raised the question whether these protons are translocated sequentially along the same pathway, or through different routes. To address this question we constructed a 3-D homology model of LmrP based on the high-resolution structure of the glycerol-3P/Pi antiporter GlpT from Escherichia coli. Interestingly, LmrP is predicted to contain an internal cavity formed at the interface between the two halves of the transporter (each containing 6 transmembrane helices). On the surface of this cavity lie two clusters of polar, aromatic and carboxylate residues with potentially important function in proton shuttling and interactions with cationic drugs (Fig. 3). We tested by mutagenesis the possible proton conduction points suggested by this model, and found that there are two proton conduction pathways at play in the drug transport mechanism, of which one might be dedicated to proton transport and the other to drug transport. Proton translocation pathways in LmrP are subject of further research.
- Figure 3. Internal cavity in the proton motive force-dependent MFS member LmrP. (A) The predicted LmrP structure represents an inward facing conformation, exposing an internal cavity to the inner membrane leaflet. Out and In refer to the outside and inside of the plasma membrane, respectively. (B) Residues lining the surface of the internal cavity. Carboxylates (in red) exposed at this surface are organized into two distinct clusters containing additional neutral-polar (in green) and aromatic residues (in blue). The role of these clusters in proton and drug transport by LmrP are being investigated.
RND multidrug transporters are being studied in detail in Gram-negative bacteria, where they interact with a membrane fusion protein and outer membrane porin to enable transport from the periplasm across the outer membrane. However, relatively little is known about the mechanism of their homologues in Gram-positive bacteria (such as Mmpl7 in the pathogen Mycobacterium tuberculosis), which lack a periplasm and outer membrane. In spite of significant differences in the primary structure of Mmpl and LmrP, both systems have overlapping drug specificities. The molecular basis of this similarity is not well understood, but the determinants of drug specificity must somehow be contained in the structure of the proteins. We aim to unravel the molecular mechanism of multidrug transport by Mmpl.