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Multi-drug resistance

Multi-drug resistance

Multidrug resistance (MDR) in cancer cells is one of the major challenges, which have to be overcome in order to improve chemotherapy and fight the disease. Several mechanisms of multidrug resistance, such as decreased uptake of drugs, alteration of the drugs, increased DNA repair activity and increased efflux of cytotoxic compounds by ATP binding cassette (ABC) transporters, combine to obstract an efficient treatment.

Likewise, in pathogenic fungi such as Candida albicans, which is the main cause of opportunistic mycosis, multidrug resistance against a broad variety of antifungals like azoles expounds the problem of an efficient treatment. The plant and fungal homologue of MDR is called pleiotropic drug resistance (PDR). The PDR network acts as a first line of defense. It consists of several ABC transporters and together with the secondary transporters, they are located in the plasma membrane and regulated by transcription factors such as Pdr1 and Pdr3 (Figure 1), providing a broad spectrum of resistance against a variety of toxic compounds. One of the most well studied PDR proteins is the ATP-binding cassette (ABC) transporter Pdr5 from Saccharomyces cerevisiae, which was first discovered in 1990 as a DNA sequence conferring resistance towards cytotoxic compounds such as azoles, ionophores, antibiotics, detergents, dyes and many others and later classified as an MDR ABC transporter. Pdr5 and its homologue from C. albicans Cdr1 show unique characteristics that might be transferable to mammalian ABC transporters.

We use a Saccharomyces cerevisiae strain that bears a mutation in Pdr1 to overexpress Pdr5 constitutively in high levels for in vivo and in vitro studies. This strain can also be used for the expression of other membrane proteins as demonstrated by us and many other laboratories. Our aim is to understand the capability of a single membrane protein to bind and transport an extremely broad range of structurally unrelated compound across the plasma membrane of yeast on a molecular level. For this purpose we established several biochemically and biophysically methods e.g. fluorescence based transport, ATPase activity and drug resistance assay to evaluate functionality.

One has to stress that Pdr5 is a so-called ‘asymmetric’ or ‘degenerated’ ABC transporters. In general, an ATP or nucleotide binding site (NBS) is formed by the two NBDs of the transporter. This composite binding site is build up by the Walker A and B motifs and the D- and H-loops of one NBD and the C-loop of the opposing NBD. In the case of Pdr5, all conserved amino acids of one NBS are exchanged against non-functional amino acids rendering this NBS inactive with respect to ATP hydrolysis.


Mutational studies revealed that a mutation within the NBD changed the specificity of the substrate spectrum while ATPase activity was preserved. The H1068A mutation abolished rhodamine transport in vivo and in vitro, while leaving the transport of other substrates unaffected. By contrast to mammalian P-glycoprotein (P-gp), the ATPase activity of yeast Pdr5 is not stimulated by the addition of substrates, indicating that Pdr5 is an uncoupled ABC transporter that constantly hydrolyses ATP to ensure active substrate transport. Taken together, our data provide new and important insights into the molecular mechanism of Pdr5, and suggest that not solely the transmembrane domains dictate substrate selection.

These findings let to the proposal of the ‘kinetic selection’ model to account for this molecular communication. We proposed that both the kinetics of transporter-substrate and transporter-nucleotide interactions affect the substrate selectivity of the MDR transporter Pdr5, and perhaps also the basal mode of P-gp and other ABC transporters. For the sake of simplicity, one can imagine a very simple situation: A MDR transporter like Pdr5 with only basal ATPase activity switches between its inward-facing and outward-facing conformation paralleling the rate of ATP-binding and -hydrolysis. Clearly, a prerequisite for such a behavior is a strict mechanical coupling between NBDs and TMDs. Let us assume further that two different drugs have identical affinities to the inward-facing substrate binding site, but do not compete with each other for binding. One drug, called FAST displays fast on kinetics and fast off kinetics, while the other drug, called SLOW, has slow on and off kinetics. At time-point zero, the transporter switches to its drug-accepting, inward-facing conformation and both drugs start to equilibrate with the substrate binding site. Thus, it becomes obvious that the rate at which the transporter switches back to the outward-facing conformation determines which of the two substrates is transported more efficient, even though both compounds have the same affinity. If the transporter remains only for a short period of time in the inward facing conformation, the FAST substrate is transported several-fold more efficiently than the SLOW substrate. By contrast, if the transporter switches much slower, both drugs will be transported with virtually identical efficiencies. This simplistic model does not yet include aspects of multidrug binding and transport, such as binding of multiple competing drugs, drug release and so forth.

As pointed out above, Pdr5 is a degenerated, asymmetric ABC transporter. This raised the question about the function of this particular site. In other words, would Pdr5 gain more efficiency and / or activity if the catalytic relevant amino acids are re-introduced? As shown in Figure 5, formation of three or more conserved motifs in the degenerated sites completely abolished ATPase and substrate transport activity. This clearly points towards an essential function of the degenerated site.

Research on Pdr5 was hampered for more than 25 years by the lack of a functional in vitro system. All detergents that were used to solubilize Pdr5 inactivated the transporter with respect to ATPase and transport activity. We could only recently identify PCC as the detergent of choice rendering Pdr5 in the detergent-solubilized state in a functional form.

This now opens the way to study Pdr5 under defined and tunable conditions to finally understand how a single membrane protein can recognize and transport a myriad of structurally unrelated compounds.

Relevant Publications:

Wagner, M., S.H.J. Smits & L. Schmitt, (2019) In vitro NTPase activity of highly purified Pdr5, a major yeast ABC multidrug transporter. Sci Rep 9: 7761.

Wagner, M., K. Doehl & L. Schmitt, (2016) Transmitting the energy: Interdomain cross-talk in Pdr5. Biol Chem.

Gupta, R.P., P. Kueppers & L. Schmitt, (2014) New examples of membrane protein expression and purification using the yeast based Pdr1-3 expression strategy. J Biotechnol 191: 158-164.

Gupta, R.P., P. Kueppers, N. Hanekop & L. Schmitt, (2014) Generating symmetry in the asymmetric ABC transporter Pdr5 from Saccharomyces cerevisiae. J Biol Chem 289: 15272- 15279.

Kueppers, P., R.P. Gupta, J. Stindt, S.H. Smits & L. Schmitt, (2013) Functional impact of a single mutation within the transmembrane domain of the multidrug ABC transporter Pdr5. Biochemistry 52: 2184-2195.

Gupta, R.P., P. Kueppers, L. Schmitt & R. Ernst, (2011) The multidrug transporter Pdr5: a molecular diode? Biological chemistry 392: 53-60.

Ernst, R., P. Kueppers, J. Stindt, K. Kuchler & L. Schmitt, (2010) Multidrug efflux pumps: Substrate selection in ATP-binding cassette multidrug efflux pumps - first come, first served? FEBS J 277: 540-549.

Ernst, R., P. Kueppers, C.M. Klein, T. Schwarzmueller, K. Kuchler & L. Schmitt, (2008) A mutation of the H-loop selectively affects rhodamine transport by the yeast multidrug ABC transporter Pdr5. Proc Natl Acad Sci U S A 105: 5069-5074.

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