ABC transporters constitute a ubiquitous superfamily of membrane pumps containing an evolutionary conserved ATP-binding cassette domain. They mediate energy-driven efflux of a great variety of substrates, including amino acids, ions, sugars, as well as synthetic and naturally occurring xenobiotics or toxins. A subset of yeast pumps plays a major role in the so-called pleiotropic drug resistance (PDR) phenomenon, where overexpressed ABC transporters such as S. cerevisiae Pdr5 or Snq2 confer resistance to a vast variety of drugs. This phenomenon resembles anti-tumor resistance in cancer cells, which is mediated by proteins such as P-glycoprotein, BCRP or MRP. However, very little is known about the molecular mechanism of function of yeast PDR transporters, mainly due to a lack of structural information. To gain insight into the principles underlying transport through yeast ABC proteins, we have initiated a structural modelling approach yielding improved homology models of yeast PDR transporters. The models enabled the prediction of the structural organization of putative transmembrane regions and their connection to the dimeric nucleotide binding domains (NBDs). Remarkably, we have identified residues in the transmission interface, which are critical for both function and formation of the NBD dimer, which is essential for ATP hydrolysis and substrate translocation. A mutational analysis of the Pdr5 and Snq2 transporters identifies further residues critical for ATP-binding, hydrolysis as well as drug substrate specificity, and defines the molecular basis of substrate specificity in the context the interplay of NBD dimerization and ATP consumption. We will present modeling data along with mutational validation, and discuss how homology modeling can facilitate genetic structure-function analysis to reveal possible mechanisms of yeast PDR transporters.
This work was supported through the SFB035-P20 project from the Austrian Science Foundation to KK.