Multidrug resistance (MDR) transporters belonging to either the ATP-Binding Cassette (ABC) or Major Facilitator Superfamily (MFS) groups are major determinants of clinical drug resistance in fungi. ...The overproduction of these proteins enables the extrusion of incoming drugs at rates that prevent lethal effects. The promiscuity of these proteins is intriguing because they export a wide range of structurally unrelated molecules. Research in the last two decades has used multiple approaches to dissect the molecular basis of the polyspecificity of multidrug transporters. With large numbers of drug transporters potentially involved in clinical drug resistance in pathogenic yeasts, this review focuses on the drug transporters of the important pathogen
. This organism harbors many such proteins, several of which have been shown to actively export antifungal drugs. Of these, the ABC protein
Cdr1 and the MFS protein
Mdr1 are the two most prominent and have thus been subjected to intense site-directed mutagenesis and suppressor genetics-based analysis. Numerous results point to a common theme underlying the strategy of promiscuity adopted by both
Cdr1 and
Mdr1. This review summarizes the body of research that has provided insight into how multidrug transporters function and deliver their remarkable polyspecificity.
ATP-binding cassette (ABC) superfamily comprises a large group of ubiquitous transmembrane proteins that play a crucial role in transporting a diverse spectrum of substrates across cellular ...membranes. They participate in a wide array of physiological and pathological processes including nutrient uptake, antigen presentation, toxin elimination, and drug resistance in cancer and microbial cells. ABC transporters couple ATP binding and hydrolysis to undergo conformational changes allowing substrate translocation. Within this superfamily, a set of ABC transporters has lost the capacity to hydrolyze ATP at one of their nucleotide-binding sites (NBS), called the non-catalytic NBS, whose importance became evident with extensive biochemistry carried out on yeast pleiotropic drug resistance (PDR) transporters. Recent single-particle cryogenic electron microscopy (cryo-EM) advances have further catapulted our understanding of the architecture of these pumps. We provide here a comprehensive overview of the structural and functional aspects of catalytically asymmetric ABC pumps with an emphasis on the PDR subfamily. Furthermore, given the increasing evidence of efflux-mediated antifungal resistance in clinical settings, we also discuss potential grounds to explore PDR transporters as therapeutic targets.
Candida albicans and C. glabrata express exporters of the ATP-binding cassette (ABC) superfamily and address them to their plasma membrane to expel azole antifungals, which cancels out their action ...and allows the yeast to become multidrug resistant (MDR). In a way to understand this mechanism of defense, we describe the purification and characterization of Cdr1, the membrane ABC exporter mainly responsible for such phenotype in both species. Cdr1 proteins were functionally expressed in the baker yeast, tagged at their C-terminal end with either a His-tag for the glabrata version, cgCdr1-His, or a green fluorescent protein (GFP) preceded by a proteolytic cleavage site for the albicans version, caCdr1-P-GFP. A membrane Cdr1-enriched fraction was then prepared to assay several detergents and stabilizers, probing their level of extraction and the ATPase activity of the proteins as a functional marker. Immobilized metal-affinity and size-exclusion chromatographies (IMAC, SEC) were then carried out to isolate homogenous samples. Overall, our data show that although topologically and phylogenetically close, both proteins display quite distinct behaviors during the extraction and purification steps, and qualify cgCdr1 as a good candidate to characterize this type of proteins for developing future inhibitors of their azole antifungal efflux activity.
•This paper describes the purification of a clinically relevant drug transporter of pathogenic fungi•It includes a detailed methodology on working with membrane proteins, including detergent screening, purification and characterization•The use of fluorescence quenching allows to measure the affinity of the protein for its ligands•NanoDifferential Scanning Fluorimetry showed a differential stabilization of the protein, thus making it an interesting tool to screen for future inhibitors•The purification of an ATPase active protein paves the way for structural studies of these transporters.
ATP-binding cassette (ABC) transporters couple ATP binding and hydrolysis to the translocation of allocrites across membranes. Two shared nucleotide-binding sites (NBS) participate in this cycle. In ...asymmetric ABC pumps, only one of them hydrolyzes ATP, and the functional role of the other remains unclear. Using a drug-based selection strategy on the transport-deficient mutant L529A in the transmembrane domain of the Candida albicans pump Cdr1p; we identified a spontaneous secondary mutation restoring drug-translocation. The compensatory mutation Q1005H was mapped 60 Å away, precisely in the ABC signature sequence of the non-hydrolytic NBS. The same was observed in the homolog Cdr2p. Both the mutant and suppressor proteins remained ATPase active, but remarkably, the single Q1005H mutant displayed a two-fold reduced ATPase activity and a two-fold increased drug-resistance as compared to the wild-type protein, pointing at a direct control of the non-hydrolytic NBS in substrate-translocation through ATP binding in asymmetric ABC pumps.
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•Function of the non-catalytic NBS in asymmetric ABC transporters is uncertain.•Q1005H mutation acts as a suppressor for debilitating mutant, L529A in TMD of Cdr1p.•Q1005H is part of the ABC signature sequence in the non-catalytic NBS.•Q1005H mutation in the suppressor re-synchronizes the ATPase engine with the TMDs.
ATP-binding cassette (ABC) superfamily comprises membrane transporters that power the active transport of substrates across biological membranes. These proteins harness the energy of nucleotide ...binding and hydrolysis to fuel substrate translocation via an alternating-access mechanism. The primary structural blueprint is relatively conserved in all ABC transporters. A transport-competent ABC transporter is essentially made up of two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). While the NBDs are conserved in their primary sequence and form at their interface two nucleotide-binding sites (NBSs) for ATP binding and hydrolysis, the TMDs are variable among different families and form the translocation channel. Transporters catalyzing the efflux of substrates from the cells are called exporters. In humans, they range from A to G subfamilies, with the B, C and G subfamilies being involved in chemoresistance. The recently elucidated structures of ABCG5/G8 followed by those of ABCG2 highlighted a novel structural fold that triggered extensive research. Notably, suppressor genetics in the orthologous yeast Pleiotropic Drug Resistance (PDR) subfamily proteins have pointed to a crosstalk between TMDs and NBDs modulating substrate export. Considering the structural information provided by their neighbors from the G subfamily, these studies provide mechanistic keys and posit a functional role for the non-hydrolytic NBS found in several ABC exporters. The present chapter provides an overview of structural and functional aspects of ABCG proteins with a special emphasis on the yeast PDR systems.
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