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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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

A significant portion of clinically observed antifungal resistance is mediated by ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transport pumps that reside in the plasma membrane. We review the mechanisms responsible for this phenomenon. Hyper resistance is often brought about by several kinds of DNA amplification or by gain-of-function mutations in a variety of transcription factors. Both of these result in overexpression of ABC and MFS transporters. Recently, however, several additional modes of resistance have been observed. These include mutations in non-conserved nucleotides leading to altered mRNA stability and a mutation in yeast transporter Pdr5, which improves cooperativity between drug-binding sites., (© 2022 John Wiley & Sons Ltd.)

The production and purification are the first steps required in any functional or structural study of a protein of interest. In the case of membrane proteins, these tasks can be difficult due to low expression levels and the necessity to extract them from their membrane environment. This chapter describes a convenient method based on GFP tagged to the membrane protein to facilitates these steps. Production is carried out in the yeast S. cerevisiae and purification steps are carried out and monitored taking advantage of an anti-GFP nanobody. We show how GFP can be a very helpful tool for controlling the correct addressing of the protein and for probing and optimizing purification. These methods are described here for producing and purifying CaCdr1p, an ABC exporter conferring multiantifungal resistance to C. albicans. This purification method can be amenable to any other GFP-tagged protein., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Antifungal resistance mediated by overexpression of ABC transporters is one of the primary roadblocks to effective therapy against Candida infections. Thus, identification and characterization of the ABC transporter repertoire in Candida species are of high relevance. The method described in the chapter is based on our previously developed bioinformatic pipeline for identification of ABC proteins in Candida species. The methodology essentially involves the utilization of a hidden Markov model (HMM) profile of the nucleotide-binding domain (NBD) of ABC proteins to mine these proteins from the proteome of Candida species. Further, a widely used tool to predict membrane protein topology is exploited to identify the true transporter candidates out of the ABC proteins. Even though the chapter specifically focuses on a method to identify ABC transporters in Candida auris , the same can also be applied to any other Candida species., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In view of the multiple clinical and physiological implications of ABC transporter proteins, there is a considerable interest among researchers to characterize them functionally. However, such characterizations are based on the premise that ABC proteins are accurately identified in the proteome of an organism, and their topology is correctly predicted. With this objective, we have developed ABC-finder, i.e., a Docker-based package for the identification of ABC proteins in all organisms, and visualization of the topology of ABC proteins using a web browser. ABC-finder is built and deployed in a Linux container, making it scalable for many concurrent users on our servers and enabling users to download and run it locally. Overall, ABC-finder is a convenient, portable, and platform-independent tool for the identification and topology prediction of ABC proteins. ABC-finder is accessible at http://abc-finder.osdd.jnu.ac.in., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

ABC transporters of the Pleiotropic Drug Resistance (PDR) family are the main actors of antifungal resistance in pathogenic fungi. While their involvement in clinical resistant strains has been proven, their transport mechanism remains unclear. Notably, one hallmark of PDR transporters is their asymmetry, with one canonical nucleotide-binding site capable of ATP hydrolysis while the other site is not. Recent publications reviewed here show that the so-called "deviant" site is of crucial importance for drug transport and is a step towards alleviating the mystery around the existence of non-catalytic binding sites., (Copyright © 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

ABC transporter proteins are involved in active transport, both in prokaryotes and eukaryotes. Sequence analysis of nucleotide binding domains (NBDs) of ABC proteins from all taxa revealed a well-conserved new motif having the signature: xT/ShxE/DNhxF, located between Q-loop and ABC signature sequence. A recent structure of an ABC transporter, ABCG5/G8 highlighted the motif as an essential structural determinant of inter-domain crosstalk and termed it as E-helix. We carried out an extensive computational analysis to unravel important structural entities alongside E-helix which plausibly play role in the interlocking mechanism of NBD with TMD. We identified E-helix to be a central structural moiety which interacts with three helices and an intracellular loop that leads to the transmembrane domain. Considering its wide occurrence, we examined the importance of this motif in one representative multidrug ABC transporter of Candida albicans, Cdr1p. The motif residues were replaced by alanines both individually as well as in combinations. The GFP-tagged versions of mutant proteins were overexpressed in Saccharomyces cerevisiae. Overall, our mutational data suggested that this motif plays a role in the maintenance of proper structural fold and/or inter-domain contacts in Cdr1p. We, thus, unveil an essential structural motif in ABC superfamily transporters., (Copyright © 2019. Published by Elsevier B.V.)

Drug resistance mechanisms in human pathogenic Candida species are continually evolving. Over the time, Candida species have acquired diverse strategies to vanquish the effects of various classes of drugs thereby, emanating as a serious life threat. Apart from the repertoire of well-established strategies, which predominantly comprise alteration, overexpression of drug targets, and chromosome duplication, Candida species have evolved a number of permeability constraints for antifungal drugs, via compromised drug import or increased drug efflux. For the latter, genome of Candida species harbour battery of exporters designated as Candida drug resistance genes. These genes predominantly encode membrane efflux transporters, which expel the incoming drugs and thus prevent toxic intracellular accumulation of drugs to manifest multidrug resistance. Such a phenomenon is restricted not only to Candida species but has been observed among many other pathogenic fungal species as well. Notably, the existence of large number of drug exporters in genomes of Candida species posits other pivotal roles for these efflux transporter proteins. The brief review discusses as to how the whole gamut of antifungal research has since been changed to include these new observations wherein reduced permeability of azoles across cell membrane of Candida cells is being implicated as one of the major determinants of antifungal susceptibilities, which all began with the identification of the first multidrug resistance gene CDR1, in Andre Goffeau's laboratory back in 1995., (© 2018 John Wiley & Sons, Ltd.)

ABC transporters are membrane-bound pumps composed of two major domains: the transmembrane domain(s) (TMDs) and the nucleotide-binding domain(s) (NBDs). Sequence analyses of the NBDs of key ABC exporters revealed a residue position within the H-loop to be differentially conserved in the ABCG family, wherein there lies glutamine instead of positively charged arginine/lysine as in non-ABCG members. Consequently, contrasting NBD sequences of fungal Pleiotropic Drug Resistance transporters (PDR/ABCG) with that of Cholesterol/Phospholipid and Retinal (CPR/ABCA) Flippase family revealed a high Cumulative Relative Entropy (CRE) score of this residue position implying its family-specific functional significance. Further, substitution of the glutamine by arginine in both the NBDs of a representative PDR/ABCG member, (Candida drug resistance 1 protein) Cdr1p led to selective susceptibility of the Saccharomyces cerevisiae strains overexpressing the corresponding mutant proteins (Q362R and Q1060R) towards antifungal substrates without any impact on the ATPase activity. Consistent with the findings from previous studies on H-loop motif of fungal PDR transporters, the current report points towards a role of the glutamine residue within both canonical and divergent H-loop of Cdr1p in conferring substrate selection in a precisely identical manner., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Throughout its evolution, the ATP-binding cassette (ABC) transporter superfamily has experienced a rapid expansion in its substrate repertoire and functions. Of the diverse functions that these pumps offer, their drug transport properties have attracted considerable attention primarily owing to their clinical significance. Despite this fact, emerging evidence suggests that physiological substrates of transporters also affect the overall functioning of an organism. Lipids, as substrates of ABC transporters, constitute one feature found in all representative groups of the living kingdom. Due to the importance of lipid species in the cellular physiology of an organism, their proper distribution within cells is crucial. This fact is well exemplified by the vast number of medical conditions that have been caused as a result of perturbations in ABC transporter-mediated lipid transport in higher organisms. In yeasts, apart from providing transport functions, ABC transporters also coordinate regulatory networks with lipids. This review focuses on yeast ABC transporters involved in the transport of lipids and briefly discusses the integration of their regulatory network with that of the lipid species., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The ABC transporter Cdr1 protein of Candida albicans, which plays a major role in antifungal resistance, has two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). The 12 transmembrane helices of TMDs that are interconnected by extracellular and intracellular loops (ICLs) mainly harbor substrate recognition sites where drugs bind while cytoplasmic NBDs hydrolyze ATP which powers drug efflux. The coupling of ATP hydrolysis to drug transport requires proper communication between NBDs and TMDs typically accomplished by ICLs. This study examines the role of cytoplasmic ICLs of Cdr1p by rationally predicting the critical residues on the basis of their interatomic distances. Among nine pairs that fall within a proximity of <4 Å, an ion pair between K577 of ICL1 and E315 of NBD1 was found to be critical. The substitution, swapping and changing of the length or charge of K577 or E315 by directed mutagenesis led to a misfolded, non-rescuable protein entrapped in intracellular structures. Furthermore, the equipositional ionic pair-forming residues from ICL3 and NBD2 (R1260 and E1014) did not impact protein trafficking. These results point to a new role for ICL/NBD interacting residues in PDR ABC transporters in protein folding and trafficking., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Multidrug resistance (MDR) transporters belonging to either the ATP-Binding Cassette(ABC) or Major Facilitator Superfamily (MFS) groups are major determinants of clinical drug re-sistance in fungi. The overproduction of these proteins enables the extrusion of incoming drugs atrates that prevent lethal effects. The promiscuity of these proteins is intriguing because they export awide range of structurally unrelated molecules. Research in the last two decades has used multipleapproaches to dissect the molecular basis of the polyspecificity of multidrug transporters. With largenumbers of drug transporters potentially involved in clinical drug resistance in pathogenic yeasts,this review focuses on the drug transporters of the important pathogenCandida albicans. This organ-ism harbors many such proteins, several of which have been shown to actively export antifungaldrugs. Of these, the ABC proteinCaCdr1 and the MFS proteinCaMdr1 are the two most prominentand have thus been subjected to intense site-directed mutagenesis and suppressor genetics-basedanalysis. Numerous results point to a common theme underlying the strategy of promiscuity adoptedby bothCaCdr1 andCaMdr1. This review summarizes the body of research that has provided insightinto how multidrug transporters function and deliver their remarkable polyspecificity. [ABSTRACT FROM AUTHOR]

Over-expression of the human P-glycoprotein (P-gp) in tumor cells is a classic example of an ABC protein serving as a hindrance to effective chemotherapy. The existence of proteins homologous to P-gp in organisms encompassing the entire living kingdom highlights extrusion of drugs as a general mechanism of multidrug resistance. Infections caused by opportunistic human fungal pathogens such as Candida species are very common and has intensified in recent years. The typical hosts, who possess suppressed immune systems due to conditions such as HIV and transplantation surgery etc., are prone to fungal infections. Prolonged chemotherapy induces fungal cells to eventually develop tolerance to most of the antifungals currently in clinical use. Amongst other prominent mechanisms of antifungal resistance such as manipulation of the drug target, rapid efflux achieved through overexpression of multidrug transporters has emerged as a major resistance mechanism for azoles. Herein, the azole-resistant clinical isolates of Candida species utilize a few select efflux pump proteins belonging to the ABC and MFS superfamilies, to deter the toxic accumulation of therapeutic azoles and thus, facilitating cell survival. In this article, we summarize and discuss the clinically relevant mechanisms of azole resistance in Candida albicans and non- albicans Candida (NAC) species, specifically highlighting the role of multidrug efflux proteins in the phenomenon. [ABSTRACT FROM AUTHOR]