{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,10]],"date-time":"2025-12-10T06:49:35Z","timestamp":1765349375001,"version":"3.46.0"},"reference-count":57,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,12,10]],"date-time":"2025-12-10T00:00:00Z","timestamp":1765324800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001509","name":"Royal Society of New Zealand","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001509","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100009193","name":"Marsden Fund","doi-asserted-by":"publisher","award":["MFP-UOO2210"],"award-info":[{"award-number":["MFP-UOO2210"]}],"id":[{"id":"10.13039\/501100009193","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>\n                    The ATP-binding cassette (ABC) transporter Cdr1 is a key mediator of antifungal drug resistance in\n                    <jats:italic>Candida albicans<\/jats:italic>\n                    , a major pathogen responsible for invasive candidiasis. Cdr1 belongs to the pleiotropic drug resistance (PDR) ABC transporter subfamily, exhibits broad substrate specificity, and plays a central role in protecting\n                    <jats:italic>C. albicans<\/jats:italic>\n                    cells from azole drugs and many other xenobiotics. Although high-resolution structures of Cdr1 and its homologs are now available, the transport cycle of Cdr1 remains incompletely understood. Furthermore, although these structures provide valuable information about the spatial arrangements of amino acids, they do not reveal how, and which, amino acid interactions affect pump function and how to relieve drug resistance. Drug resistance\u2013based gain-of-function suppressor screening has emerged as a powerful tool to identify inhibitor binding hotspots, and conformational adaptation mechanisms. These studies reveal residues critically important for the structural and functional integrity of Cdr1, offering deeper mechanistic insights into substrate\/inhibitor recognition and transport. Importantly, these insights will aid in the rational development of Cdr1 inhibitors that, when combined with azoles, may reverse efflux pump-mediated drug resistance. Inhibitors that stabilize discrete conformations of Cdr1 offer additional tools for the structural and mechanistic characterization of this important efflux pump. Here, we demonstrate the power of gain-of-function suppressor screening for determining functionally important residues and validating structural models of the prototype fungal PDR transporter,\n                    <jats:italic>C. albicans<\/jats:italic>\n                    Cdr1.\n                  <\/jats:p>","DOI":"10.3389\/fmicb.2025.1721050","type":"journal-article","created":{"date-parts":[[2025,12,10]],"date-time":"2025-12-10T06:48:12Z","timestamp":1765349292000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Use of gain-of-function suppressor screening to elucidate Candida albicans Cdr1 efflux mechanism, inhibition, and drug resistance"],"prefix":"10.3389","volume":"16","author":[{"given":"Mengcun","family":"Zhao","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Masakazu","family":"Niimi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Richard D.","family":"Cannon","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Erwin","family":"Lamping","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2025,12,10]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"102689","DOI":"10.1016\/j.jbc.2022.102689","article-title":"Residues forming the gating regions of asymmetric multidrug transporter Pdr5 also play roles in conformational switching and protein folding","volume":"298","author":"Alhumaidi","year":"2022","journal-title":"J. Biol. Chem."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"4440","DOI":"10.1021\/bi100394j","article-title":"The signaling interface of the yeast multidrug transporter Pdr5 adopts a cis conformation, and there are functional overlap and equivalence of the deviant and canonical Q-loop residues","volume":"49","author":"Ananthaswamy","year":"2010","journal-title":"Biochemistry"},{"key":"ref3","doi-asserted-by":"publisher","first-page":"1131","DOI":"10.1111\/mmi.14351","article-title":"An A666G mutation in transmembrane helix 5 of the yeast multidrug transporter Pdr5 increases drug efflux by enhancing cooperativity between transport sites","volume":"112","author":"Arya","year":"2019","journal-title":"Mol. Microbiol."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"183131","DOI":"10.1016\/j.bbamem.2019.183131","article-title":"Cdr1p highlights the role of the non-hydrolytic ATP-binding site in driving drug translocation in asymmetric ABC pumps","volume":"1862","author":"Banerjee","year":"2020","journal-title":"Biochim. Biophys. Acta Biomembr."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"163","DOI":"10.1016\/bs.apcsb.2020.09.006","article-title":"ABCG: a new fold of ABC exporters and a whole new bag of riddles!","volume":"123","author":"Banerjee","year":"2021","journal-title":"Adv. Protein Chem. Struct. Biol."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"965","DOI":"10.1016\/j.bbamem.2018.01.022","article-title":"W1038 near D-loop of NBD2 is a focal point for inter-domain communication in multidrug transporter Cdr1 of Candida albicans","volume":"1860","author":"Banerjee","year":"2018","journal-title":"Biochim. Biophys. Acta Biomembr."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"e0276123","DOI":"10.1128\/mbio.02761-23","article-title":"The American Academy of microbiology discusses gain-of-function research of concern (GOFROC) and enhanced potential pandemic pathogens (ePPP)","volume":"15","author":"Casadevall","year":"2024","journal-title":"MBio"},{"key":"ref8","doi-asserted-by":"publisher","first-page":"932","DOI":"10.1016\/j.bbamem.2013.12.002","article-title":"Mutations adjacent to the end of transmembrane helices 6 and 7 independently affect drug efflux capacity of yeast ABC transporter Pdr5p","volume":"1838","author":"Chen","year":"2014","journal-title":"Biochim. Biophys. Acta"},{"key":"ref9","doi-asserted-by":"publisher","first-page":"811","DOI":"10.1111\/j.1365-2958.2006.05562.x","article-title":"Subcellular trafficking of the yeast plasma membrane ABC transporter, Pdr5, is impaired by a mutation in the N-terminal nucleotide-binding fold","volume":"63","author":"de Thoz\u00e9e","year":"2007","journal-title":"Mol. Microbiol."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"1156","DOI":"10.1101\/gr.184901","article-title":"The human ATP-binding cassette (ABC) transporter superfamily","volume":"11","author":"Dean","year":"2001","journal-title":"Genome Res."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"12612","DOI":"10.1074\/jbc.273.20.12612","article-title":"ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p","volume":"273","author":"Decottignies","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"1025","DOI":"10.1128\/aac.02133-12","article-title":"The transmission interface of the Saccharomyces cerevisiae multidrug transporter Pdr5: Val-656 located in intracellular loop 2 plays a major role in drug resistance","volume":"57","author":"Downes","year":"2013","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"523","DOI":"10.1091\/mbc.9.2.523","article-title":"Genetic separation of FK506 susceptibility and drug transport in the yeast Pdr5 ATP-binding cassette multidrug resistance transporter","volume":"9","author":"Egner","year":"1998","journal-title":"Mol. Biol. Cell"},{"key":"ref14","doi-asserted-by":"publisher","first-page":"69","DOI":"10.1016\/j.ijantimicag.2006.02.016","article-title":"Drug susceptibilities of yeast cells are affected when expressing mutant Candida albicans drug resistance protein","volume":"28","author":"Gao","year":"2006","journal-title":"Int. J. Antimicrob. Agents"},{"key":"ref15","doi-asserted-by":"publisher","first-page":"jkae148","DOI":"10.1093\/g3journal\/jkae148","article-title":"Experimental evolution of Saccharomyces cerevisiae for caffeine tolerance alters multidrug resistance and target of rapamycin signaling pathways","volume":"14","author":"Geck","year":"2024","journal-title":"G3 (Bethesda)"},{"key":"ref16","doi-asserted-by":"publisher","first-page":"53","DOI":"10.1515\/bc.2011.011","article-title":"The multidrug transporter Pdr5: a molecular diode?","volume":"392","author":"Gupta","year":"2011","journal-title":"Biol. Chem."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"5254","DOI":"10.1038\/s41467-021-25574-8","article-title":"Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5","volume":"12","author":"Harris","year":"2021","journal-title":"Nat. Commun."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"63","DOI":"10.1016\/j.femsyr.2004.07.002","article-title":"ABC multidrug transporter Cdr1p of Candida albicans has divergent nucleotide-binding domains which display functional asymmetry","volume":"5","author":"Jha","year":"2004","journal-title":"FEMS Yeast Res."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"869","DOI":"10.1016\/j.bbrc.2003.09.094","article-title":"Covalent modification of cysteine 193 impairs ATPase function of nucleotide-binding domain of a Candida drug efflux pump","volume":"310","author":"Jha","year":"2003","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"13767","DOI":"10.1038\/s41598-017-11794-w","article-title":"The structure of the human ABC transporter ABCG2 reveals a novel mechanism for drug extrusion","volume":"7","author":"Khunweeraphong","year":"2017","journal-title":"Sci. Rep."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"5433","DOI":"10.1038\/s41467-019-13302-2","article-title":"The ABCG2 multidrug transporter is a pump gated by a valve and an extracellular lid","volume":"10","author":"Khunweeraphong","year":"2019","journal-title":"Nat. Commun."},{"key":"ref22","doi-asserted-by":"publisher","first-page":"479","DOI":"10.1016\/j.bbamem.2012.10.024","article-title":"A conserved interdomain communication pathway of pseudosymmetrically distributed residues affects substrate specificity of the fungal multidrug transporter Cdr1p","volume":"1828","author":"Kolaczkowski","year":"2013","journal-title":"Biochim. Biophys. Acta"},{"key":"ref23","doi-asserted-by":"publisher","first-page":"127","DOI":"10.1016\/j.fgb.2009.10.007","article-title":"Fungal PDR transporters: phylogeny, topology, motifs and function","volume":"47","author":"Lamping","year":"2010","journal-title":"Fungal Genet. Biol."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"1150","DOI":"10.1128\/ec.00091-07","article-title":"Characterization of three classes of membrane proteins involved in fungal azole resistance by functional hyperexpression in Saccharomyces cerevisiae","volume":"6","author":"Lamping","year":"2007","journal-title":"Eukaryot. Cell"},{"key":"ref25","doi-asserted-by":"publisher","first-page":"561","DOI":"10.1038\/nature17666","article-title":"Crystal structure of the human sterol transporter ABCG5\/ABCG8","volume":"533","author":"Lee","year":"2016","journal-title":"Nature"},{"key":"ref26","doi-asserted-by":"publisher","first-page":"3390","DOI":"10.1021\/acs.chemrev.0c00199","article-title":"Antifungal drug resistance: molecular mechanisms in Candida albicans and beyond","volume":"121","author":"Lee","year":"2021","journal-title":"Chem. Rev."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"e01318-20","DOI":"10.1128\/mSphere.01318-20","article-title":"Engineering a cysteine-deficient functional Candida albicans Cdr1 molecule reveals a conserved region at the cytosolic apex of ABCG transporters important for correct folding and trafficking of Cdr1","volume":"6","author":"Madani","year":"2021","journal-title":"mSphere"},{"key":"ref28","doi-asserted-by":"publisher","first-page":"426","DOI":"10.1038\/s41586-018-0680-3","article-title":"Cryo-EM structures of a human ABCG2 mutant trapped in ATP-bound and substrate-bound states","volume":"563","author":"Manolaridis","year":"2018","journal-title":"Nature"},{"key":"ref29","doi-asserted-by":"publisher","first-page":"e4792","DOI":"10.1002\/pro.4792","article-title":"UCSF ChimeraX: tools for structure building and analysis","volume":"32","author":"Meng","year":"2023","journal-title":"Protein Sci."},{"key":"ref30","doi-asserted-by":"publisher","first-page":"747","DOI":"10.1111\/j.1365-2958.2012.08140.x","article-title":"Specific interactions between the Candida albicans ABC transporter Cdr1p ectodomain and a d-octapeptide derivative inhibitor","volume":"85","author":"Niimi","year":"2012","journal-title":"Mol. Microbiol."},{"key":"ref31","doi-asserted-by":"publisher","first-page":"e0174821","DOI":"10.1128\/aac.01748-21","article-title":"Inhibitor-resistant mutants give important insights into Candida albicans ABC transporter Cdr1 substrate specificity and help elucidate efflux pump inhibition","volume":"66","author":"Niimi","year":"2022","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"29768675241304684","DOI":"10.1177\/29768675241304684","article-title":"A narrative review of invasive candidiasis in the intensive care unit","volume":"19","author":"Nopp\u00e8","year":"2024","journal-title":"Ther Adv Pulm Crit Care Med"},{"key":"ref33","doi-asserted-by":"publisher","first-page":"e1","DOI":"10.1093\/cid\/civ933","article-title":"Clinical practice guideline for the management of candidiasis: 2016 update by the infectious diseases society of America","volume":"62","author":"Pappas","year":"2016","journal-title":"Clin. Infect. Dis."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"577","DOI":"10.1128\/MMBR.00020-09","article-title":"ABC transporters in Saccharomyces cerevisiae and their interactors: new technology advances the biology of the ABCC (MRP) subfamily","volume":"73","author":"Paumi","year":"2009","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"7722","DOI":"10.1038\/s41467-024-52107-w","article-title":"Cryo-EM structures of Candida albicans Cdr1 reveal azole-substrate recognition and inhibitor blocking mechanisms","volume":"15","author":"Peng","year":"2024","journal-title":"Nat. Commun."},{"key":"ref36","doi-asserted-by":"publisher","first-page":"S79","DOI":"10.1093\/ofid\/ofy358","article-title":"Twenty years of the SENTRY antifungal surveillance program: results for Candida species from 1997-2016","volume":"6","author":"Pfaller","year":"2019","journal-title":"Open Forum Infect. Dis."},{"key":"ref37","doi-asserted-by":"publisher","first-page":"357","DOI":"10.1534\/g3.119.400863","article-title":"Nonsynonymous mutations in linker-2 of the Pdr5 multidrug transporter identify a new RNA stability element","volume":"10","author":"Rahman","year":"2020","journal-title":"G3 (Bethesda)"},{"key":"ref38","doi-asserted-by":"publisher","first-page":"14726","DOI":"10.1021\/bi061535t","article-title":"Conserved Asp327 of walker B motif in the N-terminal nucleotide binding domain (NBD-1) of Cdr1p of Candida albicans has acquired a new role in ATP hydrolysis","volume":"45","author":"Rai","year":"2006","journal-title":"Biochemistry"},{"key":"ref39","doi-asserted-by":"publisher","first-page":"27132","DOI":"10.1038\/srep27132","article-title":"Newly identified motifs in Candida albicans Cdr1 protein nucleotide binding domains are pleiotropic drug resistance subfamily-specific and functionally asymmetric","volume":"6","author":"Rawal","year":"2016","journal-title":"Sci. Rep."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"24480","DOI":"10.1074\/jbc.M113.488353","article-title":"Insight into pleiotropic drug resistance ATP-binding cassette pump drug transport through mutagenesis of Cdr1p transmembrane domains","volume":"288","author":"Rawal","year":"2013","journal-title":"J. Biol. Chem."},{"key":"ref41","doi-asserted-by":"publisher","first-page":"4982","DOI":"10.1128\/AAC.00925-15","article-title":"Structural insights into binding of the antifungal drug fluconazole to Saccharomyces cerevisiae lanosterol 14alpha-demethylase","volume":"59","author":"Sagatova","year":"2015","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref42","doi-asserted-by":"publisher","first-page":"10","DOI":"10.1099\/jmm.0.045054-0","article-title":"Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options","volume":"62","author":"Sardi","year":"2013","journal-title":"J. Med. Microbiol."},{"key":"ref43","doi-asserted-by":"publisher","first-page":"35010","DOI":"10.1074\/jbc.M806446200","article-title":"Mutations define cross-talk between the N-terminal nucleotide-binding domain and transmembrane helix-2 of the yeast multidrug transporter Pdr5: possible conservation of a signaling interface for coupling ATP hydrolysis to drug transport","volume":"283","author":"Sauna","year":"2008","journal-title":"J. Biol. Chem."},{"key":"ref44","doi-asserted-by":"publisher","first-page":"11211","DOI":"10.1038\/srep11211","article-title":"Mutational analysis of intracellular loops identify cross talk with nucleotide binding domains of yeast ABC transporter Cdr1p","volume":"5","author":"Shah","year":"2015","journal-title":"Sci. Rep."},{"key":"ref45","doi-asserted-by":"publisher","first-page":"867","DOI":"10.1038\/nchembio.2165","article-title":"Dual action antifungal small molecule modulates multidrug efflux and TOR signaling","volume":"12","author":"Shekhar-Guturja","year":"2016","journal-title":"Nat. Chem. Biol."},{"key":"ref46","doi-asserted-by":"publisher","first-page":"1361","DOI":"10.1128\/ec.2.6.1361-1375.2003","article-title":"Functional characterization of Candida albicans ABC transporter Cdr1p","volume":"2","author":"Shukla","year":"2003","journal-title":"Eukaryot. Cell"},{"key":"ref47","doi-asserted-by":"publisher","first-page":"520","DOI":"10.1016\/j.bbrc.2004.07.151","article-title":"Disulfiram is a potent modulator of multidrug transporter Cdr1p of Candida albicans","volume":"322","author":"Shukla","year":"2004","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref48","doi-asserted-by":"publisher","first-page":"1569","DOI":"10.1093\/jac\/dkad139","article-title":"Invasive candidiasis: current clinical challenges and unmet needs in adult populations","volume":"78","author":"Soriano","year":"2023","journal-title":"J. Antimicrob. Chemother."},{"key":"ref49","doi-asserted-by":"publisher","first-page":"183","DOI":"10.1007\/s11274-019-2762-2","article-title":"Evolutionary engineering and molecular characterization of a caffeine-resistant Saccharomyces cerevisiae strain","volume":"35","author":"Surmeli","year":"2019","journal-title":"World J. Microbiol. Biotechnol."},{"key":"ref50","doi-asserted-by":"publisher","first-page":"e01146-18","DOI":"10.1128\/aac.01146-18","article-title":"FK506 resistance of Saccharomyces cerevisiae Pdr5 and Candida albicans Cdr1 involves mutations in the transmembrane domains and extracellular loops","volume":"63","author":"Tanabe","year":"2019","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref51","doi-asserted-by":"publisher","first-page":"504","DOI":"10.1038\/nature22345","article-title":"Structure of the human multidrug transporter ABCG2","volume":"546","author":"Taylor","year":"2017","journal-title":"Nature"},{"key":"ref52","doi-asserted-by":"publisher","first-page":"3767","DOI":"10.1002\/1873-3468.13935","article-title":"Structural and functional diversity calls for a new classification of ABC transporters","volume":"594","author":"Thomas","year":"2020","journal-title":"FEBS Lett."},{"key":"ref53","doi-asserted-by":"publisher","first-page":"3125","DOI":"10.1093\/jac\/dkw252","article-title":"Positive regulation of the Candida albicans multidrug efflux pump Cdr1p function by phosphorylation of its N-terminal extension","volume":"71","author":"Tsao","year":"2016","journal-title":"J. Antimicrob. Chemother."},{"key":"ref54","doi-asserted-by":"publisher","first-page":"409","DOI":"10.1111\/j.1365-2443.2005.00847.x","article-title":"Mutational analysis of the yeast multidrug resistance ABC transporter Pdr5p with altered drug specificity","volume":"10","author":"Tutulan-Cunita","year":"2005","journal-title":"Genes Cells"},{"key":"ref55","doi-asserted-by":"publisher","first-page":"151","DOI":"10.1016\/j.tplants.2008.02.001","article-title":"Plant ABC proteins--a unified nomenclature and updated inventory","volume":"13","author":"Verrier","year":"2008","journal-title":"Trends Plant Sci."},{"key":"ref56","doi-asserted-by":"publisher","first-page":"95","DOI":"10.1016\/j.ijbiomac.2019.01.030","article-title":"The E-helix is a central core in a conserved helical bundle involved in nucleotide binding and transmembrane domain intercalation in the ABC transporter superfamily","volume":"127","author":"Vishwakarma","year":"2019","journal-title":"Int. J. Biol. Macromol."},{"key":"ref57","doi-asserted-by":"publisher","first-page":"9497","DOI":"10.1021\/bi401285j","article-title":"Characterization of the role of a highly conserved sequence in ATP binding cassette transporter G (ABCG) family in ABCG1 stability, oligomerization, and trafficking","volume":"52","author":"Wang","year":"2013","journal-title":"Biochemistry"}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2025.1721050\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,10]],"date-time":"2025-12-10T06:48:13Z","timestamp":1765349293000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2025.1721050\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,10]]},"references-count":57,"alternative-id":["10.3389\/fmicb.2025.1721050"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2025.1721050","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,10]]},"article-number":"1721050"}}