Background: ATP-binding cassette (ABC) transporters are one of the largest protein super-families found in all kingdoms of life. They are active transporters that can be divided into type I, type II, and energy-coupling factor (ECF) importers (only found in prokaryotes), and type I and type II exporters. Candida albicans Cdr1 is a member of the pleiotropic drug resistance (PDR) ABC exporter sub-family unique to plants and fungi. Overexpression of Cdr1 causes life-threatening drug resistance in clinical isolates of the opportunistic human fungal pathogen, C. albicans. PDR transporters typically have two large extracellular loops, EL3 and EL6, containing PDR transporter-defining motifs, PDRA and PDRB, and EL6-motif and EL6-helix.
Methods: The role of the four PDR-specific motifs in Cdr1 structure and function was investigated by alanine scanning mutagenesis and functionally overexpressing and characterizing individual mutants in the genetically modified heterologous host Saccharomyces cerevisiae AD∆∆.
Results: We discovered seven amino acids in PDRA, PDRB and the EL6 motif critical for pump function and identified three important extracellular disulfide bonds that are conserved in all fungal PDR transporters. Cdr1 has 23 cysteines, and progress has been made towards the creation of an entirely cysteineless, but functional, Cdr1 - an essential first step for cysteine-cross-linking studies to confirm predicted structural interactions.
Conclusion: The PDR motifs together with the two large extracellular domains of fungal PDR transporters are stabilized by three unique disulfide bonds, and the interaction between these motifs and the extracellular domains contribute to a transport mechanism that is unique to PDR transporters