Abstract
Candida albicans Cdr1 plays an important role in antifungal drug resistance through the extrusion of structurally diverse substrates. However, the molecular basis of its substrate preference remains unclear. A G521R mutation at the center of transmembrane segment 1 (TMS1) was previously found to impair the efflux of large substrates and to confer resistance to large efflux pump inhibitors, suggesting a role for G521 in regulating substrate and inhibitor access. To explore the structural and functional basis of this phenotype, we analyzed a series of Saccharomyces cerevisiae strains lacking ABC transporters and overexpressing Cdr1 with G521 replaced by residues of varying size and polarity. Increasing side chain bulk at position 521 progressively reduced transport of larger substrates but had less deleterious, or even positive, effects on the transport of smaller compounds, supporting a size-selective gating function for the G521 residue. Due to the asymmetric nature of Cdr1, no opening exists at the equivalent TMS7 region on the opposite side of the transporter. Further biochemical characterization revealed that substitutions at G521 alter ATP-binding and turnover rates, indicating that this residue also modulates the ATPase activity at the catalytically active nucleotide-binding site and is, therefore, a key regulatory element during the Cdr1 transport cycle. Suppressor screening identified second-site mutations that restored efflux in bulky G521 mutants by reshaping important transmembrane domain contact regions. These findings defined G521 as the gatekeeper residue that couples substrate selection at the entry gate to the large conformational changes experienced by Cdr1 during the transport cycle. IMPORTANCE: Candida albicans is a major fungal pathogen that can cause life-threatening invasive infections in immunocompromised individuals, and the multidrug ABC transporter Cdr1 plays a key role in its antifungal resistance. While previous studies have identified the transporter's broad substrate specificity, the structural basis underlying substrate selection remains poorly understood. In this study, we identified a key amino acid residue in transmembrane segment 1 with two important biological functions: (i) as a gatekeeper and (ii) as a key transmembrane domain contact residue affecting ATP binding and hydrolysis at the catalytically active composite nucleotide-binding site 2 just underneath the efflux pump entry gate between transmembrane segments 1 and 11. This work provides a critical understanding of how substrates and inhibitors access the Cdr1 binding cavity and how ATP binding and hydrolysis are coupled to substrate transport. These discoveries open new avenues for the development of next-generation antifungal efflux pump inhibitors.