G521 is the gatekeeper and a key transmembrane domain contact residue of <i>Candida albicans</i> Cdr1

<i>Candida albicans</i> 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.