Cryo-EM structures of Cdr1 reveal snapshots of substrate transport and diverse inhibitor recognition.

Abstract

In <i>Candida albicans</i>-a World Health Organization fungal priority pathogen-overexpression of the adenosine triphosphate (ATP)-binding cassette transporter Cdr1 drives multidrug resistance. We present seven cryo-electron microscopy structures capturing substrate entry and expulsion. An inward-facing transmembrane channel with three on-off substrate binding sites defines a proposed entry pathway for a single substrate molecule. Coordinated ATP binding to both nucleotide-binding domains induces transmembrane domain closure, driving the substrate expulsion; adenosine diphosphate release following ATP hydrolysis resets the transporter to an inward-open conformation, enabling substrate entry for the next translocation cycle. Structures with three structurally diverse inhibitors resolve two distinct binding modes: one occupying all three substrate sites and another specifically binding two extracellular-proximal sites. These findings provide snapshots of the substrate translocation cycle and structural blueprints for antifungal drug design.