CryoEM of ATP-driven dynamics and itraconazole binding in a fungal ABC pump

Azole resistance in Candida species often results from overexpression of the ABC transporter Cdr1, which expels drugs via ATP-driven conformational changes. Despite its clinical relevance, the structural basis of Cdr1 function has remained elusive. Here, we present four high-resolution cryo-EM structures of Candida glabrata Cdr1 under active turnover with ATP-Mg, itraconazole, and vanadate. Itraconazole is seen to curve deeply into the drug-binding pocket. A deep exploration of the variability within these particle datasets uncovered 80 transient conformations, including two nucleotide-bound states- one closed and one open- that capture, for the first time, the conformational transitions triggered by ATP hydrolysis in both nucleotide-binding and transmembrane domains. At the catalytic site, motion initiates in the C-helix adjacent to the ABC signature motif, retracting 4 A from the gamma-phosphate/vanadate position. This movement, transmitted to nearby TMH-1, opens the drug-binding site via its lateral displacement. A second set of conformations reveals coordinated rearrangements of transmembrane helices that drive substrate extrusion. These findings provide a direct structural and dynamic framework for understanding Cdr1-mediated azole resistance, and, more broadly, illuminate the conserved chemo-mechanical cycle of nucleotide-binding domains across the ABC superfamily, including non-membranous members.