ATP-driven dynamics and itraconazole binding of the drug efflux pump Cdr1 from Candida glabrata

Azole resistance in Candida species is often caused by the overexpression of Cdr1. Despite its clinical relevance, the structural basis for its ATP-driven efflux pump function remains elusive. We present four high-resolution cryo-EM structures of Candida glabrata Cdr1 obtained in the absence and presence of ATP-Mg²⁺, itraconazole, and vanadate, capturing distinct nucleotide- and substrate-occupancy states. Additional low-population cryo-EM conformers were unveiled by 3D variability analysis, offering a detailed view of structural transitions linked with ATP-hydrolysis. The motion shows a 4 Å piston-like retraction of the C-helix from the γ-phosphate/vanadate of the hydrolyzed ATP. This causes the nearby transmembrane helix-1 (TMH-1) to open the drug-binding site via lateral displacement and unwinding of the inner-leaflet region of TMH-2. A reverse ‘squeeze-and-push’ motion of TMH-2 possibly drives substrate extrusion. High-resolution structures also reveal how itraconazole adapts its shape to fit into the drug-binding site. Our findings provide a dynamic structural framework for Cdr1-mediated azole resistance and the conserved chemo-mechanical cycle of ABC proteins, including non-membranous members.