Conformation space of a heterodimeric ABC exporter under turnover conditions

Cryo-electron microscopy (cryo-EM) has the capacity to capture molecular machines in action(1–3). ATP-binding cassette (ABC) exporters are highly dynamic membrane proteins that extrude a wide range of substances from the cytosol(4–6) and thereby contribute to essential cellular processes, adaptive immunity, and multidrug resistance(7,8). Despite their vital importance, the coupling of nucleotide binding, hydrolysis, and release to the conformational dynamics remains poorly resolved, especially for heterodimeric/asymmetric ABC exporters that abound in humans. Here, we present eight high-resolution cryo-EM structures that delineate the full functional cycle of an asymmetric ABC exporter in lipid environment. Cryo-EM analysis under active turnover conditions reveals distinct inward-facing (IF) conformations, one of them with bound peptide substrate, and previously undescribed asymmetric post-hydrolysis states with dimerized nucleotide-binding domains (NBDs) and a closed extracellular gate. Capturing an outward-facing (OF) open conformation requires a slow-down in ATP hydrolysis, indicating the transient nature of this state vulnerable to substrate re-entry. ATP-bound pre-hydrolysis and vanadate-trapped states are conformationally equivalent and both comprise co-existing OF conformations with open and closed extracelluar gates. In contrast, the post-hydrolysis states from the turnover experiment exhibit asymmetric ADP/ATP occlusion after phosphate release from the canonical site and display a progressive separation of the nucleotide-binding domains and unlocking of the intracellular gate. Our findings reveal that phosphate release, not ATP hydrolysis, triggers the return of the exporter to the IF conformation. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates to trap the key intermediates, we resolved fundamental and so-far hidden steps of the substrate translocation cycle of asymmetric ABC transporters.