Asymmetric conformations and lipid interactions shape the ATP-coupled cycle of a heterodimeric ABC transporter.

The heterodimeric class of ATP-binding cassette (ABC) transporters represents a unique paradigm of ATP energy transduction wherein a catalytically asymmetric ATP hydrolysis cycle in the nucleotide-binding domains powers alternating access in the transmembrane domains. The structural origin of the asymmetry and how it shapes the energetics of the conformational cycle are yet to be elucidated. Here, we used cryo-electron microscopy (cryo-EM), double electron-electron resonance spectroscopy (DEER) and molecular dynamics (MD) simulations, to capture and characterize multiple conformations of the heterodimeric ABC multidrug exporter BmrCD in lipid nanodiscs at near-atomic resolution. In addition to multiple ATP- and substrate-bound inward-facing (IF) conformations, we obtained the structure of an occluded (OC) conformation wherein the unique extracellular domain (ECD) twists to partially open the extracellular gate. In conjunction with DEER analysis of the populations of these conformations, the structures reveal that ATP-powered isomerization of the transporter entails changes in the relative symmetry of the BmrC and BmrD subunits that propagates from the transmembrane domain (TMD) to the nucleotide binding domain (NBD). The structures uncover asymmetric substrate and Mg 2+ binding which we hypothesize are required for triggering ATP hydrolysis preferentially in the one of the nucleotide-binding sites. MD simulations demonstrated that multiple lipid molecules, identified from the cryo-EM density maps, differentially bind the IF versus the OC conformations, thus rationalizing the reduced efficiency of coupling ATP hydrolysis in the NBD to alternating access of the TMD in lipid bilayers. In addition to establishing how lipid interactions with BmrCD modulate the energy landscape, our findings are framed in a distinct transport model that highlights the role of asymmetric conformations in the ATP-coupled cycle with implications to the mechanism of ABC transporters in general.