BPS2026 – Bilateral lipid wedges shape outward drug release: Molecular dynamics insights into the outward facing states of the heterodimeric ABC exporter

ATP-binding cassette (ABC) exporters harness ATP hydrolysis in dimeric nucleotide binding domains to drive alternating access of their transmembrane vestibules, enabling efflux of chemically diverse substrates and contributing to multidrug resistance. Here, we focus on the heterodimeric exporter BmrCD and present two drug bound outward facing (OF) conformations in lipid nanodiscs that differ only by the position of a co-bound lipid relative to the substrate: in OF1, a POPA lipid (POV15) poised at one side of the V shaped extracellular vestibule that houses two antiparallel Hoechst molecules; in OF2, a lipid occupies the opposite side and directly contacts the substrate. Unbiased molecular dynamics (MD) simulation from OF1 captured rapid ingress of POV15 into the vestibule, where the anionic headgroup induces strong tilting of the Hoechst molecules and breaks stabilizing drug protein contacts, providing direct evidence that lipid entry destabilizes substrate binding prior to release. Biased simulations from OF2, specifically 2D umbrella sampling that uses the outward progress and the lipid-substrate distance as collective variables, capture lipid-mediated dissociation from the binding pocket when the contralateral lipid stays in long contact with the drug. Together, the OF1 and OF2 simulations establish a unified bilateral lipid assisted release mechanism in which lipids can wedge from either side to force open or loosen drug-protein contacts and, in OF2, directly chaperone dissociation, yielding predictions for mutational (D141/D377) and membrane composition perturbations.