Cryoelectron Microscopy Structures of AdeB Illuminate Mechanisms of Simultaneous Binding and Exporting of Substrates

Acinetobacter baumannii is a Gram-negative pathogen that has emerged as one of the most highly antibiotic-resistant bacteria worldwide. Multidrug efflux within these highly drug-resistant strains and other opportunistic pathogens is a major cause of failure of drug-based treatments of infectious diseases. The best-characterized multi-drug efflux system in A. baumannii is the prevalent Acinetobacter drug efflux B (AdeB) pump, which is a member of the resistance-nodulation-cell division (RND) superfamily. Here, we report six structures of the trimeric AdeB multidrug efflux pump in the pres-ence of ethidium bromide using single-particle cryoelectron microscopy (cryo-EM). These structures allow us to directly observe various novel conformational states of the AdeB trimer, including the transmembrane region of trimeric AdeB can be associated with form a trimer assembly or dissociated into dimer plus monomer and monomer plus monomer plus monomer configurations. We also discover that a single AdeB pro-tomer can simultaneously anchor a number of ethidium ligands and that different AdeB protomers can bind ethidium molecules simultaneously. Combined with molecular dy-namics (MD) simulations, we reveal a drug transport mechanism that involves multiple multidrug-binding sites and various transient states of the AdeB membrane protein. Our data suggest that each AdeB protomer within the trimer binds and exports drugs independently. IMPORTANCE Acinetobacter baumannii has emerged as one of the most highly antibi-otic-resistant Gram-negative pathogens. The prevalent AdeB multidrug efflux pump mediates resistance to a broad spectrum of clinically relevant antimicrobial agents. Here, we report six cryo-EM structures of the trimeric AdeB pump in the presence of ethidium bromide. We discover that a single AdeB protomer can simultaneously anchor a number of ligands, and different AdeB protomers can bind ethidium mole-cules simultaneously. The results indicate that each AdeB protomer within the trimer recognizes and extrudes drugs independently. Superscript/Subscript Available

Automated summary

Acinetobacter baumannii, a highly antibiotic-resistant Gram-negative pathogen, utilizes the AdeB multidrug efflux pump to resist a wide range of clinically relevant antimicrobial agents. The study of AdeB pump structures in the presence of ethidium bromide reveals that each AdeB protomer within the trimer can independently recognize and extrude drugs. This sheds light on the drug transport mechanism involving multiple multidrug-binding sites and various transient states of the AdeB membrane protein.