One-step drug transport across two membranes of Gram-negative bacteria by an MFS tripartite assembly

Transport of proteins and small molecules across cellular membrane is crucial for bacterial interaction with the environment and survival against antibiotics. In Gram-negative bacteria that possess two layers of membranes, specialized macromolecular machines are required to transport substrates across the envelope, often via an indirect stepwise procedure. The major facilitator superfamily (MFS)-type tripartite efflux pumps and type I secretion systems likely employ a direct one-step transport approach, but the structural mechanisms remain elusive. A representative MFS-type tripartite efflux pump, EmrAB-TolC, mediates resistance to multiple antimicrobial drugs through proton-coupled EmrB, a member of the DHA2 transporter family. Here, we report the high-resolution (3.13 Å) structure of the EmrAB-TolC pump, revealing a unique, asymmetric architecture emerging from the assembly of TolC:EmrA:EmrB with a ratio of 3:6:1. This structure reveals two critical subdomains AssA and AssB that are essential for the pump assembly. Key residues involved in drug recognition, proton translocation and coupling are identified and corroborated by mutagenesis and antibiotic sensitivity assays. The delineation of the complete translocation pathway unravels the one-step drug transport mechanism of the MFS pump across the entire envelope of Gram-negative bacteria.