Abstract
Transport of proteins and small molecules across the complex cell envelope of Gram-negative bacteria is crucial for their survival and interaction with their environment and is facilitated by specialized macromolecular machines that enable direct one-step or indirect two-step translocation of substrates. Major facilitator superfamily (MFS)-type tripartite efflux pumps and type I secretion systems likely employ a similar one-step mechanism for substrate transport across cell membranes, but the structural details remain elusive. A representative MFS-type tripartite efflux pump, EmrAB-TolC, mediates multidrug resistance through proton-coupled EmrB, a member of the DHA2 transporter family. Here, we demonstrate that the EmrAB-TolC pump confers resistance to clinical antibiotics, including polymyxin B and neomycin, and report the high-resolution (3.11 Å) structure of the pump, revealing a unique, asymmetric architecture emerging from the TolC:EmrA:EmrB ratio of 3:6:1. This structure identifies two critical subdomains, AssA and AssB, essential for pump assembly and key residues involved in pump assembly, drug recognition, proton translocation and coupling, which are corroborated by mutagenesis and antibiotic sensitivity assays. The delineation of the complete translocation pathway reveals the molecular mechanism for one-step drug transport process across the entire cell envelope.