Cryo-EM reveals the structural heterogeneity and conformational flexibility of multidrug efflux pumps MdtB and MdtF

Abstract

RND efflux pumps are a major cause of multidrug resistance in bacteria, particularly in Gram-negative bacteria. They are complex molecular machines forming tripartite assemblies that actively pump out a wide range of antimicrobial agents, including antibiotics, biocides and host defence molecules. However, the presence of multiple RND pumps with overlapping functions in a single bacterium raises questions about their individual functional relevance. In this study, we determined the Cryo-EM structures of two distinct HAE-RND pumps from E.coli., MdtB and MdtF. MdtF is a unique class of RND pump whose expression is regulated by oxygen availability and is crucial for the survival of E.coli in anaerobic growth conditions. Surprisingly, homotrimeric MdtB, previously reported to be inactive, exhibited activity in our experiments. Cryo-EM structure determination of MdtF at 2.8 [A] resolution revealed the flexible transmembrane domain with significant conformational changes in the core helixes, leading to the closure of the central pore. While the MdtB structure adopts a resting state, MdtF displays structural dynamics in the presence of DDM. The apo structures of both pumps unveil novel conformational states of HAE-RND efflux transporters. Our findings highlight significant structural and functional divergence among closely related HAE-RND proteins. Despite 71% sequence similarity and phylogenetic proximity to AcrB, the structure of MdtF differs from the solved AcrB structures. In contrast, the MdtB structure exhibits greater resemblance to AcrB, regardless of 32% sequence similarity. Furthermore, our structural analysis provides insights into drug-binding sites and the transport mechanism of these important transporters.