Molecular recognition, in- and out- of equilibrium, in the actions of bacterial multidrug efflux nanomachines

Abstract

The increasing number of antibiotic-resistant infections is outpacing the provision of new antibacterial treatments and leading to a growing health and economic burden. One of the main mechanisms that bacteria use to acquire drug resistance is through the expression of multidrug efflux transporters. Among the many efflux pumps present in bacterial lineages, the AcrAB-TolC assembly in Escherichia coli is one of the best studied efflux machines for conferring intrinsic multidrug resistance. This assembly is a representative of a wide family of homologous and analogous nanomachines, including those found in numerous clinically relevant pathogens. The AcrAB-TolC pump spans the cell envelope and is powered by proton gradients across the inner membrane to energise the active efflux of an array of chemically unrelated compounds. How such diverse compounds are recognised, while critical metabolites and other molecules needed by the cells are retained, is one intriguing puzzle of this system. Structural information and dynamics are crucial for elucidating the functionality of this molecular machine; however, its allosteric nature and complex conformational landscape have presented challenges to achieve this aim. Using a combination of structural, computational and biochemical approaches, mechanistic insights have been gained into substrate recognition and non-equilibrium displacement that underpins active efflux. High-resolution cryo-EM structures of the tripartite pump have been determined in membrane-mimicking scaffolds and detergents. A gallery of experimental structures was obtained, including the pump in the apo form as well as in the presence of substrates and synthetic peptide inhibitors with resolutions ranging from 2.9-4.7 Å showing different transport-active states. Comparison of apo- and ligand- bound forms implicate a pathway of allosteric communication that favours transition of TolC in the outer membrane from a closed resting state to an open state required for substrate efflux. Allostery has also been proposed in a model of transport involving obligate cycling of the subunits of the AcrB transporter between three states. Computational analysis of the experimental tripartite assembly using metadynamics simulations captures transition states for the process and provides insight into the inter-subunit allosteric pathways. The simulations identify critical residues in this intramolecular communication, leading to testable hypotheses for inhibiting the mechanism of drug efflux. In vivo, the pump interacts with partners in a complex milieu. Through cryo-EM analysis, the first atomic resolution model of the interactions between the pump and the peptidoglycan layer of the bacterial cell envelope was obtained. To explore the impact of electrochemical gradients on the structure and to dissect the contributions of the proton-motive force to the efflux process, a synthetic cell model was developed. To further explore the in-situ structure of the pump and its potential higher-order assembly in the cell, novel self-assembling nanostructures were engineered as a tagging strategy for tomographic reconstructions. Using cryo-ET, the assembly and organisation of this system was investigated in situ providing clues on the impact of the native bacterial environment during the transport process. Altogether, the structural and functional data obtained can serve to guide approaches for manipulating the pump’s structure and mechanism of action to ultimately, combat multidrug resistance.