“If you’ve got a problem which you think is worth tackling, you should just start doing it now.” These words, spoken by Ada E. Yonath at the 65th Lindau Nobel Laureate Meeting in 2015, left a lasting impression on University of Southampton chemist Eamonn Reading. At the time, Reading had recently completed his graduate work on membrane proteins and was interested in using his skillset for societal impact. “I never really had the confidence or the agency to start to do that, and I took that as a real kind of validation that we can, as scientists, just decide to start working on that,” he said.
As a postdoctoral fellow at King’s College London, Reading narrowed his research focus to multidrug efflux pumps, membrane proteins that recognize antibiotics and drain them out of the cell (1). Since bacteria evade antibiotic exposure by overexpressing efflux pumps, these proteins play a key role in the development of antimicrobial resistance, which is a global health and development threat according to the World Health Organization (2).
In a recent study published in Nature Communications, Reading and his collaborators revealed a new mechanism for an E. coli efflux pump called AcrAB-TolC (3). Their study identified regions of the protein that could be targeted to inhibit the pump with the goal of overcoming efflux pump-based antibiotic resistance.
The AcrAB-TolC pump is named for its three components: AcrB, the inner membrane transporter that recognizes antibiotics in the cytoplasm and draws them into the pump, TolC, the outer membrane pore that pushes the antibiotics into the extracellular environment, and AcrA, the periplasmic linker. To keep antibiotics in the cell, scientists have tried to shut down the pump by administering compounds that inhibit AcrB. However, AcrB adapts to recognize these inhibitors as substrates and draws them into the pump, rendering them ineffective.
To overcome this limitation, Helen Zgurskaya, a biochemist at the University of Oklahoma and collaborator in the study, identified an inhibitor called NCS 60339 that instead binds to AcrA (4). To probe this binding interaction in greater detail, Reading offered to examine AcrA using hydrogen-deuterium exchange mass spectrometry (HDX-MS). In HDX-MS, a protein is placed in a buffer that allows its hydrogen atoms to exchange with heavier deuterium atoms (5). Since structural bonds prevent hydrogen atoms in more rigid parts of the protein from being replaced, a higher rate of hydrogen-deuterium exchange indicates a more flexible part of the protein.
Based on the HDX-MS data, the team found that AcrA’s most flexible portions are the NCS 60339 binding site and a pocket adjacent to the AcrA-AcrB interface. They also observed that these regions became more rigid when they added NCS 60339, but not when they added novobiocin, a compound that binds AcrA but does not inhibit efflux. The researchers therefore suspected that NCS 60339 halts the activity of the multidrug efflux pump by restricting the movement of AcrA and blocking its ability to interact with AcrB.
Zgurskaya’s team then sought to study this mechanism in bacteria. The researchers designed a series of AcrA variants, each containing a cysteine residue at a different position and expressed them as AcrAB-TolC mutants in E. coli. They treated these cells with a methanethiosulfonate molecule, which AcrB recognized as a substrate and drew into the pump. This molecule formed covalent bonds with the cysteine residues, restricting the movement of AcrA at each position. Finally, they added a fluorescent dye compound that is expelled via the AcrAB-TolC pump and measured the rate at which the dye built up within the bacteria. The dye accumulated significantly faster in cells expressing proteins that were conformationally restricted at the NCS 60339 binding site and the interface-adjacent pocket, confirming that free motion in these flexible regions is critical to the efflux pump’s activity.
We're excited to see the community reaction to this new mechanism — this new way of thinking about inhibition.
- Eamonn Reading, University of Southampton
Previous research revealed that AcrB cycles through various conformational states depending on whether it has an antibiotic to transport (6). The results of these experiments suggest that the flexible regions in AcrA shift in response to AcrB’s conformational changes, signaling the TolC pore to open and close. NCS 60339 appears to act as a molecular wedge that locks these regions in place to inhibit the pump. Another collaborator in the study, Georgia Institute of Technology physicist James Gumbart, ran molecular dynamics simulations that supported this mechanism.
“We need to have more mechanistic studies that give insight like this,” said Brenda Wilson, a microbiologist at the University of Illinois Urbana-Champaign who was not involved in the study. Wilson envisions that this new information on the inner workings of AcrAB-TolC can guide the development of combinatorial drugs with both pump inhibition and antibacterial activity. “Now you actually have an inhibitor that can be combined with many different kinds of antibiotics and allow them to work,” she said.
Reading’s team is now further validating their mechanism with in situ studies and aims to soon begin screening inhibitor candidates. They hope to discover compounds that readily cross the bacteria’s outer membrane to reach AcrA and bind one or both of the flexible regions they identified. “We're excited to see the community reaction to this new mechanism — this new way of thinking about inhibition,” said Reading. “Hopefully, that is going to yield these next exciting steps.”
- Sun, J.J. et al. Bacterial multidrug efflux pumps: mechanisms, physiology and pharmacological exploitations. Biochem Biophys Res Commun 453, 254-267 (2014).
- World Health Organization. Antimicrobial resistance.
- Lewis, B.R. et al. Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein. Nature communications. Nat Commun 14, 3900 (2023).
- Abdali, N. et. al. Reviving antibiotics: efflux pump inhibitors that interact with AcrA, a membrane fusion protein of the AcrAB-TolC multidrug efflux pump. ACS Infect Dis 3, 89-98 (2017).
- Masson, G.R. et. al. Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments. Nat Methods 15, 595-602 (2019).
- Chen, M.Y. et. al. In situ structure of the AcrAB-TolC efflux pump at subnanometer resolution. Structure 30, 107-113.e3 (2022).