Using nuclear magnetic resonance (NMR) spectroscopy, chemists have discovered how the structure of E. coli’s EmrE efflux pump—a transporter that can pump toxic molecules like antibiotics out of bacterial cells—changes as a compound moves through it. The scientists found that, as the pH drops, the helices begin to tilt so that the channel is more open toward the outside of the cell, guiding the compound out. Knowledge of this detailed structure may make it possible to design drugs that could block these transport proteins and help resensitize drug-resistant bacteria to existing antibiotics.
“Knowing the structure of the drug-binding pocket of this protein, one might try to design competitors to these substrates, so that you could block the binding site and prevent the protein from removing antibiotics from the cell,” said Mei Hong, PhD, professor of chemistry at MIT.
This work appears in Nature Communications in the paper, “High-pH Structure of EmrE Reveals the Mechanism of Proton-Coupled Substrate Transport.”
Pumping drugs out through their cell membranes is one of many strategies that bacteria can use to evade antibiotics. The membrane-bound protein EmrE belongs to a family of proteins known as small multidrug resistance (SMR) transporters. Although EmrE is not directly involved in resistance to antibiotics, other members of the family have been found in drug-resistant forms of Mycobacterium tuberculosis and Acinetobacter baumanii.
“The SMR transporters have high sequence conservation across key regions of the protein. EmrE is by far the best-studied member of the family, both in vitro and in vivo, which makes it an ideal model system to investigate the structure that supports SMR activity,” said Katherine Henzler-Wildman, PhD, professor of biochemistry at the University of Wisconsin at Madison.
Using an improved NMR technique, and the ligand F4-TPP+, the researchers set out to determine an atomic-resolution structure of EmrE. It was already known that each EmrE molecule contains four transmembrane helices that are roughly parallel. Two EmrE molecules assemble into a dimer, so that eight transmembrane helices form inner walls that interact with the ligand as it moves through the channel.
EmrE transports toxic molecules from the inside of a bacterial cell, which is at neutral pH, to the outside, which is acidic. This change in pH across the membrane affects the structure of EmrE. In a 2021 paper, Hong and Henzler-Wildman discovered the structure of the protein as it binds to F4-TPP+ in an acidic environment. In the new study, they analyzed the structure at a neutral pH, allowing them to determine how the structure of the protein changes as the pH changes.
At neutral pH, the four helices that make up the channel are relatively parallel to one another, creating an opening that the ligand can easily enter. As the pH drops, moving toward the outside of the membrane, the helices begin to tilt so that the channel is more open toward the outside of the cell. This helps to push the ligand out of the channel. At the same time, several rings found in the protein side chains shift their orientation in a way that also helps to guide the ligand out of the channel.
The acidic end of the channel is also more welcoming to protons, which enter the channel and help it to open further, allowing the ligand to exit more easily.
“This paper really completes the story,” Hong said. “One structure is not enough. You need two, to figure out how a transporter can actually open to both sides of the membrane, because it’s supposed to pump the ligand or the antibiotic compound from inside the bacteria out of the bacteria.”
The EmrE channel is believed to transport many different toxic compounds, so Hong and her colleagues now plan to study how other molecules travel through the channel.
