Chasing the tail: Biochemists zone in on bacterial transporter’s tip as its secret to antibiotic resistance

Photo of Katie Henzler-Wildman
Professor Katherine Henzler-Wildman.

Nutrients in. Waste out. Everyone and almost every living thing does it, even at the cellular level. It seems like a simple enough concept: to survive, cells have to have a way to move different types of molecules in and out of themselves. It’s how they take in food, get rid of waste, send signals to each other, sense their environment, and more. But the reality is much more complex. Transmembrane proteins — those embedded in the cell membrane and of which some are responsible for this transmission of molecules — are notoriously hard to study and understand.

University of Wisconsin–Madison associate professor of biochemistry Katherine Henzler-Wildman has dedicated her lab to doing a deep dive into these transporters and channels. In particular, part of the lab has been focusing on one called EmrE, which is present in the bacteria E. coli. It’s part of a family of bacterial transporters called multidrug resistance transporters because most confer resistance to antibiotics. Through a series of studies that exemplify the incremental process of basic scientific discovery, her lab is uncovering how exactly this transporter works — and the secret could lie in its tiny molecular “tail.”

“I’m fascinated by membrane proteins, channels and transporters in particular, because that’s how molecules move in and out across the membrane barrier and that’s a super important process,” Henzler-Wildman says. “These relatively big conformational changes are regulated by protons, something so small. These transporters can be a bit of a black box so it’s important to understand how they work.”

Schematic showing a membrane closing and opening according to specific actions.
This schematic shows the bacterial membrane transporter EmrE, nestled in the inner membrane of E. coli. The blue and purple structures represent the “rocking bananas” of the transporter that rocks back and forth across the membrane to move protons (H+) into the cell (cytoplasm) and drugs (red molecule) out. Image courtesy of Nathan Thomas.

EmrE is small, among the smallest transporter of its kind. Made of just eight main parts called helices, they rock back and forth, connecting at one end to close and moving apart to open the other end. Scientists have a fun shorthand name for transporters that move this way — rocking bananas. Other transporters open and close in different ways to move molecules across the membrane through “elevator” or rotary mechanisms.

EmrE is found only in bacteria and transports all kinds of molecules. Some of these molecules happen to be drugs used to kill the bacteria. These transporters confer resistance to the cell by pumping the antibiotic drugs out. This may or may not be EmrE’s original, native function, which is something that still eludes researchers.

The transporter moves molecules using what is called the proton motive force. Other membrane transporters maintain an environment where there are more protons outside the cell than inside. This means protons naturally want to move into the cell — to spread out from higher concentration to lower concentration. This “downhill” movement of protons can be used to do work the same way that the downhill flow of water can be used to power machinery or create electricity – although the exact mechanism for harnessing the energy and using it to drive another process is a bit different at the molecular level. The proton motive force provides the chemical fuel that EmrE uses to pump molecules, such as antibiotics, to the outside of the cell. The transporter physically moves bound protons in and drugs out through a series of conformational changes.

For years, researchers thought this exchange of protons and drugs was very strict. However, Henzler-Wildman and her team of researchers showed this was not the case in a paper published in 2017 in the Proceedings of the Natural Academy of Sciences (PNAS). The study found that proton and drug movements are not as strictly coupled as they thought in EmrE. This transporter can actually also move drugs and protons across the membrane in the same direction, as well as the opposite direction — introducing the option of moving molecules both into or out of the cell.

While their new model did a better job of explaining the observed activity of EmrE, they acknowledged it wasn’t perfect.

Illustration of hypothesis in molecular form.
This illustration highlights the researchers’ hypothesis of the transporter’s activity. The largely ignored “tail” of the transporter is depicted by a chain that stops the flow of protons when drug is not present. The gate can be opened when drug (red molecule) “unlocks” it. Illustration by Laura Vanderploeg.

“We think the model we proposed did a better job of describing EmrE behavior than the traditional line of thought but it wasn’t perfect,” says Nathan Thomas, the first author on the study and an Integrated Program in Biochemistry (IPiB) graduate student in the Henzler-Wildman Lab. “If you look at the model at face value, it seems as if the cell would continually take in protons, which would be detrimental to the cell. So we dug deeper.”

Now in a new paper in the Journal of Biological Chemistry published in late 2018, the team presented their next step in the project. Here they describe how a largely ignored “tail” on the end of the transporter could be serving as a gate to stop the flow of protons when drug isn’t present. The lab uses nuclear magnetic resonance (NMR) spectroscopy in their work, a technique that allows them to take very precise measurements at the molecular level to piece together the structure or movement of a molecule or complex. The paper was recently selected as an Editors’ Pick from the journal, a distinction that places it in the top 2% of the more than 6,600 papers they publish each year.

“It’s part of the protein no one really had ever thought of before because in general we think of these tails as being unstructured and kind of flopping around,” Thomas explains. “What our data point to and we will try to confirm next is somehow the tail is tightly coupled to the binding site down inside the transporter, which is very surprising. And we start to look at published results from others that have been going on for about two decades and this idea starts to make sense. Does this mean tails in other transporters are important? Some could be but not all. This tail is highly evolutionarily conserved so we knew it might have a purpose but it might be worth looking at other transporter tails, too.”

The implications of their work cover many areas. They say their first paper especially points to the importance of understanding how this transporter confers antibiotic resistance and might point to a way to actively transport drugs into bacteria. Their second paper adds more to the story of EmrE as a model for other membrane transporters. Since it is small and easy to study, its inner workings can give valuable insights into larger and more complex transporters.

“There are many other important and larger transporters in other larger organisms that are involved in important biological processes or implicated in disease,” Henzler-Wildman says. “Having the details from this simple model allows scientists to start designing experiments to specifically probe those larger and more complex transporters. If we don’t have these details on these smaller systems we don’t even know what to look for in the larger ones. The tail isn’t just the throw away bit we once thought and we will continue to test this idea.”

This research was funded by the National Institutes of Health Grant 1R01GM095839.