Protein structure offers clues to drug-resistance mechanism
A new study sheds light on how a protein pumps toxic molecules out of bacterial cells

Date:
February 18, 2022
Source:
Massachusetts Institute of Technology
Summary:
Chemists discovered the structure of a protein that can pump toxic
molecules out of bacterial cells. Knowledge of this structure
may make it possible to design drugs that could block transport
proteins and help resensitize drug-resistant bacteria to existing
antibiotics.



FULL STORY ==========================================================================
MIT chemists have discovered the structure of a protein that can pump
toxic molecules out of bacterial cells. Proteins similar to this one,
which is found in E. coli, are believed to help bacteria become resistant
to multiple antibiotics.


========================================================================== Using nuclear magnetic resonance (NMR) spectroscopy, the researchers were
able to determine how the structure of this protein changes as a drug-like molecule moves through it. 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,
says Mei Hong, an MIT professor of chemistry.

"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," says Hong, who is the senior author of the paper.

MIT graduate student Alexander Shcherbakov is the lead author of the
study, which appears today in Nature Communications. The research team
also includes MIT graduate student Aurelio Dregni and two researchers
from the University of Wisconsin at Madison: graduate student Peyton
Spreacker and professor of biochemistry Katherine Henzler-Wildman.

Drug-resistance transporters Pumping drugs out through their cell
membranes is one of many strategies that bacteria can use to evade
antibiotics. For several years, Henzler-Wildman's group at the University
of Wisconsin has been studying a membrane-bound protein called EmrE,
which can transport many different toxic molecules, including herbicides
and antimicrobial compounds.



==========================================================================
EmrE belongs to a family of proteins called the 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," Henzler-Wildman says.

A few years ago, Hong's lab developed a technique that allows researchers
to use NMR to measure the distances between fluorine probes and hydrogen
atoms in proteins. This makes it possible to determine the structure of
a protein as it binds to a molecule that contains fluorine.

After Hong gave a talk about the new technique at a conference,
Henzler-Wildman suggested that they team up to study EmrE. Her lab has
spent many years studying how EmrE transports a drug-like molecule, or
ligand, across the phospholipid membrane. This ligand, known as F4-TPP+,
is a tetrahedral molecule with four fluorine atoms attached to it,
one at each corner.

Using this ligand with Hong's new NMR technique, 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. Previous studies have revealed
the overall topology of the helices, but not of the protein side chains
that extend into the channel interior, which are like arms that grab
the ligand and help guide it 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 Nature Communications
study, they analyzed the structure at a neutral pH, allowing them to
determine how the structure of the protein changes as the pH changes.

A complete structure At neutral pH, the researchers found in this study,
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 says. "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.

The research was funded by the National Institutes of Health and the
MIT School of Science Camplan Fund.

========================================================================== Story Source: Materials provided by
Massachusetts_Institute_of_Technology. Original written by Anne
Trafton. Note: Content may be edited for style and length.


========================================================================== Journal Reference:
1. Alexander A. Shcherbakov, Peyton J. Spreacker, Aurelio J. Dregni,
Katherine A. Henzler-Wildman, Mei Hong. High-pH structure of EmrE
reveals the mechanism of proton-coupled substrate transport. Nature
Communications, 2022; 13 (1) DOI: 10.1038/s41467-022-28556-6 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2022/02/220218080243.htm

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