Protein structure provides clues to drug resistance mechanisms

MIT chemists have discovered how the structure of the EmrE transporter changes as the compound moves. The left side is a transporter structure at high pH. As the pH drops (right), the helix begins to tilt and the channels open more towards the outside of the cell, leading to the compound. Credit: MIT

MIT chemists have discovered the structure of proteins that can pump toxic molecules from bacterial cells. A protein similar to this is E. coliIt is believed to help the bacteria become resistant to multiple antibiotics.

use Nuclear magnetic resonance (NMR) spectroscopy, researchers were able to determine the structure of this protein It changes as a molecule, such as a drug, moves through it.Knowledge of this detailed structure may make it possible to design drugs that may block these. Transport proteins May Hong, a professor of chemistry at the Massachusetts Institute of Technology, said it helps resensitize drug-resistant bacteria to existing antibiotics.

“Knowing the structure of the drug-binding pockets of this protein allows us to design competitors for these substrates to block binding sites and prevent the protein from removing antibiotics from cells,” Hong said. Says. Senior author of the dissertation.

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

Drug resistance transporter

Ejecting drugs from cell membranes is one of many strategies that bacteria can use to avoid antibiotics. Hentsler Wildman’s group at the University of Wisconsin has been researching a membrane-binding protein called EmrE that can transport a variety of toxic molecules, such as herbicides and antibacterial compounds, for several years.

EmrE belongs to a family of proteins called small multidrug resistance (SMR) transporters. EmrE is not directly involved in antibiotic resistance, but other members of the family have been found in the form of drug resistance Mycobacterium tuberculosis When Acinetobacter Baumani..

“SMR transporters have high sequence conservation across major regions of the protein. EmrE is by far the most well-studied member of the family. In vitro When In vivoIt will be an ideal model system for investigating the structures that support SMR activities, “says Hentsler Wildman.

A few years ago, Hong’s lab developed a technique that allowed researchers to use NMR to measure the distance between a fluorine probe and a hydrogen atom in a protein. This allows you to determine the structure of the protein that binds to the fluorine-containing molecule.

After Hong talked about the new technology at the conference, Hentsler-Wildman suggested that they team up to study EmrE. Her lab has spent many years studying how EmrE transports drug-like molecules or ligands across phospholipid membranes. This ligand known as FFour-TPP+ +Is a tetrahedral molecule with four fluorine atoms bonded, one at each corner.

Using this ligand in conjunction with Hong’s new NMR technology, researchers set out to determine the atomic resolution structure of EmrE. It is already known that each EmrE molecule contains four transmembrane helices that are nearly parallel. As the two EmrE molecules assemble into a dimer, eight transmembrane helices form the inner wall and interact with the ligand as it moves through the channel. Previous studies have revealed the overall topology of the helix, but not the topology of the protein side chains that extend inside the channel. It’s like an arm that grabs a ligand and guides it through a channel.

EmrE transport Toxic molecule From the inside of bacterial cells 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 a protein that binds to F.Four-TPP+ + In an acidic environment.New Nature Communications In the study, they analyzed the structure at neutral pH, allowing them to determine how the structure of the protein changes as the pH changes.

Complete structure

At neutral pH, researchers in this study found that the four helices that make up the channel were relatively parallel to each other, creating an opening through which the ligand could easily enter. As the pH drops and moves towards the outside of the membrane, the helix begins to tilt and the channels open more towards the outside of the cell. This helps push the ligand out of the channel. At the same time, some rings found in the protein side chains shift their orientation in a way that also helps derive ligands from the channels.

The acidic end of the channel also welcomes the proton more, allowing the proton to enter the channel, help the channel open further, and allow the ligand to exit more easily.

“This paper really completes the story,” says Hong. “One structure is not enough. To understand how the transporter actually opens on both sides of the membrane, we need two. Ligand Or an antibiotic compound from the inside of the bacterium to the outside of the bacterium. “

Since the EmrE channel is thought to transport many different toxic compounds, Hong and her colleagues are currently planning to study other methods. molecule Move the channel.

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For more information:
The high pH structure of EmrE reveals the mechanism of proton-bound substrate transport. Nature Communications (2022). DOI: 10.1038 / s41467-022-28556-6

Quote: The protein structure is the drug resistance mechanism obtained on February 18, 2022 from https: // (February 18, 2022). Provides clues for the day)

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Protein structure provides clues to drug resistance mechanisms

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