New insights into the link between protein dynamics and antibiotic resistance

ABC transporters are molecular pumps embedded in the cell membrane, which are gatekeepers of the cell. They recognize and translocate a wide range of substrates and thus regulate the general bioavailability of molecules.

In normal conditions, they generally help to detoxify cells against xenobiotics, such as drugs, pollutants and toxins. However, this protective role can have a negative impact when we try to get rid of a bacterial infection, as the transporter will protect the bad bacteria against the medicine, making the therapies ineffective. This resistance mechanism can be found in all kingdoms of life, protecting bacteria against antibiotics, in pathogenic yeast against antifungal agents, and in cancer settings upon chemotherapy treatments. The mechanisms of action remain somewhat similar across these large classes of living beings, with some local specificities that make each case unique.

It is crucial to understand the interplay of these transporters with these drugs to decipher their transport mechanisms, opening the way to rational design of inhibitors that can effectively block them and restore drug sensitivity.

CM01 to track how BmrA pumps out drugs

Scientists led by the CNRS in France have investigated the mechanism by which the ABC transporter BmrA, found in Bacillus subtilis, pumps antibiotics out. In the bacterial warfare for a habitat, B. subtilis uses BmrA to defend itself against antibiotic secreted by competitors.  

In particular, they focused on how the transporter binds ATP, then breaks it down to release energy to remove drugs or toxins from inside the cell to outside. They came to the ESRF’s cryo- electron microscope CM01 to track how BmrA pumps out drugs, using two dyes. In particular, they used drugs Rhodamine 6G and Hoechst 33342, which act like a substrate of the transporter (meaning that they can be recognized, bound and moved out of the cell by the transporter).

BmrA’s action starts in an open conformation towards the cell (inward-facing), which allows the drug to bind. When ATP attaches, BmrA changes shape and opens outward, pushing the drug out. CryoEM experiments provided important details on the transition of BmrA from inward to outward conformations.

The team then extracted structural dynamics information, and complemented them with an integrative structural enzymology approach, which revealed how drugs influence ATP-binding a long distance away.

“The novelty of our approach resides in how we extract the conformational space exploration of our transporter”, says Vincent Chaptal, researcher at the CNRS- University of Lyon-1 and corresponding author of the publication. “It is amazing how we can now visualise protein movements in response to ligand binding and measure it. We used to solve structures in defined states, we now have access to their deformations between states, it is a dream come true”, he adds. “This revolution is possible thanks to cryoEM. The transporter is frozen ‘in action’ and cryoEM visualises all these conformations. The quality of the data that we obtained from CM01 at the ESRF is essential for the success of such an investigation”

Whilst this research is academic, its results may inspire pharmaceutical companies to design drugs that can block ABC transporters in the future.

The next step for the team is to explore this flexibility in many various settings to link more observations to functional studies.

Reference:

Gobet, A. et al, Nat Commun 16, 1745 (2025). https://doi.org/10.1038/s41467-025-56849-z

Text by Montserrat Capellas Espuny

Top image: Variability analysis of BmrA in the IF conformation with several ligands. A/ 3DVA analysis of E504Aapo in several components, resulting in 20 maps each. Models built by variability refinement in the maps are represented in cartoon and colored from blue to cyan, with view from the NBDs. The main movement is represented on the structure with black lines representing the NBD rotation and the colored arrows depict the movement and its amplitude. B/ same as A/ for E504AR6G, colored from red to yellow. C/ same as A/ for E504AH33342, colored from green to white.