High-Resolution Cryo-EM Imaging Reveals a Hidden Secondary Drug Binding Site on Bacterial Ribosomes

A new Nature Communications study, published May 19, redefines scientists' understanding of how a popular class of antibiotics work.

For decades, doctors have widely used tetracyclines for conditions ranging from acne to tick-borne illnesses. Using high-resolution imaging technology, researchers in the laboratory of Christopher Bunick, MD, PhD, associate professor of dermatology at Yale School of Medicine (YSM), captured a never-before-seen look into how different kinds of tetracyclines bind to and kill bacteria.

The findings could help scientists learn how to develop new therapies that are more potent, safer for the gut microbiome, and less susceptible to antibiotic resistance.

"We as dermatologists have to be tetracycline experts because we write so many tetracycline prescriptions," says Bunick, who is the study's principal investigator. "And that means we need to better understand these drugs."

How Tetracyclines Bind to Bacteria

Scientists knew that tetracyclines work by binding to the bacterial ribosome, a molecule that synthesizes proteins. Specifically, they bind to a region known as the mRNA decoding center where they block protein production, preventing bacteria from growing.

In the new study, scientists used a technique called single particle cryo-electron microscopy (cryo-EM) to visualize how common kinds of tetracyclines-including doxycycline, sarecycline, and minocycline-bound with the ribosomes of common gut microbe Escherichia coli and acne-causing Cutibacterium acnes.

The researchers discovered that in addition to the mRNA decoding center, all of the tetracyclines also bound to a second site on the bacterial ribosome called the nascent peptide exit tunnel (NPET), where newly synthesized proteins leave the ribosome.

The team also observed the unique ways through which the different tetracyclines interacted with this secondary binding site. Doxycycline, for example, is one of the most commonly prescribed antibiotics due to its high potency. Unlike most antibiotics, which bind as single molecules, doxycycline can stick to itself and form pairs, or dimers, in the NPET of both E. coli and C. acnes. These dimers more effectively block the tunnel and prevent proteins from getting through.

"Every year, there are around 11 million prescriptions for doxycycline, and yet, for decades, people didn't know how it was working," says Swapnil Chandrakant Devarkar, PhD, associate research scientist at YSM and the study's co-first author. "Our study provides a structural and mechanistic basis for why this one drug works so well."

Sarecycline received approval from the U.S. Food and Drug Administration (FDA) for treating moderate to severe acne in 2018. Unlike its predecessors, the drug is narrow-spectrum-it targets C. acnes while preserving beneficial gut bacteria.

How it could function as an antibiotic and not significantly harm the gut microbiome-no one knew why."

Christopher Bunick, Associate Professor, Dermatology, Yale School of Medicine

The new findings offer an explanation. Biologists classify bacteria as gram-positive or gram-negative based on key structural differences. C. acnes, for example, are gram-positive bacteria, and E. coli are gram-negative. In gram-negative E. coli, NPETs offered less space for sarecycline to bind, as it has a bulkier molecular structure than other tetracyclines. This forced the drug to flip its position 180 degrees in order to fit into the binding pocket. As a result, sarecycline was less likely to bind to NPET in E. coli, reducing its potency against the bacteria.

Developing Improved Antibiotics

The study offers a roadmap to creating stronger antibiotics, the researchers say. They plan to use these new insights to create future versions of tetracycline that dimerize, as seen in doxycycline, and are thus more potent against harmful bacteria.

"This dimerization in the ribosome could lead to the development of antibiotics with very powerful activity," says Ivan Lomakin, PhD, research scientist in dermatology at YSM and the study's co-first author and corresponding author.

The study also provides the first structural evidence for why sarecycline preferentially targets gram-positive organisms like C. acnes while preserving gram-negative organisms in the gut. This can help scientists understand ways to create new narrow-spectrum tetracyclines that target pathogenic species while sparing the gut microbiome. Not only would this reduce side effects associated with the loss of healthy gut microbes, but also mitigate risk of bacteria becoming antibiotic resistant.

"We want to leverage our new knowledge to innovate new therapeutics that address microbiome concerns, as well as resistance concerns," Bunick says.