Selective sabotage: How macrolide antibiotics jam the ribosome, but only for certain proteins

Original article on work by the Polikanov lab can be found at the APS website.

In the arms race that is the life of bacteria, crafty microbial combatants have developed chemical weaponry that targets the cellular machinery of their enemies and kills them. For the last 100 years, scientists have studied various strains of bacteria to identify and isolate these antibiotic chemicals to be used as treatments for our own bacterial infections.

This approach has yielded many of our most effective medicines and detailed study of the mechanisms of action of antibiotics has led to efforts to adapt their chemical structures to make them even more effective. However, as we have used these drugs, those shrewd bacterial chemists have found ways to circumvent antibiotic action and develop resistance, so new drugs are needed.

One important class of antibiotics, called macrolides, was originally isolated from soil bacteria and found to target the protein-producing machines, called ribosomes, of many Gram-positive bacterial species that can cause serious infections in humans. Researchers who study macrolide antibiotics have thought for many years that they act by simply clogging the tunnel in the bacterial ribosome where peptides emerge as they are made.

However, recent studies have shown that bacterial protein synthesis is not completely blocked in the presence of macrolides. In fact, it appears that most peptides are allowed through the tunnel even in the presence of “tunnel-clogging” macrolides except those with a specific sequence of amino acids. This interesting discovery that, rather than being indiscriminate blockers of protein synthesis, macrolides act in a context-specific fashion, has been recently built upon by research conducted by a team from the University of Illinois, Chicago who sought to elucidate the structural basis for this new understanding of the mechanism of action of macrolides.

The team used the resources of the Northeastern Collaborative Access Team (NE-CAT) beamlines at 24-ID-C and 24-ID-E of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science user facility at DOE’s Argonne National Laboratory.

The goal of the research project was to provide detailed structural information to potentially inform structure-based drug design efforts to overcome macrolide resistance mechanisms and develop new antibiotics. The research question was driven by the recent advances in our understanding of the mechanism of action of macrolides that showed that, although they acted by binding to a critical region of the tunnel, translation of some proteins was not blocked by macrolide binding.

Cryo-electron microscopy data showed that many protein chains can fit comfortably in the ribosomal tunnel in the presence of macrolide antibiotics and ribosome profiling data showed that the majority of proteins for which translation is blocked by macrolides have stalled at a sequence with a lysine or arginine amino acid, followed by another amino acid, and then followed again by lysine or arginine. This is called the +X+ motif.

To understand how macrolide antibiotics block the translation of proteins with +X+ motifs, the research team developed in vitro tools to recapitulate different scenarios with purified components.

They then solved structures that mimicked ribosomal translation with different peptides to capture the stalled and unstalled states in the presence or absence of a macrolide antibiotic. The X-ray crystal structures showed that the macrolide bound to the ribosome did not allosterically reconfigure the tunnel as was thought before. It appears, instead, that the key to macrolide action is a partial block of the first tRNA binding site, called the A site, that causes crowding of bulky arginine or lysine amino acids. When these are in the +X+ configuration, the macrolide molecule causes the first lysine/arginine already in the peptide to lean over a bit into the A site and prevent the other one from coming in because they are both so bulky.

In other words, when the macrolide is there, the next lysine/arginine residue cannot be accommodated and translation is stalled. Other amino acid combinations don’t have this problem, so they don’t stall. The structures show that the crowded alignment of the two arginine/lysine amino acids in the peptidyl transferase center when the macrolide is there means they are not close enough to undergo the nucleophilic attack needed for peptide bond formation.

Interestingly, modeling of the site with other bulky amino acids in place of lysine or arginine suggests that amino acids such as tyrosine or phenylalanine are too rigid to move over and block ribosome-catalyzed peptide transfer reaction in the presence of the macrolide. Instead, they likely just push out the drug. This enhanced understanding of the nuances of ribosome/antibiotic interactions with the growing peptide chain will be important for informing the design of new macrolide-based antibiotics in the ongoing battle between microbes and humans. – Sandy Field

E.A. Syroegin1, E.V. Aleksandrova1, A.A. Kruglov1, M.N. Paranjpe1, M.S. Svetlov1, Y.S. Polikanov1, “Structural insights into context-specific inhibition of bacterial translation by macrolides,” Nat Commun 16, 9685 (2025). https://doi.org/10.1038/s41467-025-64692-5