Author interview: Haaris Safdari has completed his PhD at the University of Hamburg, Germany under Prof. Daniel Wilson, studying translational machinery using biochemical and structural analysis (cryo-EM).
Lab: Prof. Daniel Wilson, University of Hamburg, Germany
Research Summary: Our study explores how kasugamycin, edeine, and GE81112, chemically distinct compounds, bind a conserved site on the 30S ribosomal subunit to uniquely inhibit translation initiation.
What was the core problem you aimed to solve with this research?
Translation initiation is the rate-limiting step during protein synthesis and is critical for selecting the correct reading frame of the mRNA. Unsurprisingly, it is targeted by several potent antibiotics such as kasugamycin (Ksg), edeine (Ede), and the recently discovered tetrapeptide GE81112 (GE). With the rise in multidrug-resistant bacteria, understanding the mechanisms of these inhibitors is crucial for developing improved antimicrobial agents. Although structures of these compounds bound to the 30S subunit have been reported, none were determined in the context of translation initiation complexes, and the resolutions were limited (3.3–13 Å). Additionally, discrepancies between the available structures and biochemical data, especially for edeine and GE81112 left their mechanisms unclear.
a Overall location of three antibiotics relative to the mRNA (green) with the initiator tRNA (grey) in the P-site shown for reference. b 30S is joined by IF1, IF2, IF3 and mRNA to form 30S-IFs complex. c Ksg, Ede, and GE interfere with the mRNA placement across the mRNA path, though it allows the SD-aSD interaction. d tRNA joins the 30S-IFs complex to form 30S-PIC1 complex in which tRNA is non-accommodated (with 30S head open). e Conformation of tRNA with simultaneous head movement to form 30S-PIC2 complex with head closed and ordered (and tRNA accommodated). This transition from 30S-PIC1 to 30S-PIC2 is also inhibited by Ksg and Ede. f IF3-CTD moves away from the P site and forms 30S initiation complex (30S IC). GE81112 prevents IF3-CTD from leaving the P site, hindering the formation of the 30S-IC. g 50S subunit joins the 30S-IC to form the 70S-IC. h 70S-IC formed to ready to be matured into 70S elongation-competent complex (70S-EC).
How did you go about solving this problem?
We assembled E. coli 30S ribosomal subunits in vitro with all three initiation factors (IF1, IF2, IF3), mRNA, tRNA, GTP, and each antibiotic (Ksg or Ede or GE) to form 30S initiation intermediate complexes. These were then used to determine cryo-EM structures of the antibiotics bound to 30S. A key advantage of cryo-EM over X-ray crystallography is its ability to capture various conformational states from a single dataset which helped us to decipher a detailed mechanism of these antibiotics.
We also performed light scattering and FRET experiments in collaboration with Prof. Pohl Milon’s lab at UPC, Peru, to complement our structural findings.
How would you explain your research outcomes (Key findings) to the non-scientific community?
Our research explores how certain antibiotics (kasugamycin, edeine, and GE81112) interfere with bacterial growth by targeting their ribosomes, the molecular machines that make proteins. Just like a chef follows a recipe, bacteria rely on ribosomes to read genetic instructions and build proteins essential for survival. We found that these antibiotics bind to specific parts of the ribosome, disrupting this process. As a result, the bacteria cannot make the proteins they need and stop growing.
Interestingly, although all three drugs bind to the same general area on the ribosome, they block protein synthesis in different ways. By identifying precisely how and where they bind, we gain valuable insights that could help design better antibiotics.
What are the potential implications of your findings for the field and society?
Our study shows that, contrary to earlier models, all three translation initiation inhibitors bind to a highly conserved site on the 30S subunit. However, their distinct chemical structures lead them to block different steps of the initiation process. Beyond clarifying the actions of kasugamycin, edeine, and GE81112, our findings also enhance the general understanding of translation initiation.
Importantly, we describe how these drugs interact with the ribosome in detail, including water-mediated interactions. This knowledge may aid in designing improved antibiotics. For example, since the binding sites of Ksg and GE are adjacent, hybrid compounds linking both molecules could be developed to better evade antibiotic resistance.
What was the exciting moment during your research?
The most exciting moment was discovering completely different binding sites for edeine and GE81112 in our cryo-EM structures compared to earlier X-ray studies. We noticed inconsistencies in those previous models, likely due to their lower resolution and crystallographic artifacts. Notably, our binding site for edeine aligns with that seen in eukaryotic ribosomes, supporting its role as a universal translation inhibitor. Additionally, our observations match previous biochemical data unlike the older structural models.
Reference: Safdari et al. The translation inhibitors kasugamycin, edeine and GE81112 target distinct steps during 30S initiation complex formation. Nat Commun 16, 2470 (2025). https://doi.org/10.1038/s41467-025-57731-8
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