⭐️ Present in the Cell, Yet Absent from the Binding Site

The conventional view of antibiotic toxicity often assumes that once a molecule enters the cell, it directly binds and exerts its effect. However, neomycin exhibits a more complex behavior: while it binds tightly to ribosomes in cell-free biochemical assays, it acts differently within the complex environment of an intact cell.

Utilizing high-resolution cryo-electron microscopy (cryo-EM), our team captured near-atomic resolution structures of human ribosomes extracted directly from cells. The results revealed an unexpected finding: despite the intracellular presence of neomycin, the functional decoding center of a major ribosome population remained in an ligand-free state (Apo state).

Based on this, we proposed a "Conditional Engagement Model." The intracellular physicochemical environment appears to act as a regulatory barrier, meaning that cellular entry does not automatically guarantee ribosome binding. This discovery offers a nuanced perspective that refines conventional paradigms in the field.

⭐️ The Toxicity Mechanism: Disrupting Fidelity Rather Than Halting Translation

If the drug does not fully lock down the ribosome, where does neomycin’s toxicity stem from? By integrating biochemical assays with omics analyses, our team investigated the underlying mechanism: neomycin does not completely halt protein synthesis, but rather impairs its accuracy.

Our study shows that neomycin interferes with translational fidelity via a subtle "induced-fit" mechanism, leading to widespread "stop codon readthrough." In short, instead of stopping work, the ribosomes begin producing aberrant, mistranslated proteins, which ultimately induces severe cellular stress.

⭐️ Scientific Significance and Outlook

This work introduces a new perspective for studying the toxicity of aminoglycosides and provides structural insights for designing next-generation antibiotics with higher efficacy and reduced toxicity. Understanding how drugs behave in the actual cellular context is essential for engineering therapeutics that precisely target pathogens while sparing human cells.

⭐️ The Team and Collaborators Behind the Work

A great team effort from everyone involved (＾－＾)V :

• Meng Wu, Lingfan, Keqin, along with our graduated alumni Xinyi and Changfeng, contributed equally as co-first authors!

• Excellent support was provided by our teammates Shuangjie, Professor Zhe Zhang, Chuansen, and Yuanjie.

• Professor Xuben Hou from the School of Pharmaceutical Sciences and Professor Guolei from the Children's Hospital served as co-corresponding authors for this project.

  We would like to express our sincere gratitude to Professor Jamie Doudna Cate (UC Berkeley) and Professor Alexey Amunts (Max Planck Institute) for their invaluable support and inspiring discussions throughout the development of this project.

  
  

  Paper Link: https://www.biorxiv.org/content/10.1101/2025.07.22.666027v3