Human ribosomes interactions reveal the binding landscape of neomycin

Aminoglycosides such as neomycin are clinically limited by toxicity, commonly attributed to the inhibition of mitoribosomes. Here, we investigate this model by integrating Cryo-EM and functional assays to assess neomycin's binding landscape and functional consequences for human ribosomes. Cryo-EM reveals extensive multi-site binding of neomycin on both human mitochondrial and cytosolic ribosomes in vitro, including conserved regions of the decoding center. In contrast, ribosomes isolated from neomycin-treated cells show markedly different patterns, with no detectable neomycin density on the mitoribosomes and reduced occupancy at a subset of cytosolic ribosome sites. Functional assays indicate that neomycin remained active in cells, inducing widespread stop-codon readthrough while having minimal effects on global mitochondrial and cytosolic protein synthesis. Together, these findings highlight a disconnect between the structural binding landscape and ribosome-associated neomycin recovered following cellular exposure, while linking neomycin exposure to altered translational fidelity with minimal effects on global protein synthesis.