Khawaja, A. | bioRxiv Science

bioRxiv2022

Structural basis of streptomycin off-target binding to human mitoribosome

The ribosome in mitochondria regulates cellular energy production, and its deactivation is associated with pathologies and ageing. Inhibition of human mitoribosome can be caused by antimicrobial off-target binding, which leads to clinical appearances. The anti-tuberculosis drug aminoglycoside streptomycin targets the small subunit and was shown to be coupled with a bilateral decreased visual acuity with central scotomas and an altered mitochondrial structure. Previously, we reported mitochondria-specific aspects of translation related to specialties of the human mitoribosome (Aibara et al., 2020). In this Research advance article, we report 2.23-[A] resolution structure of the human mitoribosomal small subunit in complex with streptomycin. The structural data reveals new details of the streptomycin interactions, including specific water molecules and metal ions involved in the coordination. The density for the streptose moiety reveals that previously modeled aldehyde group appears as a loosely bound density, and the hydroxyl group is not resolved. The density replacing the aldehyde group is within hydrogen bonding distance of four phosphate groups of rRNA, suggesting that the ribosome-bound streptomycin is likely to be in the hydrated gem-diol form rather than in the free aldehyde form. Since streptomycin is a widely used drug for treatment, the newly resolved fine features can serve as determinants for targeting.

molecular biology↗

bioRxiv2021

Structural basis for late maturation steps of the human mitoribosomal large subunit

Mitochondrial ribosomes (mitoribosomes) synthezise a critical set of proteins essential for oxidative phosphorylation. Therefore, their function is vital to cellular energy supply and mitoribosomal defects give rise to a large and diverse group of human diseases 1. The architecture of mitoribosomes is strikingly different from that of their bacterial and eukaryotic cytosolic counterparts and display high divergence between species 2-6. Mitoribosome biogenesis follows distinct molecular pathways that remain poorly understood. Here, we determined the cryo-EM structures of mitoribosomes isolated from human cell lines with either depleted or overexpressed mitoribosome assembly factor GTPBP5. This allowed us to capture consecutive steps during mitoribosomal large subunit (mt-LSU) biogenesis that involve normally short-lived assembly intermediates. Our structures provide important insights into the last steps of 16S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, which require the coordinated action of nine assembly factors. We show that mammalian-specific MTERF4 contributes to the folding of 16S rRNA, allowing 16S rRNA methylation by MRM2, while GTPBP5 and NSUN4 promote fine-tuning rRNA rearrangments leading to PTC formation. Moreover, our data reveal an unexpected role for the elongation factor mtEF-Tu in mt-LSU assembly, in which mt-EF-Tu interacts with GTPBP5 in a manner similar to its interaction with tRNA during translational elongation. Together, our approaches provide detailed understanding of the last stages of mt-LSU biogenesis that are unique to mammalian mitochondria.

biochemistry↗