Structural characterization of cephaeline binding to the eukaryotic ribosome using Cryo-Electron Microscopy

The eukaryotic ribosome is emerging as a promising target against human pathogens, includ- ing amoeba, protozoans, and fungi. Among the eukaryotic-specific families of inhibitors, al- kaloids are known to bind to the eukaryotic ribosome and inhibit translocation. However, these inhibitors have varying medical indications and toxicity to humans. Structural information is available for only two of them, cryptopleurine and emetine. Aim. In our work, we aimed to elucidate the binding mechanism of another alkaloid, cephaeline, to the eukaryotic ribosome. Methods. We used cryogenic electron microscopy and cell-free assays to reveal its mechanism of action. Results. Our results indicate that cephaeline binds to the E-tRNA binding site on the small subunit of the eukaryotic ribosome. Similar to emetine, cephaeline forms a stacking interaction with G889 of 18S rRNA and L132 of the protein uS11. We propose the hypothesis of cephaeline specificity to eukaryotes by comparing the interaction pattern of cephaeline with other inhibitors binding to the E-site of the mRNA tunnel. Conclusions. The high-resolution structure of ribosome-bound cephaeline (2.45 Å) allowed us to precisely determine the in- hibitor’s position in the binding site, which holds potential for the development of the next generation of drugs targeting the mRNA tunnel of the ribosome.