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

E-site drug specificity of the human pathogen Candida albicans ribosome

International audienceCandida albicans is a widespread commensal fungus with substantial pathogenic potential and steadily increasing resistance to current antifungal drugs. It is known to be resistant to cycloheximide (CHX) that binds to the E–transfer RNA binding site of the ribosome. Because of lack of structural information, it is neither possible to understand the nature of the resistance nor to develop novel inhibitors. To overcome this issue, we determined the structure of the vacant C. albicans 80 S ribosome at 2.3 angstroms and its complexes with bound inhibitors at resolutions better than 2.9 angstroms using cryo–electron microscopy. Our structures reveal how a change in a conserved amino acid in ribosomal protein eL42 explains CHX resistance in C. albicans and forms a basis for further antifungal drug development

Bulk-solvent correction in large macromolecular structures.

International audienceThe estimation of the bulk-solvent contribution to the diffraction of a macromolecular crystal makes use of a solvent mask which delimits the bulk-solvent regions in the crystal. It is shown that the way this mask is usually defined in CNS contains a bias which can lead to absurd results in the case of very large structures, where the calculations can only be made on relatively coarse grids. A modified procedure is described and applied to 70S ribosome data at 5.5 A resolution. The B factor affecting the bulk solvent is also discussed. Even in this case of very high and widely variable atomic B factors, it seems sufficient to consider a constant and isotropic B factor for the bulk solvent. This is initially set to the average value of the atomic B factor, but can be refined