Inhibition of the Eukaryotic 80S Ribosome as a Potential Anticancer Therapy: A Structural Perspective.

Protein biosynthesis is a vital process for all kingdoms of life. The ribosome is the massive ribonucleoprotein machinery that reads the genetic code, in the form of messenger RNA (mRNA), to produce proteins. The mechanism of translation is tightly regulated to ensure that cell growth is well sustained. Because of the central role fulfilled by the ribosome, it is not surprising that halting its function can be detrimental and incompatible with life. In bacteria, the ribosome is a major target of inhibitors, as demonstrated by the high number of small molecules identified to bind to it. In eukaryotes, the design of ribosome inhibitors may be used as a therapy to treat cancer cells, which exhibit higher proliferation rates compared to healthy ones. Exciting experimental achievements gathered during the last few years confirmed that the ribosome indeed represents a relevant platform for the development of anticancer drugs. We provide herein an overview of the latest structural data that helped to unveil the molecular bases of inhibition of the eukaryotic ribosome triggered by small molecules

Abstract P-31: Assembly of the Complex of the 30S Ribosomal Subunit and the Ribosome Maturation Factor P from Staphylococcus aureus for Structural Studies by Cryo-Electron Microscopy

Background: Staphylococcus aureus (S. aureus) is one of the main human pathogens causing numerous nosocomial soft tissue infections and is among the best-known causes of bacterial infections. The bacterial 70S ribosome consists of two subunits, designated the 30S (small) and 50S (large) subunits. The small subunit (30S) consists of 16S ribosomal RNA (rRNA), from which the assembly of 30S begins, and 21 ribosomal proteins (r-proteins). The ribosome maturation factor P (RimP protein) binds to the free 30S subunit. Strains lacking RimP accumulate immature 16S rRNA, and fewer polysomes and an increased amount of unassociated 30S and 50S subunits compared to wild-type strains are observed in the ribosomal profile. Structural studies of the 30S subunit complex and the ribosome maturation factor RimP will make it possible in the future to develop an antibiotic that slows down or completely stops the translation of Staphylococcus aureus, which will complicate the synthesis and isolation of its pathogenic factors. Here we present the protocol of the in vitro reconstruction of S. aureus 30S ribosome subunit in a complex with RimP for further structural studies by cryo-electron microscopy. Methods: Recombinant RimP protein from S. aureus was expressed in E. coli and purified by Ni-NTA chromatography and size exclusion chromatography. Reconstitution of the 30S–RimP complex was performed by mixing RimP protein with 30S ribosome. Unbound RimP protein was removed by Amicon Ultra Concentration (Merk KGaA, Darmstadt, Germany) with a cut-off limit of 100 kDa. The presence of RimP protein in the resulting 30S-RimP complex was confirmed by SDS-PAGE, and the quality of the final sample was analyzed by the negative staining EM. Results: Finally, by in vitro reconstruction, the 30S-RimP complex from S. aureus was obtained for further structural studies by cryo-electron microscopy

Abstract P-25: High-Resolution Cryo-Electron Microscopy Structure of the Staphylococcus Aureus Ribosome Brings to Light New Possible Drug Targets

Background: Antibiotic resistance is a growing worldwide problem. One of the major resistant bacterial pathogens is Staphylococcus aureus, which became a burden of healthcare systems around the world. To overcome the issue, more drug discovery studies are needed. One of the main antibiotic targets is a ribosome – the central hub of protein synthesis. Structural data of the ribosome and its features are a crucial milestone for the effective development of new drugs, especially using structure-based drug design approaches. Apart from many small structural features, ribosome possesses rRNA modifications that play a role in the fine-tuning of protein synthesis. Detailed species-specific structural data of the S. aureus ribosome is also a useful model for understanding the resistance mechanisms. This information could help with the design of new antibiotics and the upgrading of old ones. The data on S. aureus ribosomal RNA modifications and corresponding modification enzymes are very limited. Our aim was to improve the current models of the S. aureus ribosome by determining its structure with functional ligands at a much higher resolution - thereby creating a foundation for structure-based drug design experiments and research of new drug targets. Methods: The S. aureus ribosome complex consists of three components: ribosome, fMet-tRNAfMet, mRNA and 70S ribosome. The complex from purified components was formed in vitro and applied to cryo-EM grids. Data was collected at Titan Krios with Gatan K2 detector (IGBMC, France). The data was processed and modeled in Relion 2.1, Chimera, Coot, and Phenix. Results: We determined the cryo-EM reconstruction at 3.2 Å resolution of the S. aureus ribosome with P-site tRNA, messenger RNA. Based on the experimental map and existing bioinformatic data, we at the first time identified and assigned 10 modifications of S. aureus rRNA. We analyzed the positions of rRNA modifications and their possible functions. Conclusion: In this study, we describe our structure of S. aureus ribosome with functional ligands. The present model is the highest resolution and most precise that is available at the moment. We propose a set of methyltransferases as targets for future drug discovery studies. The proposed methyltransferases and corresponding modifications may play an important role in protein synthesis and its regulation

Abstract P-26: Staphylococcus Aureus 30S Ribosomal Subunit in a Complex with the Era GTPase: Sample Preparation for Cryo-EM

Background: An essential in bacteria GTPase Era is a multifunctional protein that is involved in cell cycle regulation and appears to play a significant role in ribosome biogenesis. It is required for the maturation of the 30S ribosomal subunit. Era consists of two domains: the GTPase N-terminal domain, conserved in the GTPase family, and a C-terminal RNA-binding KH domain. Era specifically binds to the 16S rRNA and stimulates processing of the small ribosomal subunit to its mature form. Precise determination of nucleotide and amino acid sequences in the active site of binding will help in finding specific ways to prevent this interaction. In this way, it will be possible to disrupt the biogenesis of the ribosome and, thereby, stop or slow down protein synthesis in the bacterial cell. It is very important in the fight against pathogenic bacteria, such as Staphylococcus aureus (S. aureus). Methods: The His-tagged Era (His–Era) protein from S. aureus was expressed in E. coli BL21 strain and purified by Ni-NTA and SEC. The 30S ribosomal subunits were collected after dissociation of the S. aureus 70S ribosomes in sucrose gradient (0 – 30%). Complex 30S-Era was obtained by mixing in vitro 30S subunits and His–Era, incubated for 15 min at 37°C and followed by Ni-NTA purification to remove unbound 30S subunits. The presence of a stable 30S-Era complex has been confirmed by SDS-PAGE and agarose gel electrophoresis. The final sample quality was analyzed by negative staining EM. Results: For the first time in vitro 30S-Era complex from S. aureus was assembled and a sample was prepared for further structural studies by cryo-electron microscopy

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

Abstract P-30: Structure of the 80S Ribosome From Candida Albicans Revealed by Integrative Structural Biology Approach

Background: The fungus Candida albicans is one of the most common fungal human pathogens, causing numerous mucocutaneous infections. About 80% of the world population is colonized with this pathogen, and in many cases, it does not reveal its pathogenic properties. However in the events when the suppression of normal bacterial flora occurs, e.g. as a consequence of chemotherapy or antibiotics treatment, multi-resistant strains of Candida albicans can rapidly colonize the host organism and prevent the normal flora recovery, which in turn can trigger many other diseases especially in the case of immunocompromised patients. Methods: We used an integrated structural biology approach based on the single-particle cryo-EM reconstruction and macromolecule X-ray crystallography. Results: One of the most promising targets for antibiotic action is a protein synthesis apparatus and ribosomes in the cell. In this study, we characterized structurally the protein synthesis machinery of Candida albicans using the state-of-the-art techniques of single-particle Cryo-electron microscopy and macromolecular X-ray crystallography. Conclusion: We obtained the 2.4 Å resolution structure of the 80S ribosome from Candida albicans and the 4.2 Å resolution structure of the vacant C. albicans ribosome by X-ray crystallography. We believe that this study will shed light on mechanisms of antimicrobial resistance in C. albicans and improve candidiasis treatment in the future