Elongation factor P: mechanism of action and opportunities for drug design

by Vitalii Mudryi
Doctoral thesis
Date of Examination:2022-11-03
Date of issue:2023-07-24
Advisor:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Kai Tittmann
Referee:Dr. Alex Caspar Faesen
Referee:Prof. Dr. Wolfgang Wintermeyer
Referee:Prof. Dr. Markus Bohnsack
Referee:Dr. Ricarda Richter-Dennerlein
Persistent Address: http://dx.doi.org/10.53846/goediss-10005

 

 

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Abstract

English

EF-P is a conserved protein that has shape similarity to the tRNAs and binds to the E site of the ribosome. EF-P enhances the rate of peptide bond formation during the synthesis of polyPro sequences. In E. coli one third of proteins have at least one polyPro sequence. The effect of EF-P knockouts differs between different bacteria species. In some organisms EF-P is essential, whereas its deletion can reduce growth or virulence in others. The eukaryotic analog of EF-P is eIF5A, which lacks one domain in comparison to the prokaryotic factor and is strictly essential. EF-P is chemically modified and these modifications are important for the factor’s function, but the modification varies between organisms, for example ß-Lys, rhamnose or 5-aminopentanol modifications have been described, while in some organisms EF-P is not modified. eIF5A is modified with hypusine. Here I studied the interaction of EF-P with ribosomes at different stages of translation to determine which elements of the translating ribosome contribute to the factor binding. The association rate constants are very similar for all complexes, however, the dissociation rate constants of EF-P from the ribosome differ depending on the identity of the P-site tRNA and on the codon presented in the E site. This argues for a scanning mechanism in which EF-P can bind to any type of the ribosome as long as the E site is empty. Interactions with specific P-site tRNA ensure a longer residence time on its substrate complexes, thereby providing the time window for EF-P to enhance peptide bond formation, whereas the factor is rapidly released from non-substrate complexes. Also I have shown that the ß-Lys modification greatly contributes to EF-P binding. In Pseudomonas aeruginosa EF-P is modified with rhamnose by the enzyme EarP. Pseudomonas aeruginosa is a clinically relevant pathogen and the rhamnose modification is crucially important for the bacteria’s virulence. Therefore, the enzyme was chosen as a promising target for the screening for new antimicrobials. I developed an assay that can be used for a high throughput screening to identify small molecule compounds inhibiting EarP. In parallel, I performed virtual screening in silico and 8 of the highest scoring compounds were subsequently tested with the established assay in vitro. The highest inhibition of a compound was 45% under standard conditions, making it a potential lead compound for further studies.
Keywords: EF-P; elongation factor P; elongation; ribosome