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Fusidic acid (FA) is an antibiotic that inhibits protein synthesis by trapping elongation factor G (EF-G) on the ribosome during elongation and ribosome recycling. In Staphylococcus aureus, there are three main mechanisms of resistance (FusA-, FusB- and FusE-type). FusA- and FusE-type resistance arises from mutations on EF-G or ribosomal protein uL6. The more widespread FusB-type resistance involves a resistance protein, FusB, that releases EF-G from the ribosome without directly interacting with FA.

In this thesis, I first determined high-resolution structures of S. aureus EF-G trapped on the ribosome by FA and an FA analog (FA-CP), providing insights into FusA- and FusE-type resistance and a foundation for structure-guided antibiotic design. I then elucidated the molecular mechanism of FusB-type resistance. Using time-resolved cryo-EM, I obtained a structure of FusB bound to EF-G on the ribosome prior to rescue. This structure shows that FusB causes a major conformational change of EF-G that triggers loss of interaction with the ribosome, promoting its release even though FA remains bound. Furthermore, I show that FusB binds to the ribosome independently of EF-G, which perhaps represents an unknown additional function of FusB.

Next, I characterized a putative FA resistance operon from Streptomyces canus, which encodes for a C-terminal domain homolog of FusB (Sc-cFusB), a TetR-family regulator (Sc-TFR) and a predicted esterase (Sc-Esterase). Our structural and biochemical data suggest that Sc-cFusB has the same function as S. aureus FusB, Sc-Esterase enzymatically inactivates FA and Sc-TFR represses the transcription of the operon and is regulated by FA.

Finally, I provide new structural insights into ribosome recycling and its inhibition by FA. Using FA, I obtained a high-resolution cryo-EM structure of EF-G bound to ribosome recycling factor (RRF) on the 50S ribosome. In addition, I characterize a previously unobserved ribosome recycling intermediate containing both RRF and EF-G on the 70S ribosome with disrupted intersubunit bridges.