Structural Studies on the Mechanism of Argyrin B and the L12 – L11 Ribosomal Protein Interface

The mechanism of action of argyrin B and the molecular interactions of L11 and L12 has been undetermined and underrepresented in the research of bacterial translation. This work seeks to examine the mechanism of action of argyrin B by performing in vitro structural studies of its interaction with its specific target protein elongation factor-G. We demonstrate that argyrin B inhibits translation, and allows GTPase activity to proceed, contrary to assumptions made in prior research. We determine that ribosome recycling is the likely step through which argyrin B acts as an antibiotic, since translocation is unaffected by argyrin B and association with post-termination complexes are increased in the presence of argyrin B. We propose that the mechanism of action involves the ratcheting of EF-G 45 ° from a normal binding conformation with the ribosome, which sterically hinders the positioning of loop II of domain IV of EF-G and the cooperativity of EF-G and RRF in interrupting Bridge 2a, the largest intersubunit bridge on the ribosome. Furthermore, we provide preliminary insight on the molecular interactions present between L11 and the L12 C Terminal Domain by performing alanine scanning mutations on select glutamic acid and aspartic acid residues around an electronegatively charged pocket. These mutations while not effecting L11 binding in a tangible way, strongly influenced select GTPase activity