Insights Into Protein Synthesis - Structural Aspects of Elongation Factor G and Fusidic Acid Inhibition

University dissertation from Molecular Biophysics, Lund University

Abstract: Elongation factor G (EF-G) catalyses the translocation step in protein synthesis on the ribosome. During this event the antibiotic Fusidic acid (FA) binds to and irreversably inhibits the release of the EF-G:GDP complex from the ribosome. The difficulties to elucidate the exact binding mechanism of FA to the EF-G:GDP:ribosome complex are due to the fact that FA only binds to EF-G when the factor is on the ribosome and in complex with GDP. In solution EF-G has decreased ability to form a complex with FA due to the low solubility of FA in a solution that enables further structural characterization. The role of Phe90 in EF-G from T. thermophilus has been highlighted as a regulator that act as a gatekeeper promoting the binding site for FA. These observations came from direct structural interpretation of the highly sensitive G16V and highly resistant T84A EF-G mutants. Further support of this putative binding site came from mutational studies and further structural characterization of the two F90L and F90A mutants of EF-G. The F90L mutant also revealed the highest resolution (2.1 Å) of EF-G to date. To further establish and implement the binding site of FA in EF-G the structure of the D109K mutant was solved. These five mutants all support the putative binding site of FA being located at the interface between switch II from the G domain and domain III. The possible role of Lys25 in nucleotide regulation was proposed in the G16V and T84A study. Characterization of the T84A mutant with a GDPNP molecule revealed the structural conformational changes that EF-G undergoes between nucleotide discrimination between GDP and GTP in solution. From this observation Phe90 again displays a central role in modulating the conformaitons of the whole of EF-G. Also, the role of Lys25 as a key residue in nucleotide regulation was further established. The crystal structure of T84A in complex with GDPCP was solved to 2.9 Å resolution. The remarkable in this structure was the coordination of Lys25 to the nucleotide. From these results the role of Lys25 was thoroughly investigated and a proposed mechanism for its involvement in nucleotide regulation, nucleotide binding and GTPase mechanism was elaborated.

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