Insights Into Protein Synthesis - Structural Aspects of Elongation Factor G and Fusidic Acid Inhibition
(2005)- 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... (More) - 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. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/545310
- author
- Hansson, Sebastian LU
- supervisor
-
- Anders Liljas LU
- Derek Logan LU
- opponent
-
- PhD Aevarsson, Arnthor, Prokaria Ltd, Iceland
- organization
- publishing date
- 2005
- type
- Thesis
- publication status
- published
- subject
- keywords
- Molekylär biofysik, Structure chemistry, Strukturkemi, Molecular biophysics, Biologi, Biology, Biomedicinska vetenskaper, Biomedical sciences, conformational change, fusidic acid, protein synthesis, elongation factor G, crystal structures
- pages
- 171 pages
- publisher
- Molecular Biophysics, Lund University
- defense location
- Lecture hall D Center for Chemistry and Chemical Engineering Lund University
- defense date
- 2005-09-30 10:00:00
- ISBN
- 91-7422-090-X
- language
- English
- LU publication?
- yes
- id
- 9be2f4a9-988b-4aea-8f2d-32940ae15f0b (old id 545310)
- date added to LUP
- 2016-04-04 12:07:43
- date last changed
- 2018-11-21 21:09:09
@phdthesis{9be2f4a9-988b-4aea-8f2d-32940ae15f0b, 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.<br/><br> <br/><br> 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.<br/><br> <br/><br> 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.<br/><br> <br/><br> 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.}}, author = {{Hansson, Sebastian}}, isbn = {{91-7422-090-X}}, keywords = {{Molekylär biofysik; Structure chemistry; Strukturkemi; Molecular biophysics; Biologi; Biology; Biomedicinska vetenskaper; Biomedical sciences; conformational change; fusidic acid; protein synthesis; elongation factor G; crystal structures}}, language = {{eng}}, publisher = {{Molecular Biophysics, Lund University}}, school = {{Lund University}}, title = {{Insights Into Protein Synthesis - Structural Aspects of Elongation Factor G and Fusidic Acid Inhibition}}, year = {{2005}}, }