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

Hansson, Sebastian LU (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)
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author
supervisor
opponent
  • PhD Aevarsson, Arnthor, Prokaria Ltd, Iceland
organization
publishing date
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
ISBN
91-7422-090-X
language
English
LU publication?
yes
id
9be2f4a9-988b-4aea-8f2d-32940ae15f0b (old id 545310)
date added to LUP
2007-10-13 12:14:08
date last changed
2016-09-19 08:45:14
@misc{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},
  keyword      = {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},
  pages        = {171},
  publisher    = {ARRAY(0x7314c18)},
  title        = {Insights Into Protein Synthesis - Structural Aspects of Elongation Factor G and Fusidic Acid Inhibition},
  year         = {2005},
}