Functional analysis of the GTPases EngA and YhbZ encoded by Salmonella typhimurium.

Lamb, HK; Thompson, P; Elliott, C; Charles, IG; Richards, J; Lockyer, M; Watkins, N; Nichols, C; Stammers, DK; Bagshaw, CR; Cooper, A; Hawkins, AR

Functional analysis of the GTPases EngA and YhbZ encoded by Salmonella typhimurium.

Lamb, HK Thompson, P Elliott, C Charles, IG Richards, J Lockyer, M Watkins, N Nichols, C Stammers, DK Bagshaw, CR Cooper, A Hawkins, AR

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Publication Type:: Journal Article
Citation:: Protein Sci, 2007, 16 (11), pp. 2391 - 2402
Issue Date:: 2007-11

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Field	Value	Language
dc.contributor.author	Lamb, HK	en_US
dc.contributor.author	Thompson, P	en_US
dc.contributor.author	Elliott, C	en_US
dc.contributor.author	Charles, IG	en_US
dc.contributor.author	Richards, J	en_US
dc.contributor.author	Lockyer, M	en_US
dc.contributor.author	Watkins, N	en_US
dc.contributor.author	Nichols, C	en_US
dc.contributor.author	Stammers, DK	en_US
dc.contributor.author	Bagshaw, CR	en_US
dc.contributor.author	Cooper, A	en_US
dc.contributor.author	Hawkins, AR	en_US
dc.date.issued	2007-11	en_US
dc.identifier.citation	Protein Sci, 2007, 16 (11), pp. 2391 - 2402	en_US
dc.identifier.issn	0961-8368	en_US
dc.identifier.uri	http://hdl.handle.net/10453/13409
dc.description.abstract	The S. typhimurium genome encodes proteins, designated EngA and YhbZ, which have a high sequence identity with the GTPases EngA/Der and ObgE/CgtAE of Escherichia coli. The wild-type activity of the E. coli proteins is essential for normal ribosome maturation and cell viability. In order to characterize the potential involvement of the Salmonella typhimurium EngA and YhbZ proteins in ribosome biology, we used high stringency affinity chromatography experiments to identify strongly binding ribosomal partner proteins. A combination of biochemical and microcalorimetric analysis was then used to characterize these protein:protein interactions and quantify nucleotide binding affinities. These experiments show that YhbZ specifically interacts with the pseudouridine synthase RluD (KD=2 microM and 1:1 stoichiometry), and we show for the first time that EngA can interact with the ribosomal structural protein S7. Thermodynamic analysis shows both EngA and YhbZ bind GDP with a higher affinity than GTP (20-fold difference for EngA and 3.8-fold for YhbZ), and that the two nucleotide binding sites in EngA show a 5.3-fold difference in affinity for GDP. We report a fluorescence assay for nucleotide binding to EngA and YhbZ, which is suitable for identifying inhibitors specific for this ligand-binding site, which would potentially inhibit their biological functions. The interactions of YhbZ with ribosome structural proteins that we identify may demonstrate a previously unreported additional function for this class of GTPase: that of ensuring delivery of rRNA modifying enzymes to the appropriate region of the ribosome.	en_US
dc.language	eng	en_US
dc.relation.ispartof	Protein Sci	en_US
dc.relation.isbasedon	10.1110/ps.072900907	en_US
dc.subject.classification	Biophysics	en_US
dc.subject.mesh	Ribosomes	en_US
dc.subject.mesh	Escherichia coli	en_US
dc.subject.mesh	Salmonella typhimurium	en_US
dc.subject.mesh	GTP Phosphohydrolases	en_US
dc.subject.mesh	Monomeric GTP-Binding Proteins	en_US
dc.subject.mesh	Nucleotides	en_US
dc.subject.mesh	Escherichia coli Proteins	en_US
dc.subject.mesh	Ribosomal Proteins	en_US
dc.subject.mesh	Guanosine Diphosphate	en_US
dc.subject.mesh	Guanosine Triphosphate	en_US
dc.subject.mesh	Calorimetry	en_US
dc.subject.mesh	Chromatography, Thin Layer	en_US
dc.subject.mesh	Proteomics	en_US
dc.subject.mesh	Binding Sites	en_US
dc.subject.mesh	Molecular Conformation	en_US
dc.subject.mesh	Protein Binding	en_US
dc.subject.mesh	Kinetics	en_US
dc.subject.mesh	Thermodynamics	en_US
dc.subject.mesh	Models, Molecular	en_US
dc.title	Functional analysis of the GTPases EngA and YhbZ encoded by Salmonella typhimurium.	en_US
dc.type	Journal Article
utslib.citation.volume	11	en_US
utslib.citation.volume	16	en_US
utslib.location.activity	United States	en_US
utslib.for	0707 Veterinary Sciences	en_US
utslib.for	0601 Biochemistry and Cell Biology	en_US
utslib.for	0802 Computation Theory and Mathematics	en_US
utslib.for	0899 Other Information and Computing Sciences	en_US
dc.location.activity	ISI:000250444400008	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Strength - ithree - Institute of Infection, Immunity and Innovation
utslib.copyright.status	closed_access
pubs.issue	11	en_US
pubs.publication-status	Published	en_US
pubs.volume	16	en_US

Abstract:

The S. typhimurium genome encodes proteins, designated EngA and YhbZ, which have a high sequence identity with the GTPases EngA/Der and ObgE/CgtAE of Escherichia coli. The wild-type activity of the E. coli proteins is essential for normal ribosome maturation and cell viability. In order to characterize the potential involvement of the Salmonella typhimurium EngA and YhbZ proteins in ribosome biology, we used high stringency affinity chromatography experiments to identify strongly binding ribosomal partner proteins. A combination of biochemical and microcalorimetric analysis was then used to characterize these protein:protein interactions and quantify nucleotide binding affinities. These experiments show that YhbZ specifically interacts with the pseudouridine synthase RluD (KD=2 microM and 1:1 stoichiometry), and we show for the first time that EngA can interact with the ribosomal structural protein S7. Thermodynamic analysis shows both EngA and YhbZ bind GDP with a higher affinity than GTP (20-fold difference for EngA and 3.8-fold for YhbZ), and that the two nucleotide binding sites in EngA show a 5.3-fold difference in affinity for GDP. We report a fluorescence assay for nucleotide binding to EngA and YhbZ, which is suitable for identifying inhibitors specific for this ligand-binding site, which would potentially inhibit their biological functions. The interactions of YhbZ with ribosome structural proteins that we identify may demonstrate a previously unreported additional function for this class of GTPase: that of ensuring delivery of rRNA modifying enzymes to the appropriate region of the ribosome.

Please use this identifier to cite or link to this item:

http://hdl.handle.net/10453/13409