Exploring the correlation between the sequence composition of the nucleotide binding G5 loop of the FeoB GTPase domain (NFeoB) and intrinsic rate of GDP release.
GDP release from GTPases is usually extremely slow and is in general assisted by external factors, such as association with guanine exchange factors or membrane-embedded GPCRs (G protein-coupled receptors), which accelerate the release of GDP by several orders of magnitude. Intrinsic factors can also play a significant role; a single amino acid substitution in one of the guanine nucleotide recognition motifs, G5, results in a drastically altered GDP release rate, indicating that the sequence composition of this motif plays an important role in spontaneous GDP release. In the present study, we used the GTPase domain from EcNFeoB (Escherichia coli FeoB) as a model and applied biochemical and structural approaches to evaluate the role of all the individual residues in the G5 loop. Our study confirms that several of the residues in the G5 motif have an important role in the intrinsic affinity and release of GDP. In particular, a T151A mutant (third residue of the G5 loop) leads to a reduced nucleotide affinity and provokes a drastically accelerated dissociation of GDP.
*Structural Biology Program, Centenary Institute, Locked Bag 6, Sydney, New South Wales 2042, Australia.
Amy P
AP
Guilfoyle
*Structural Biology Program, Centenary Institute, Locked Bag 6, Sydney, New South Wales 2042, Australia.
Chandrika N
CN
Deshpande
‡School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Queensland 4072, Australia.
Gerhard
G
Schenk
§La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.
Megan J
MJ
Maher
*Structural Biology Program, Centenary Institute, Locked Bag 6, Sydney, New South Wales 2042, Australia.
Mika
M
Jormakka
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002151:Calorimetry / Q000379:methods; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; C470580:FeoB protein, E coli; D009711:Nucleotides; D006153:Guanosine Diphosphate; D020558:GTP Phosphohydrolases