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.

Amy P Guilfoyle,Mika Jormakka,Chandrika N Deshpande,Megan J Maher,Gerhard Schenk

doi:10.1042/bsr20140152

Amy P Guilfoyle, Mika Jormakka + Show 3 more

Open Access

https://doi.org/10.1042/bsr20140152

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Abstract

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.

Highlights

GTPases are involved in essential cellular processes in both prokaryotes and eukaryotes
Despite the lack of overall sequence conservation, several studies have shown that the sequence composition of the G5 loop plays an important role in influencing the intrinsic or spontaneous GDP release rate; the second residue of the motif is generally conserved as an alanine (Figure 1B), and mutation to this residue in the human Gαs- protein (A366S) results in a constitutively active state through a drastically accelerated GDP release, causing testotoxicosis and pseudohypoparathyroidism in young men [18]
Our studies reveal that the intrinsic GDP release rate is highly influenced by the sequence composition of the loop, with the third residue (T151) in particular having an important role in nucleotide affinity

Summary

INTRODUCTION

GTPases are involved in essential cellular processes in both prokaryotes and eukaryotes. Despite the lack of overall sequence conservation, several studies have shown that the sequence composition of the G5 loop plays an important role in influencing the intrinsic or spontaneous GDP release rate; the second residue of the motif is generally conserved as an alanine (Figure 1B), and mutation to this residue in the human Gαs- protein (A366S) results in a constitutively active state through a drastically accelerated GDP release, causing testotoxicosis and pseudohypoparathyroidism in young men [18]. Our studies reveal that the intrinsic GDP release rate is highly influenced by the sequence composition of the loop, with the third residue (T151) in particular having an important role in nucleotide affinity

MATERIALS AND METHODS

Nucleotide release in NFeoB

RESULTS AND DISCUSSION