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Structural and functional analysis of a FeoB A143S G5 loop mutant explains the accelerated GDP release rate
Author(s) -
Guilfoyle Amy P.,
Deshpande Chandrika N.,
Vincent Kimberley,
Pedroso Marcelo M.,
Schenk Gerhard,
Maher Megan J.,
Jormakka Mika
Publication year - 2014
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12779
Subject(s) - guanosine diphosphate , gtpase , gtp' , biochemistry , g protein , heterotrimeric g protein , protein structure , biology , chemistry , microbiology and biotechnology , signal transduction , guanosine triphosphate , enzyme
GTP ases (G proteins) hydrolyze the conversion of GTP to GDP and free phosphate, comprising an integral part of prokaryotic and eukaryotic signaling, protein biosynthesis and cell division, as well as membrane transport processes. The G protein cycle is brought to a halt after GTP hydrolysis, and requires the release of GDP before a new cycle can be initiated. For eukaryotic heterotrimeric G αβγ proteins, the interaction with a membrane‐bound G protein‐coupled receptor catalyzes the release of GDP from the G α subunit. Structural and functional studies have implicated one of the nucleotide binding sequence motifs, the G5 motif, as playing an integral part in this release mechanism. Indeed, a G αs G5 mutant (A366S) was shown to have an accelerated GDP release rate, mimicking a G protein‐coupled receptor catalyzed release state. In the present study, we investigate the role of the equivalent residue in the G5 motif (residue A143) in the prokaryotic membrane protein FeoB from Streptococcus thermophilus , which includes an N‐terminal soluble G protein domain. The structure of this domain has previously been determined in the apo and GDP ‐bound states and in the presence of a transition state analogue, revealing conformational changes in the G5 motif. The A143 residue was mutated to a serine and analyzed with respect to changes in GTP ase activity, nucleotide release rate, GDP affinity and structural alterations. We conclude that the identity of the residue at this position in the G5 loop plays a key role in the nucleotide release rate by allowing the correct positioning and hydrogen bonding of the nucleotide base.