title: Structural and functional analysis of a FeoB A143S G5 loop mutant explains the accelerated GDP release rate.
abstract: GTPases (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 Gαβγ proteins, the interaction with a membrane-bound G protein-coupled receptor catalyzes the release of GDP from the Gα 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 Gαs 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 GTPase 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.
authors.affiliation: Structural Biology Program, Centenary Institute, Sydney, New South Wales, Australia; Faculty of Medicine, Central Clinical School, University of Sydney, New South Wales, Australia.
authors.forename: Amy P
authors.initials: AP
authors.lastname: Guilfoyle
authors.forename: Chandrika N
authors.initials: CN
authors.lastname: Deshpande
authors.forename: Kimberley
authors.initials: K
authors.lastname: Vincent
authors.forename: Marcelo M
authors.initials: MM
authors.lastname: Pedroso
authors.forename: Gerhard
authors.initials: G
authors.lastname: Schenk
authors.forename: Megan J
authors.initials: MJ
authors.lastname: Maher
authors.forename: Mika
authors.initials: M
authors.lastname: Jormakka
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D005453:Fluorescence; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics; D048249:Streptococcus thermophilus / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D006153:Guanosine Diphosphate; D012694:Serine; D019204:GTP-Binding Proteins; D000409:Alanine
keywords: GDP release; GTPase; crystal structure; isothermal titration calorimetry; protein motifs