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 G abc proteins, the interaction with a membrane-bound G protein-coupled receptor catalyzes the release of GDP from the G a 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 as G5 mutant (A366S) was shown to have an accelerated GDP release rate, mimicking a G proteincoupled 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.

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

“…Furthermore, the ITC experimental results showed that the S150A mutant protein had the greatest GDP affinity ( K d =2.0 μM), approximately five times that of wild-type Ec NFeoB, which is also in agreement with a decelerated GDP release rate ( Figure 3 and Table 1 ). These molecular phenotypes conform to previous studies of Ec NFeoB [ 16 ], as well as with studies of the equivalent residue in St NFeoB [ 23 ], Gα s [ 18 ] and Gα i 1 [ 24 ]. The rationale for the altered nucleotide-binding and release properties can be inferred from our recent studies of the inverse mutant of St NFeoB [ 23 ].…”

“…These molecular phenotypes conform to previous studies of Ec NFeoB [ 16 ], as well as with studies of the equivalent residue in St NFeoB [ 23 ], Gα s [ 18 ] and Gα i 1 [ 24 ]. The rationale for the altered nucleotide-binding and release properties can be inferred from our recent studies of the inverse mutant of St NFeoB [ 23 ]. The St NFeoB protein has, as most other GTPases, an alanine residue at this position ( Figure 1 B).…”

“…In addition, mutation to the equivalent residue in human HRas leads to rapid GDP release rates, is clinically manifested as the Costello syndrome [ 19 , 20 ] and is also present at high frequency in colorectal cancer [ 21 , 22 ]. A recent mutational analysis of the equivalent residue in the prokaryotic NFeoB from St NFeoB, Streptococcus thermophilus FeoB also illustrated similar acceleration of the GDP release rate [ 23 ]. To understand the mechanism of the accelerated release, crystal structures of a Gα i 1 (β 1 γ 2 ) A326S mutant and a St NFeoB A143S mutant (both equivalent to the Gα s A366S mutation) in complex with GDP were determined.…”

“…To understand the mechanism of the accelerated release, crystal structures of a Gα i 1 (β 1 γ 2 ) A326S mutant and a St NFeoB A143S mutant (both equivalent to the Gα s A366S mutation) in complex with GDP were determined. These structures revealed that the mutation at this position of the G5 loop causes a displacement of the nucleotide base, an altered hydrogen-bonding network, and reduced GDP affinity [ 23 , 24 ].…”

A GTPase Chimera Illustrates an Uncoupled Nucleotide Affinity and Release Rate, Providing Insight into the Activation Mechanism

“…Recently, we reported mutational studies of the G5 loop of Escherichia coli NFeoB, which illustrated a correlation be-tween the sequence composition of the loop and the intrinsic GDP release rate (13). However, despite these observations, it is unclear whether the observed conformational changes in the G5 loop are a prerequisite for GDP release, or if the movement is a consequence of GDP release.…”