Site‐directed mutagenesis of <i>Thermus thermophilus</i> EF‐Tu: the substitution of threonine‐62 by serine or alanine

Ahmadian, Mohammad Réza; Kreutzer, Roland; Blechschmidt, Bernd; Sprinzl, Mathias

doi:10.1016/0014-5793(95)01354-7

Cited by 12 publications

(11 citation statements)

References 37 publications

(11 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…S2C). Consistently, substitution of the homologous Thr with an Ala residue results in a dramatic reduction in GTP hydrolysis activity of both T. thermophilus and E. coli EF-Tu (23,24). Thus, phosphorylation could affect binding and/or nucleotide hydrolysis.…”

Section: Phosphorylation Of Ef-tu Inhibits Gtp Hydrolysis and Translamentioning

confidence: 57%

See 1 more Smart Citation

Protein synthesis during cellular quiescence is inhibited by phosphorylation of a translational elongation factor

Pereira

Gonzalez

Dworkin

2015

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

In nature, most organisms experience conditions that are suboptimal for growth. To survive, cells must fine-tune energy-demanding metabolic processes in response to nutrient availability. Here, we describe a novel mechanism by which protein synthesis in starved cells is down-regulated by phosphorylation of the universally conserved elongation factor Tu (EF-Tu). Phosphorylation impairs the essential GTPase activity of EF-Tu, thereby preventing its release from the ribosome. As a consequence, phosphorylated EF-Tu has a dominant-negative effect in elongation, resulting in the overall inhibition of protein synthesis. Importantly, this mechanism allows a quick and robust regulation of one of the most abundant cellular proteins. Given that the threonine that serves as the primary site of phosphorylation is conserved in all translational GTPases from bacteria to humans, this mechanism may have important implications for growth-rate control in phylogenetically diverse organisms.A daptation to nutrient availability is a fundamental cellular process. From unicellular prokaryotes to complex multicellular organisms, cells sense and adjust their metabolism to respond to variations in nutrient levels. Protein synthesis is one of the most energy-intensive cellular processes, and both the initiation and elongation stages of translation are down-regulated in response to nutrient limitation (1). Proteins that mediate translation, such as the essential and universally conserved GTPase Elongation factor Tu (EF-Tu), are observed in the phosphoproteomes of diverse organisms (2), suggesting that they are subject to regulatory phosphorylation. However, EF-Tu is the most abundant protein in bacteria, present at ∼100,000 copies per cell of growing Escherichia coli (3), but less than 10% of the EF-Tu molecules are phosphorylated (4). Thus, a key question is how this relatively small fraction of phosphorylated EF-Tu can cause a down-regulation of protein synthesis.GTP-bound EF-Tu binds and delivers an aminoacyl-tRNA (aa-tRNA) molecule to the translating ribosome (3). Upon formation of a correctly base-paired mRNA codon-aa-tRNA anticodon interaction, the ribosome activates the GTPase activity of EF-Tu, followed by accommodation of the aa-tRNA into the ribosomal aa-tRNA binding (A) site, and release of the inactive GDP-bound EF-Tu from the ribosome. EF-Tu belongs to the GTPase superfamily which comprises molecular switches that share a core catalytic domain and mechanism but have evolved to perform diverse roles in many cellular processes (5). Central to their function is the hydrolysis of GTP, which controls the switch between the ON, GTP-bound, and the OFF, GDP-bound, states. Hydrolysis of GTP is followed by large conformational changes in two flexible regions known as "switch I" and "switch II." These regions are composed of highly conserved motifs that surround and contact the nucleotide and are involved in interactions with both exchange factors and effectors that regulate GTPase function.GTPases also can be regulated by direct inhibiti...

show abstract

Section: Phosphorylation Of Ef-tu Inhibits Gtp Hydrolysis and Translamentioning

confidence: 57%

“…S2). However, because this residue is essential for EF-Tu function (23,24), it is difficult or impossible to interpret experiments involving mutations at this site unambiguously. Therefore we cannot irrefutably ascribe the reduced GTPase activity of the phosphorylated protein to phosphorylation of this Thr residue.…”

Section: Discussionmentioning

confidence: 99%

Protein synthesis during cellular quiescence is inhibited by phosphorylation of a translational elongation factor

Pereira

Gonzalez

Dworkin

2015

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…1A). Arg 58(59) and Thr 61(62) , two conserved residues important for GTPase activity and interaction with the ribosome (21,22), are moved away by ϳ15 Å toward the vacant binding pocket for the aminoacyl residue of tRNA. Fig.…”

Section: Table 2 Connections Between Enacyloxin Iia and Its Ef-tu Binmentioning

confidence: 99%

Enacyloxin IIa Pinpoints a Binding Pocket of Elongation Factor Tu for Development of Novel Antibiotics

Parmeggiani

Krab

Watanabe

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

Elongation factor (EF-) Tu⅐GTP is the carrier of aminoacyl-tRNA to the programmed ribosome. Enacyloxin IIa inhibits bacterial protein synthesis by hindering the release of EF-Tu⅐GDP from the ribosome. The crystal structure of the Escherichia coli EF-Tu⅐guanylyl iminodiphosphate (GDPNP)⅐enacyloxin IIa complex at 2.3 Å resolution presented here reveals the location of the antibiotic at the interface of domains 1 and 3. The binding site overlaps that of kirromycin, an antibiotic with a structure that is unrelated to enacyloxin IIa but that also inhibits EF-Tu⅐GDP release. As one of the major differences, the enacyloxin IIa tail borders a hydrophobic pocket that is occupied by the longer tail of kirromycin, explaining the higher binding affinity of the latter. EF-Tu⅐GDPNP⅐enacyloxin IIa shows a disordered effector region that in the PhetRNA Phe ⅐EF-Tu (Thermus aquaticus)⅐GDPNP⅐enacyloxin IIa complex, solved at 3.1 Å resolution, is stabilized by the interaction with tRNA. This work clarifies the structural background of the action of enacyloxin IIa and compares its properties with those of kirromycin, opening new perspectives for structure-guided design of novel antibiotics. Elongation factor (EF)5 Tu is a GTPase that, as all members of this superfamily, cycles between the active GTP-bound and the inactive GDP-bound state. In bacterial protein synthesis EF-Tu is the carrier of aa-tRNA to the mRNA-programmed ribosome (for review see Refs. 1 and 2). The codon-anticodon interaction triggers the hydrolysis of the bound GTP, inducing the release of EF-Tu⅐GDP, the positioning of aa-tRNA into the ribosomal A-site, and peptide bond formation with the P-site-bound peptidyl-tRNA. EF-Tu folds in three distinct domains: the nucleotide-binding domain 1 (residues 1-199 in Escherichia coli (Ec) EF-Tu⅐GDP) shows the classical GTPase mixed ␣/␤ Rossman fold, whereas domains 2 and 3 (residues 209 -299 and 300 -393, respectively) are ␤-barrels. The association of the domains in EF-Tu⅐GDP with reduced interfaces and a central hole becomes more compact in EF-Tu⅐GTP with extensive interfaces and no hole (3-5). EF-Tu is the target of four structurally unrelated families of antibiotic inhibitors of protein synthesis (for review see Ref.2), of which kirromycin and enacyloxin IIa hinder the release of EF-Tu⅐GDP from the ribosome after GTP hydrolysis, thus inhibiting its recycling and peptide bond formation. The high-resolution crystal structure of Thermus thermophilus (Tt) EF-Tu⅐GDP in complex with methylkirromycin (also called aurodox) showed that the antibiotic binds in the interface of domains 1 and 3, inducing a unique, compact EF-Tu-GTP-like conformation, which is the reason for its inhibition of the EF-Tu⅐GDP release (6). These conclusions have been further confirmed in studies of the Phe-tRNA Phe ⅐ EF-Tu⅐GDPNP⅐kirromycin. 6 Specific differences between the enacyloxin IIa and kirromycin complexes with EF-Tu concern various aspects such as the electrophoretic migration on native gel, binding affinity, GTPase activity, different effects of...

show abstract

“…The eRF3C domain that is sufficient for binding to eRF1 does not include the G-domain motifs+ This is in sharp contrast with other translational G proteins, elongation factors EF-Tu and EF-1a, or initiation factors IF2 and eIF-2, whose aminoacyl-tRNA or N-formylmethionyl-tRNA binding is controlled by G-domain function: GTP stimulates the association and GDP dissociates the complex+ There have been numerous reports that the N-terminal domain, including the G domain, of EF-Tu and EF-1a plays a crucial role in the binding of aminoacyl-tRNA directly or indirectly: the binding is diminished by mutations of Lys-4 (Laurberg et al+, 1998), Arg-7 (Mansilla et al+, 1997), Lys-9 (Laurberg et al+, 1998), Arg-58 , Lys-89 (Wiborg et al+, 1996), Asn-90 (Wiborg et al+, 1996), Gly-94 , His-118 (Jonak et al+, 1994), and Glu-259 (Pedersen et al+, 1998) of E. coli EF-Tu; Thr-62 of T. thermophilus EF-Tu (Ahmadian et al+, 1995); and Gly-280 of Salmonella typhimurium EF-Tu (Tubulekas & Hughes, 1993)+ Some of these substitutions, however, are known to affect the stability of the GTP form of EF-Tu/EF-1a relative to the GDP form, and thereby diminish the binding of aminoacyl-tRNA+ Because of the functional requirement for continuous delivery of aminoacyl-tRNA during protein elongation, the G-domain activity influences, directly or indirectly, the binding of aminoacyl-tRNA+ On the other hand, guanine nucleotides do not seem to influence the eRF1-eRF3 interaction+ They form a complex in vitro both in the presence (Zhouravleva et al+, 1995) or absence (Stansfield et al+, 1995;Frolova et al+, 1998) of GTP+ Therefore, the G-domain function of eRF3 may not be to change the binding of eRF1, but instead to change the binding of the ribosome or to catalyze final translocation of the ribosome+ Once eRF3 is associated with eRF1 before or after binding to the ribosome, the two probably remain associated via their C-termini interaction until their release from the ribosome, showing a clear functional difference between eRF3 and EF-Tu/EF-1a+ FIGURE 6. Comparison of the amino acid sequences of eRF3s and elongation factors EF-Tu and EF-1a+ The similarity alignments of eRF1s were accomplished using the PILEUP program from the GCG program package (Devereux et al+, 1984)+ Identical and similar amino acids are boxed in black and gray, respectively+ Asterisks indicate amino acids of T. aquaticus EF-Tu that are involved in tRNA binding in the three-dimensional structure (Nissen et al+, 1996)+ Daggers represent amino acids of S. pombe eRF3 that were mutated to alanine+ The number refers to the amino acid position counted from the N-terminal Met+ FIGURE 7.…”

Section: Uncoupling Between Erf1 Binding and G-domain Functionmentioning

confidence: 99%

C-terminal interaction of translational release factors eRF1 and eRF3 of fission yeast: G-domain uncoupled binding and the role of conserved amino acids

Ebihara

Nakamura

1999

RNA

View full text Add to dashboard Cite

Translation termination in eukaryotes requires a stop codon-responsive (class-I) release factor, eRF1, and a guanine nucleotide-responsive (class-II) release factor, eRF3. Schizosaccharomyces pombe eRF3 has an N-terminal polypeptide similar in size to the prion-like domain of Saccharomyces cerevisiae eRF3 in addition to the EF-1a-like catalytic domain. By in vivo two-hybrid assay as well as by an in vitro pull-down analysis using purified proteins of S. pombe as well as of S. cerevisiae, eRF1 bound to the C-terminal one-third domain of eRF3, named eRF3C, but not to the N-terminal two-thirds, which was inconsistent with the previous report by Paushkin et al. (1997, Mol Cell Biol 17:2798-2805). The activity of S. pombe eRF3 in eRF1 binding was affected by Ala substitutions for the C-terminal residues conserved not only in eRF3s but also in elongation factors EF-Tu and EF-1a. These single mutational defects in the eRF1-eRF3 interaction became evident when either truncated protein eRF3C or C-terminally altered eRF1 proteins were used for the authentic protein, providing further support for the presence of a C-terminal interaction. Given that eRF3 is an EF-Tu/EF-1a homolog required for translation termination, the apparent dispensability of the N-terminal domain of eRF3 for binding to eRF1 is in contrast to importance, direct or indirect, in EF-Tu/EF-1a for binding to aminoacyl-tRNA, although both eRF3 and EF-Tu/EF-1a share some common amino acids for binding to eRF1 and aminoacyl-tRNA, respectively. These differences probably reflect the independence of eRF1 binding in relation to the G-domain function of eRF3 (i.e., probably uncoupled with GTP hydrolysis), whereas aminoacyl-tRNA binding depends on that of EF-Tu/EF-1a (i.e., coupled with GTP hydrolysis), which sheds some light on the mechanism of eRF3 function.

show abstract

Site‐directed mutagenesis of Thermus thermophilus EF‐Tu: the substitution of threonine‐62 by serine or alanine

Cited by 12 publications

References 37 publications

Protein synthesis during cellular quiescence is inhibited by phosphorylation of a translational elongation factor

Protein synthesis during cellular quiescence is inhibited by phosphorylation of a translational elongation factor

Enacyloxin IIa Pinpoints a Binding Pocket of Elongation Factor Tu for Development of Novel Antibiotics

C-terminal interaction of translational release factors eRF1 and eRF3 of fission yeast: G-domain uncoupled binding and the role of conserved amino acids

Contact Info

Product

Resources

About