Structural dynamics of translation elongation factor Tu during aa-tRNA delivery to the ribosome

Kavaliauskas, Darius; Chen, Chunlai; Liu, Wei; Cooperman, Barry S.; Goldman, Yale E.; Knudsen, Charlotte R.

doi:10.1093/nar/gky651

Cited by 17 publications

(27 citation statements)

References 53 publications

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“…Therefore, the nucleotide release mechanism is likely the inverse in which a conformational change has to occur prior to nucleotide dissociation. This process would require a unique conformation for EF-Tu•apo and is constant with the reported conformational flexibility observed by Johansen et al and Kavaliauskas et al [3][4] . Considering the entropic landscape of EF-Tu, the EF-Tu•apo conformation is less stable than both EF-Tu•GTP and EF-Tu•GDP (Fig 4).…”

supporting

confidence: 63%

Elongation Factor Tu’s Nucleotide Binding Is Governed by a Thermodynamic Landscape Unique among Bacterial Translation Factors

et al. 2019

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Insights into the apo Conformation of EF-TuThe first structural insight into the apo conformation of EF-Tu was gleaned from the crystal structure of the EF-Tu•EF-Ts complex 1 . In this structure, switch I of EF-Tu is disordered and the domain arrangement is similar to the GDP conformation. Subsequently, Thirup and co-workers were able to crystalize an EF-Tu•EF-Ts complex with EF-Tu in the closed GTP conformation, indicating that the EF-Tu•EF-Ts complex exhibits conformational flexibility 2 . Our kinetic data reported here provides additional insight in the apo state of EF-Tu and suggests that the free apo state of EF-Tu adopts a unique conformation that is able to recognize GTP and GDP similarly. Similar association and different dissociation activation barriers support that nucleotide binding to this non-discriminatory conformation of EF-Tu is followed by conformational changes of EF-Tu that depend on the presence and absence of the gamma-phosphate in GTP. Therefore, the nucleotide release mechanism is likely the inverse in which a conformational change has to occur prior to nucleotide dissociation. This process would require a unique conformation for EF-Tu•apo and is constant with the reported conformational flexibility observed by Johansen et al and Kavaliauskas et al [3][4] . Considering the entropic landscape of EF-Tu, the EF-Tu•apo conformation is less stable than both EF-Tu•GTP and EF-Tu•GDP (Fig 4). Since EF-Tu employs water coordination to entropically stabilize the GTP conformation it is likely that water coordination stabilizes EF-Tu•GTP and EF-Tu•GDP compared to EF-Tu•apo. If GDP is removed from the structure of EF-Tu•GDP the SASA increases by 109 Å 2 . The difference in SASA between EF-Tu•GTP and EF-Tu•GDP is 1041 Å 2 ( Fig S6B) and since the entropy gap between EF-Tu•GDP and EF-Tu•GTP is similar to the entropy gap between EF-Tu•apo and EF-Tu•GDP ( Fig 4C) it is likely that SASA alone does not explain the entropy of the EF-Tu•apo conformation. Therefore, EF-Tu•apo cannot merely be a similar conformation to EF-Tu•GDP without nucleotide, but has to be a unique conformation. Another possible explanation for the entropy of EF-Tu•apo is that this state is less flexible. This is unlikely as switch I is disordered in the EF-Tu•EF-Ts crystalized complex from Kawashima et al 1 . If this is the conformation that switch I adopts in the apo state then it is likely going to be more flexible. However, this does agree with the observation that EF-Tu•apo coordinates more water molecules, as a disordered switch I would have a larger SASA. Since EF-Tu•apo is less entropically stable compared to EF-Tu•GTP or EF-Tu•GDP which cannot be explained simply by the loss of nucleotide or EF-Tu•apo being less flexible then EF-Tu•apo must adopt a unique conformation. To directly compare the thermodynamic contributions of each nucleotide bound state relative to each other we can use the law of mass-action. However, since GTP contains an additional phosphate compared to GDP mass is not conserved in the kinetic mechanism of EF-Tu nu...

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supporting

confidence: 63%

Elongation Factor Tu’s Nucleotide Binding Is Governed by a Thermodynamic Landscape Unique among Bacterial Translation Factors

et al. 2019

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“…Sites for introducing Cys residues for labeling with maleimide dyes were selected based on the analysis of the following structures: 70S with stalled Thermus thermophilus EF-Tu·GDP·kirromycin·aa-tRNA (PDB codes 1WRN, 1WRO), E. coli EF-Tu·GDPNP·kirromycin·aa-tRNA (PDB code: 1OB2) and EF-Tu·GDP (PDB code: 1DG1). Three pairs of amino acid residues, T33/M351, D47/D314 and D165/D314 ( Supplementary Figure S1 ), were chosen for labeling, and cysteines were introduced at these positions in a variant of EF-Tu, denoted EF-Tu AV , which contains only one native cysteine, as described in the accompanying paper ( 28 ). The resulting mutants, denoted EF-Tu AV-33/351 , EF-Tu AV-47/314 and EF-Tu AV-165/314 , were expressed, purified, labeled and characterized as described elsewhere ( 28 ).…”

Section: Methodsmentioning

confidence: 99%

E. coli elongation factor Tu bound to a GTP analogue displays an open conformation equivalent to the GDP-bound form

Johansen

Kavaliauskas

Pfeil

et al. 2018

Nucleic Acids Research

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According to the traditional view, GTPases act as molecular switches, which cycle between distinct ‘on’ and ‘off’ conformations bound to GTP and GDP, respectively. Translation elongation factor EF-Tu is a GTPase essential for prokaryotic protein synthesis. In its GTP-bound form, EF-Tu delivers aminoacylated tRNAs to the ribosome as a ternary complex. GTP hydrolysis is thought to cause the release of EF-Tu from aminoacyl-tRNA and the ribosome due to a dramatic conformational change following Pi release. Here, the crystal structure of Escherichia coli EF-Tu in complex with a non-hydrolysable GTP analogue (GDPNP) has been determined. Remarkably, the overall conformation of EF-Tu·GDPNP displays the classical, open GDP-bound conformation. This is in accordance with an emerging view that the identity of the bound guanine nucleotide is not ‘locking’ the GTPase in a fixed conformation. Using a single-molecule approach, the conformational dynamics of various ligand-bound forms of EF-Tu were probed in solution by fluorescence resonance energy transfer. The results suggest that EF-Tu, free in solution, may sample a wider set of conformations than the structurally well-defined GTP- and GDP-forms known from previous X-ray crystallographic studies. Only upon binding, as a ternary complex, to the mRNA-programmed ribosome, is the well-known, closed GTP-bound conformation, observed.

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“…This model specifies that aa-tRNA entry into the PTC is unassisted by EF-Tu. In contrast, ensemble fluorescence and two-color single-molecule fluorescence-resonance energy transfer (smFRET) investigations indicate that aa-tRNA accommodation occurs faster than EF-Tu• GDP dissociation (17,42,43). Such findings suggest that EF-Tu may remain bound to the ribosome during proofreading and dissociate after peptide bond formation has occurred.…”

Section: Significancementioning

confidence: 97%

Elongation factor-Tu can repetitively engage aminoacyl-tRNA within the ribosome during the proofreading stage of tRNA selection

Morse

Girodat

Burnett

et al. 2020

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

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The substrate for ribosomes actively engaged in protein synthesis is a ternary complex of elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA), and GTP. EF-Tu plays a critical role in mRNA decoding by increasing the rate and fidelity of aa-tRNA selection at each mRNA codon. Here, using three-color single-molecule fluorescence resonance energy transfer imaging and molecular dynamics simulations, we examine the timing and role of conformational events that mediate the release of aa-tRNA from EF-Tu and EF-Tu from the ribosome after GTP hydrolysis. Our investigations reveal that conformational changes in EF-Tu coordinate the rate-limiting passage of aa-tRNA through the accommodation corridor en route to the peptidyl transferase center of the large ribosomal subunit. Experiments using distinct inhibitors of the accommodation process further show that aa-tRNA must at least partially transit the accommodation corridor for EF-Tu⋅GDP to release. aa-tRNAs failing to undergo peptide bond formation at the end of accommodation corridor passage after EF-Tu release can be reengaged by EF-Tu⋅GTP from solution, coupled to GTP hydrolysis. These observations suggest that additional rounds of ternary complex formation can occur on the ribosome during proofreading, particularly when peptide bond formation is slow, which may serve to increase both the rate and fidelity of protein synthesis at the expense of GTP hydrolysis.

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Structural dynamics of translation elongation factor Tu during aa-tRNA delivery to the ribosome

Cited by 17 publications

References 53 publications

Elongation Factor Tu’s Nucleotide Binding Is Governed by a Thermodynamic Landscape Unique among Bacterial Translation Factors

Elongation Factor Tu’s Nucleotide Binding Is Governed by a Thermodynamic Landscape Unique among Bacterial Translation Factors

E. coli elongation factor Tu bound to a GTP analogue displays an open conformation equivalent to the GDP-bound form

Elongation factor-Tu can repetitively engage aminoacyl-tRNA within the ribosome during the proofreading stage of tRNA selection

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