Phosphorylation decelerates conformational dynamics in bacterial translation elongation factors

Talavera, Ariel; Hendrix, Jelle; Versées, Wim; Jurėnas, Dukas; Nerom, Katleen Van; Vandenberk, Niels; Singh, Ranjan K.; Konijnenberg, Albert; Gieter, Steven De; Castro-Roa, Daniel; Barth, Anders; Greve, Henri De; Sobott, Frank; Hofkens, Johan; Zenkin, Nikolay; Loris, Remy; García-Pino, Abel

doi:10.1126/sciadv.aap9714

Cited by 39 publications

(38 citation statements)

References 68 publications

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“…In agreement with that, Doc preferentially phosphorylates the GDP-bound state of EF-Tu (Castro-Roa et al, 2013). Phosphorylation of the Thr382 located on the loop of the beta-barrel domain III of EF-Tu locks it in an unfavorable open conformation typical of GDP-bound EF-Tu (Talavera et al, 2018). Conformational dynamics of EF-Tu are the essence of its function and GTP hydrolysis has a major effect on aa-tRNA binding and interaction with the ribosome.…”

Section: Hipa Toxinssupporting

confidence: 71%

“…Conformational dynamics of EF-Tu are the essence of its function and GTP hydrolysis has a major effect on aa-tRNA binding and interaction with the ribosome. Once locked in an open state, EF-Tu exhibits decreased affinity for aa-tRNA to a similar extent as the affinity of GDP-bound EF-Tu (Talavera et al, 2018) and is not compatible with translation (Castro-Roa et al, 2013). Fic domain toxins that perform AMPylation have also been reported to constitute type II TA modules (Harms et al, 2015).…”

Section: Hipa Toxinsmentioning

confidence: 99%

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The Variety in the Common Theme of Translation Inhibition by Type II Toxin–Antitoxin Systems

Jurėnas

Melderen

2020

Front. Genet.

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Type II Toxin-antitoxin (TA) modules are bacterial operons that encode a toxic protein and its antidote, which form a self-regulating genetic system. Antitoxins put a halter on toxins in many ways that distinguish different types of TA modules. In type II TA modules, toxin and antitoxin are proteins that form a complex which physically sequesters the toxin, thereby preventing its toxic activity. Type II toxins inhibit various cellular processes, however, the translation process appears to be their favorite target and nearly every step of this complex process is inhibited by type II toxins. The structural features, enzymatic activities and target specificities of the different toxin families are discussed. Finally, this review emphasizes that the structural folds presented by these toxins are not restricted to type II TA toxins or to one particular cellular target, and discusses why so many of them evolved to target translation as well as the recent developments regarding the role(s) of these systems in bacterial physiology and evolution.

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Section: Hipa Toxinssupporting

confidence: 71%

Section: Hipa Toxinsmentioning

confidence: 99%

The Variety in the Common Theme of Translation Inhibition by Type II Toxin–Antitoxin Systems

Jurėnas

Melderen

2020

Front. Genet.

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“…In other words, PBD moves from one of its states to the other more slowly than 10 ms. Therefore, a PDA model incorporating different FRET states was fitted to the data, with each state assuming a Gaussian distance distribution (Kalinin et al, 2008Talavera et al, 2018) Figure 3C, bottom) or moves close to IRA2 (state 1) or to WD (state 3). The fraction of molecules that occupy these states is distinct: more than half populate state 2, followed by state 1 and 3.…”

Section: Accurate Measurement Of Physical Positioning Of Pbd States Imentioning

confidence: 99%

The Preprotein Binding Domain of SecA Displays Intrinsic Rotational Dynamics

et al. 2019

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Graphical AbstractHighlights d The preprotein binding domain of SecA is mobile in solution d PBD swivels around its stem to acquire 3-4 distinct states d Monomerization, but not nucleotides, regulate PBD swiveling d smFRET provides unique dynamics analysis of SecA SUMMARY SecA converts ATP energy to protein translocation work. Together with the membrane-embedded SecY channel it forms the bacterial protein translocase. How secretory proteins bind to SecA and drive conformational cascades to promote their secretion remains unknown. To address this, we focus on the preprotein binding domain (PBD) of SecA. PBD crystalizes in three distinct states, swiveling around its narrow stem. Here, we examined whether PBD displays intrinsic dynamics in solution using single-molecule Fö rster resonance energy transfer (smFRET). Unique cysteinyl pairs on PBD and apposed domains were labeled with donor/acceptor dyes. Derivatives were analyzed using pulsed interleaved excitation and multi-parameter fluorescence detection. The PBD undergoes significant rotational motions, occupying at least three distinct states in dimeric and four in monomeric soluble SecA. Nucleotides do not affect smFRET-detectable PBD dynamics. These findings lay the foundations for single-molecule dissection of translocase mechanics and suggest models for possible PBD involvement during catalysis.

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“…The titrations were performed on an affinity ITC instrument (TA Instruments) at 10 C by injecting 2 ml of the MnCl 2 solution for each titration point at 200 s intervals with the stirring rate set to 75 rev min À1 . Data analysis and modelling were performed as described previously (Garcia-Pino et al, 2016;Talavera et al, 2018) with NanoAnalyze (TA Instruments) and MicroCal Origin 7.0. final Mg 2+ concentration of 5 mM. After pre-incubation at 40 C for 3 min, the reaction was started by the addition of preheated ATP or APPNP {adenosine 5 0 -[(,)-imido]-triphosphate} to a final concentration of 1 mM and quenched with 4 ml 70% formic acid supplemented with a cold nucleotide standard (4 mM GDP) for UV-shadowing.…”

Section: Isothermal Titration Calorimetrymentioning

confidence: 99%

The Rel stringent factor from Thermus thermophilus: crystallization and X-ray analysis

et al. 2019

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The stringent response, controlled by (p)ppGpp, enables bacteria to trigger a strong phenotypic resetting that is crucial to cope with adverse environmental changes and is required for stress survival and virulence. In the bacterial cell, (p)ppGpp levels are regulated by the concerted opposing activities of RSH (RelA/SpoT homologue) enzymes that can transfer a pyrophosphate group of ATP to the 3 0 position of GDP (or GTP) or remove the 3 0 pyrophosphate moiety from (p)ppGpp. Bifunctional Rel enzymes are notoriously difficult to crystallize owing to poor stability and a propensity for aggregation, usually leading to a loss of biological activity after purification. Here, the production, biochemical analysis and crystallization of the bifunctional catalytic region of the Rel stringent factor from Thermus thermophilus (Rel Tt NTD ) in the resting state and bound to nucleotides are described. Rel Tt and Rel Tt NTD are monomers in solution that are stabilized by the binding of Mn 2+ and mellitic acid. Rel Tt NTD crystallizes in space group P4 1 22, with unit-cell parameters a = b = 88.4, c = 182.7 Å , at 4 C and in space group P4 1 2 1 2, with unit-cell parameters a = b = 105.7, c = 241.4 Å , at 20 C. research communications Acta Cryst. (2019). F75, 561-569 Van Nerom et al. Rel stringent factor 565 research communications Acta Cryst. (2019). F75, 561-569 Van Nerom et al. Rel stringent factor 569

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Phosphorylation decelerates conformational dynamics in bacterial translation elongation factors

Abstract: Phosphorylation-induced conformational trap is an essential mechanism for phosphoregulation of bacterial metabolism.

Cited by 39 publications

References 68 publications

The Variety in the Common Theme of Translation Inhibition by Type II Toxin–Antitoxin Systems

The Variety in the Common Theme of Translation Inhibition by Type II Toxin–Antitoxin Systems

The Preprotein Binding Domain of SecA Displays Intrinsic Rotational Dynamics

The Rel stringent factor from Thermus thermophilus: crystallization and X-ray analysis

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