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Nat Chem Biol ; 16(8): 834-840, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32393900


Bifunctional Rel stringent factors, the most abundant class of RelA/SpoT homologs, are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3' of guanosine tri-/diphosphate (GTP/GDP) to synthesize the bacterial alarmone (p)ppGpp, and also catalyze the 3' pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide-switch mechanism controls catalysis by Thermus thermophilus Rel (RelTt). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of RelTt (RelTtNTD) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation.

Proteínas Proto-Oncogênicas c-rel/genética , Proteínas Proto-Oncogênicas c-rel/metabolismo , Sequência de Aminoácidos , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Regulação Bacteriana da Expressão Gênica/genética , Genes rel/genética , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/metabolismo , Hidrolases/metabolismo , Ligases/metabolismo , Ligases/fisiologia , Nucleotídeos/metabolismo , Ribossomos/metabolismo , Thermus thermophilus/enzimologia , Thermus thermophilus/metabolismo
Structure ; 27(1): 90-101.e6, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30471924


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 Förster 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.

Proteínas de Escherichia coli/química , Simulação de Dinâmica Molecular , Proteínas SecA/química , Sítios de Ligação , Proteínas de Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Nucleotídeos/química , Nucleotídeos/metabolismo , Ligação Proteica , Proteínas SecA/metabolismo
Sci Adv ; 4(3): eaap9714, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29546243


Bacterial protein synthesis is intricately connected to metabolic rate. One of the ways in which bacteria respond to environmental stress is through posttranslational modifications of translation factors. Translation elongation factor Tu (EF-Tu) is methylated and phosphorylated in response to nutrient starvation upon entering stationary phase, and its phosphorylation is a crucial step in the pathway toward sporulation. We analyze how phosphorylation leads to inactivation of Escherichia coli EF-Tu. We provide structural and biophysical evidence that phosphorylation of EF-Tu at T382 acts as an efficient switch that turns off protein synthesis by decoupling nucleotide binding from the EF-Tu conformational cycle. Direct modifications of the EF-Tu switch I region or modifications in other regions stabilizing the ß-hairpin state of switch I result in an effective allosteric trap that restricts the normal dynamics of EF-Tu and enables the evasion of the control exerted by nucleotides on G proteins. These results highlight stabilization of a phosphorylation-induced conformational trap as an essential mechanism for phosphoregulation of bacterial translation and metabolism. We propose that this mechanism may lead to the multisite phosphorylation state observed during dormancy and stationary phase.

Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/metabolismo , Modelos Moleculares , Nucleotídeos/metabolismo , Fosforilação , Fosfotreonina/metabolismo , Ligação Proteica , Conformação Proteica , Termodinâmica
J Phys Chem B ; 122(15): 4249-4266, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29543457


Förster resonance energy transfer (FRET) is a powerful tool to probe molecular interactions, activity, analytes, forces, and structure. Single-molecule (sm)FRET additionally allows real-time quantifications of conformation and conformational dynamics. smFRET robustness critically depends on the employed dyes, yet a systematic comparison of different dye pairs is lacking. Here, we evaluated blue (Atto488 and Alexa488) and far-red (Atto647N, Alexa647, StarRed, and Atto655) dyes using confocal smFRET spectroscopy on freely diffusing double-stranded (ds)DNA molecules. Via ensemble analyses (correlation, lifetime, and anisotropy) of single-labeled dsDNA, we find that Alexa488 and Atto647N are overall the better dyes, although the latter interacts with DNA. Via burstwise analyses of double-labeled dsDNA with interdye distances spanning the complete FRET-sensitive range (3.5-9 nm), we show that none of the dye pairs stands out: distance accuracies were generally <1 nm and precision was ∼0.5 nm. Finally, excitation of photoblinking dyes such as Alexa647 influences their fluorescence quantum yield, which has to be taken into account in distance measurements and leads to FRET dynamics. Although dye performance might differ in experiments on immobilized molecules, our combined ensemble and single-molecule approach is a robust characterization tool for all types of smFRET experiments. This is especially important when smFRET is used for atomic-scale distance measurements.