Crystallographic and solution structure of the N‐terminal domain of the Rel protein from <i>Mycobacterium tuberculosis</i>

Singal, Bharti; Balakrishna, A.M.; Nartey, Wilson; Manimekalai, Malathy Sony Subramanian; Jeyakanthan, Jeyaraman; Grüber, Gerhard

doi:10.1002/1873-3468.12739

Cited by 30 publications

(58 citation statements)

References 47 publications

(80 reference statements)

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“…Our previous results (Gropp et al, 2001) showed the importance of the last two domains (AA 564-744), and the importance of the three Cys residues present in the RIS domain (Loveland et al, 2016; Figure 1A), especially in protein-protein interactions. This was also reinforced with recent reports about the involvement of the CTD in the oligomerization of Rel protein in Mycobacterium (Singal et al, 2017) and also in the regulation of Bacillus Subtilis Rel synthetic activity (Pausch et al, 2020). Here, we closely examined RelA-RelA via its CTD interactions in vitro by using purified ribosomes (70S) where lack of charged tRNA in the tube mimic amino acid stress conditions.…”

Section: Discussionmentioning

confidence: 73%

“…To survive, bacteria must be able to respond to changes in their environment. Depriving Escherichia coli of one or more amino acids (AAs) triggers the stringent response (Stent and Brenner, 1961;Cashel, 1969;Cashel and Gallant, 1969;Kaspy et al, 2013). Within a few seconds after the onset of amino-acid starvation, one can observe the accumulation of phosphorylated derivatives of GTP and GDP, collectively called (p)ppGpp (Cashel, 1969;Cashel and Gallant, 1969;Fiil et al, 1972;Lund and Kjeldgaard, 1972).…”

Section: Introductionmentioning

confidence: 99%

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Escherichia coli RelA Regulation via Its C-Terminal Domain

Kaspy

Glaser

2020

Front. Microbiol.

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One of the most important stress responses in bacteria is the stringent response. The main player in this response is the signal molecule (p)ppGpp, which is synthesized by a Rel family protein. In Escherichia coli, RelA is the main synthetase of (p)ppGpp in response to amino acid starvation. Although the synthetic activity of RelA is well-understood, its regulation is not yet fully characterized. The C-terminus domain (CTD) of the E. coli RelA is responsible for the regulation of the protein and for its complete dependency on wild-type (WT) ribosome. The CTD contains three Cysteine residues, positioned in a very conserved order. Together with our previous results, we show in vitro the negative dominant effect of a part of the WT CTD (AA 564-744) named YG4 on RelA synthetic activity. This effect is abolished using mutated YG4 (YG4-638). In vitro and mass spectrometry (MS)-MS analysis of the native RelA and the mutated RelA in Cys-638 (Rel638) in the presence of the native and mutated YG4 (YG4-638) reveals that RelA forms a homodimer via its CTD by the formation of a disulfide bond between the two Cys-638 residues. This supports our previous data which showed, using a two-hybrid system, interactions between RelA proteins via the CTD. Finally, we show in vitro that excess of the native YG4 inhibited RelA synthetic activity but did not affect the amount of RelA bound to the ribosome. Our results suggest that the regulatory mechanism of RelA is by the dimerization of the protein via disulfide bonds in the CTD. Upon amino-acid starvation, the dimer changes its conformation, thus activating the stringent response in the cell.

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Section: Discussionmentioning

confidence: 73%

Section: Introductionmentioning

confidence: 99%

Escherichia coli RelA Regulation via Its C-Terminal Domain

Kaspy

Glaser

2020

Front. Microbiol.

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“…Indeed, our data show that SpoT harbours at least two multimerization domains, (i) ACT at the C-terminal end and (ii) another one located in the N-terminal catalytic domains (Figure 1 and our unpublished data). Note that the N-terminal extremity of M. tuberculosis Rel harbouring the catalytic domains was already shown to homo-dimerize (31). In addition, the ACT alone can interact with full length SpoT but not with SpoT ΔACT (Figure 1 and Supplementary Figure S1), showing that the ACT domain unlikely interferes directly with HD.…”

Section: Discussionmentioning

confidence: 99%

Regulation of (p)ppGpp hydrolysis by a conserved archetypal regulatory domain

Ronneau

Caballero-Montes

Coppine

et al. 2018

Nucleic Acids Research

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Sensory and regulatory domains allow bacteria to adequately respond to environmental changes. The regulatory ACT (Aspartokinase, Chorismate mutase and TyrA) domains are mainly found in metabolic-related proteins as well as in long (p)ppGpp synthetase/hydrolase enzymes. Here, we investigate the functional role of the ACT domain of SpoT, the only (p)ppGpp synthetase/hydrolase of Caulobacter crescentus. We show that SpoT requires the ACT domain to efficiently hydrolyze (p)ppGpp. In addition, our in vivo and in vitro data show that the phosphorylated version of EIIANtr (EIIANtr∼P) interacts directly with the ACT and inhibits the hydrolase activity of SpoT. Finally, we highlight the conservation of the ACT-dependent interaction between EIIANtr∼P and SpoT/Rel along with the phosphotransferase system (PTSNtr)-dependent regulation of (p)ppGpp accumulation upon nitrogen starvation in Sinorhizobium meliloti, a plant-associated α-proteobacterium. Thus, this work suggests that α-proteobacteria might have inherited from a common ancestor, a PTSNtr dedicated to modulate (p)ppGpp levels in response to nitrogen availability.

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“…Previously, this was impossible to do with the RelA/RSH (RelA-SpoT homolog) family of enzymes, as there were no adequate high-resolution molecular structures available. However, several RelA and related enzyme structures have been recently characterized and published: RelA ( E. coli ) [ 36 ], RelP ( Staphylococcus aureus ) [ 37 ], RelQ ( S. aureus ) [ 38 ], Rel seq ( Streptococcus equisimilis ) [ 39 ], and Rel ( Mycobacterium tuberculosis ) [ 40 ]. Thus, it has become possible through alignment and homology studies to determine the active residues within the catalytic center of these enzymes and to specifically target this region to predict and understand the ligand binding events for the rational identification of inhibitors.…”

Section: Introductionmentioning

confidence: 99%

The Development of a Pipeline for the Identification and Validation of Small-Molecule RelA Inhibitors for Use as Anti-Biofilm Drugs

et al. 2020

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Biofilm infections have no approved effective medical treatments and can only be disrupted via physical means. This means that any biofilm infection that is not addressable surgically can never be eliminated and can only be managed as a chronic disease. Therefore, there is an urgent need for the development of new classes of drugs that can target the metabolic mechanisms within biofilms which render them recalcitrant to traditional antibiotics. Persister cells within the biofilm structure may play a large role in the enhanced antibiotic recalcitrance of bacteria biofilms. Biofilm persister cells can be resistant to up to 1000 times the minimal inhibitory concentrations of many antibiotics, as compared to their planktonic envirovars; they are thought to be the prokaryotic equivalent of metazoan stem cells. Their metabolic resistance has been demonstrated to be an active process induced by the stringent response that is triggered by the ribosomally-associated enzyme RelA in response to amino acid starvation. This 84-kD pyrophosphokinase produces the “magic spot” alarmones, collectively called (p)ppGpp. These alarmones act by directly regulating transcription by binding to RNA polymerase. These transcriptional changes lead to a major shift in cellular function to both upregulate oxidative stress-combating enzymes and down regulate major cellular functions associated with growth and replication. These changes in gene expression produce the quiescent persister cells. In this work, we describe a hybrid in silico laboratory pipeline for identifying and validating small-molecule inhibitors of RelA for use in the combinatorial treatment of bacterial biofilms as re-potentiators of classical antibiotics.

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Crystallographic and solution structure of the N‐terminal domain of the Rel protein from Mycobacterium tuberculosis

Cited by 30 publications

References 47 publications

Escherichia coli RelA Regulation via Its C-Terminal Domain

Escherichia coli RelA Regulation via Its C-Terminal Domain

Regulation of (p)ppGpp hydrolysis by a conserved archetypal regulatory domain

The Development of a Pipeline for the Identification and Validation of Small-Molecule RelA Inhibitors for Use as Anti-Biofilm Drugs

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