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A) GTPase assay was performed, as described in the text, by using 1 µM EngAMS in the reaction mixtures each containing 200 µM GTP and different concentrations of GDP. Shown is the bar graph plot using values of the rate of GTP hydrolysis (µM Pi released per minute) and concentrations of GDP (µM), represented on y- and x-axis respectively. B) GTPase assay was performed by using 2–4 µM EngAMS in the reaction mixtures each containing 10 µM GDP and different concentrations of GTP and the values were plotted in a graph using GraphPad Prism software, as described in the materials and methods section. The x- and y-axes represent GTP concentrations (µM) and rate of Pi release (µM Pi released per minute) due to GTP hydrolysis, respectively. Each assay was performed in triplicate and the mean values ± s.d. were used to determine the GTPase activity. *P<0.05.
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Bacterial P-loop GTPases belong to a family of proteins that selectively hydrolyze a small molecule guanosine tri-phosphate (GTP) to guanosine di-phosphate (GDP) and inorganic phosphate, and regulate several essential cellular activities such as cell division, chromosomal segregation and ribosomal assembly. A comparative genome sequence analysis of...
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... EngA was shown to be a potential drug target against lethal diseases like Tuberculosis [12] and other bacterial diseases. Towards developing species-specific antibiotics, we set out to understand species-specific variations in this class of proteins, as they would provide insights for the design of highly specific and potent antibiotics against pathogens. ...
EngA is an essential and unique bacterial GTPase involved in ribosome biogenesis. The essentiality and species-specific variations among EngA homologues make the protein a potential target for future drug development. In this aspect, it is important to understand the variations of EngA among probiotic organisms and non-probiotic bacteria to understand species specificity. The search for variations among EngA homologues revealed a unique variant, exclusively found in Bifidobacterium and a few Actinobacteria species. Bifidobacterium possesses a multifunctional fusion protein, wherein EngA is fused with an N-terminal CMK (Cytidylate Monophosphate Kinase) domain. The resulting protein is therefore a large (70kDa size) with 3 consecutive P-loops and a 50 amino acid long linker connecting the EngA and CMK domains. EngA is known to regulate ribosome biogenesis via nucleotide-dependent conformational changes. The additional domain may introduce further intricate regulation in ribosome biogenesis or participate in newer biological processes. This study is the first attempt to characterise this novel class of bacterial EngA found in the Genus of Bifidobacteria.
... Next, the Km and Vmax were calculated using a regression line equation to link the numbers. Finally, Vmax was divided by total enzyme concentration to determine Kcat [63,67]. ...
Simple Summary
Rv1636 is a mycobacterial universal stress protein whose expression level increases in different type of stress conditions. This protein promotes the growth of Mycobacterium tuberculosis in the host derived stress conditions generated during infection. Therefore in this manuscipt, we are trying to target Rv1636 using natural inhibitor. Targeting essential Mycobacterial protein using natural prodect was hypothesized to generate a molecule with low toxic effects and high inhibitory activity. It was found that Rv1636 contains ATPase activity and its ATPase activity gets disturbed by addition of β-Amyrin in the reaction. β-Amyrin was forund to interfere with the ATP binding site of Rv1636 which was confirmed by molecular docking anad dynamic studies. In addition to the ATPase activity, Rv1636 was also contain the cAMP binding capacity and also involved in balancing the cAMP levels inside cells. So, targeting Rv1636 using β-Amyrin disrupts its ATPase activity and cAMP regulatory activity and these conditions might make Mycobacterium tuberculosis more susceptible to the host derived stress conditions.
Abstract
Mycobacterium tuberculosis has seen tremendous success as it has developed defenses to reside in host alveoli despite various host-related stress circumstances. Rv1636 is a universal stress protein contributing to mycobacterial survival in different host-derived stress conditions. Both ATP and cAMP can be bound with the Rv1636, and their binding actions are independent of one another. β-Amyrin, a triterpenoid compound, is abundant in medicinal plants and has many pharmacological properties and broad therapeutic potential. The current study uses biochemical, biophysical, and computational methods to define the binding of Rv1636 with β-Amyrin. A substantial interaction between β-Amyrin and Rv1636 was discovered by molecular docking studies, which helped decipher the critical residues involved in the binding process. VAL60 is a crucial residue found in the complexes of both Rv1636_β-Amyrin and Rv1636-ATP. Additionally, the Rv1636_β-Amyrin complex was shown to be stable by molecular dynamics simulation studies (MD), with minimal changes observed during the simulation. In silico observations were further complemented by in vitro assays. Successful cloning, expression, and purification of Rv1636 were accomplished using Ni-NTA affinity chromatography. The results of the ATPase activity assay indicated that Rv1636’s ATPase activity was inhibited in the presence of various β-Amyrin concentrations. Additionally, circular dichroism spectroscopy (CD) was used to examine modifications to Rv1636 secondary structure upon binding of β-Amyrin. Finally, isothermal titration calorimetry (ITC) advocated spontaneous binding of β-Amyrin with Rv1636 elucidating the thermodynamics of the Rv1636_β-Amyrin complex. Thus, the study establishes that β-Amyrin binds to Rv1636 with a significant affinity forming a stable complex and inhibiting its ATPase activity. The present study suggests that β-Amyrin might affect the functioning of Rv1636, which makes the bacterium vulnerable to different stress conditions.
... Perhaps this signifies that this family has probably originated from bacteria and the plant members obtained it from the pro-chloroplast symbiont via horizontal transfer (Leipe et al., 2002). EngA and its orthologs are known to consist of two GTPase domains (Leipe et al., 2002;Agarwal et al., 2012). The family of EngA has been named after essential Neisserial GTP-binding protein A since it was first discovered in Neisseria gonorrhoeae (Agarwal et al., 2012). ...
... EngA and its orthologs are known to consist of two GTPase domains (Leipe et al., 2002;Agarwal et al., 2012). The family of EngA has been named after essential Neisserial GTP-binding protein A since it was first discovered in Neisseria gonorrhoeae (Agarwal et al., 2012). Era (E. coli Ras-like protein) is widely conserved across all forms of life, was initially identified as a bacterial protein, and serves as an essential GTPase in E. coli. ...
Obg proteins belong to P‐loop guanine triphosphatase (GTPase) that are conserved from bacteria to humans. Like other GTPases, Obg cycles between guanine triphosphate (GTP) bound “on” state and guanine diphosphate (GDP)‐bound “off” state, thereby controlling various cellular processes. Different members of this group have unique structural characteristics; a conserved glycine‐rich N‐terminal domain known as obg fold, a central conserved nucleotide binding domain, and a less conserved C‐terminal domain of other functions. Obg is a ribosome dependent GTPase helps in ribosome maturation by interacting with several proteins of the 50S subunit of the ribosome. Obg proteins have been widely considered as a regulator of cellular functions, helping in DNA replication, cell division. Apart from that, this protein also takes part in various stress adaptation pathways like a stringent response, sporulation, and general stress response. In this particular review, the structural features of ObgE have been highlighted and how the structure plays important role in interacting with regulators like GTP, ppGpp that are crucial for executing biological function has been orchestrated. In particular, we believe that Obg‐like proteins can provide a link between different global pathways that are necessary for fine‐tuning cellular processes to maintain the cellular energy status. Proposed model of Obg action: Wildtype Obg binds to GTP (Guanine triphosphate) and gets conformationally active. Active Obg interacts with 50S ribosomal subunit of 70S ribosome that helps ribosome to modulate protein translation under various physiological conditions. Unlike the wild type Obg, mutated Obg fails to activate ribosome since it is unable to bind GTP and therefore fails to regulate the cellular functions of different living systems.
... The effect of stress condition on protein enzymatic activity was also evaluated by carrying out the same reaction at different temperatures (4°C, 25°C, 37°C, 50°C and 65°C), different pH (3.0, 5.5, 7.4, 8.0 and 10.0), different magnesium and manganese concentration (10 mM, 20 mM, 40mM, 60mM and 100 mM) and in the presence of glycerol (10%, 20% and 30%). The assays were performed in triplicates and the mean values were used to determine the GTPases activity of three individual experiments [40][41][42][43]. Apart from these enzymatic assays, enzymatic activity was also measured in the presence of another neighboring nucleotide (ATP, CTP and UTP). ...
Mycobacterium tuberculosis (M. tuberculosis H37Rv) utilizes the signal recognition particle pathway (SRP pathway) system for secretion of various proteins from ribosomes to the extracellular surface which plays an important role in the machinery running inside the bacterium. This system comprises of three major components FtsY, FfH and 4.5S rRNA. This manuscript highlights essential factors responsible for the optimized enzymatic activity of FtsY. Kinetic parameters include Vmax and Km for the hydrolysis of GTP by ftsY which were 20.25±5.16 μM/min/mg and 39.95±7.7 μM respectively. kcat and catalytic efficiency of the reaction were 0.012±0.003 s⁻¹ and 0.00047±0.0001 μM/s⁻¹ respectively. These values were affected upon changing the standard conditions. Cations (Mg²⁺ and Mn²⁺) play important role in FtsY enzymatic activity as increasing Mg²⁺ decrease the activity. Mn²⁺on the other hand is required at higher concentration around 60 mM for carrying optimum GTPase activity. FtsY is hydrolyzing ATP and GDP as well and GDP acts as an inhibitor of the reaction. MD simulation shows effective binding and stabilization of the FtsY complexed structure with GTP, GDP and ATP. Mutational analysis was done at two important residues of GTP binding motif of FtsY, namely, GXXXXGK (K236) and DXXG (D367) and showed that these mutations significantly decrease FtsY GTPase activity. FtsY is comprised of α helices, but this structural pattern was shown to change with increasing concentrations of GTP and ATP which symbolize that these ligands cause significant conformational change by variating the secondary structure to transduce signals required by downstream effectors. This binding favors the functional stabilization of FtsY by destabilization of α-helix integrity. Revealing the hidden aspects of the functioning of FtsY might be an essential part for the understanding of the SRP pathway which is one of the important contributors of M. tuberculosis virulence.
... Interaction of EngA with the 50S subunit [9,10,12,13] and with the 70S ribosome [11,13] was shown in E. coli, B. subtilis and Mycobacterium smegmatis, in the presence of GTP analogues. Interaction of EngA with the 30S subunit was also suggested to occur in a nucleotide-specific manner by the work of different groups [10,13], and specific interaction with the ribosomal protein S7 was shown in Salmonella typhimurium by both pulldown assays and ITC experiments [14]. ...
... When GDP is bound to GD1, however, interdomain interactions between GD1 and KH domains shift the affinity of EngA towards the 30S subunit [13]. Conversely, a study with M. segmatis EngA indicates that binding of EngA to the 30S occurs regardless of the presence of nucleotides [12]. ...
... Our data indicate that similar levels of GTP concentration are required to either induce a conformational change on EngA or increase the binding of EngA to the ribosomes. The need of 10 mM nucleotides to induce a change in conformation may seem unexpected given the effect of nucleotides on ribosome binding observed by other groups using lower concentrations (0.1-1 mM, [10][11][12][13]). The difference may come from the fact that the apo EngA produced for the present study seems to retain GDP bound to the GD2 domain. ...
... Sucrose density gradient sedimentation experiments in E. coli, B. subtilis and M. smegmatis showed cofractionation of EngA with the 50S subunit (Agarwal et al., 2012;Bharat et al., 2006;Hwang and Inouye, 2006;Tomar et al., 2009) and with the 70S ribosome Tomar et al., 2009) in the presence of GTP analogues. This interaction was strongly inhibited by GDP, suggesting that EngA is a GTP-dependent 50S-associating protein. ...
... When GDP is bound to GD1, however, inter-domain interactions between GD1 and KH domains shift the affinity of EngA towards the 30S subunit. Conversely, a study done on M. smegmatis EngA indicates that binding of EngA to the 30S occurs regardless of the presence of nucleotides (Agarwal et al., 2012). ...
... Values of kcat for Ras with and without GAP Ras are presented as a reference. Values taken from references: Agarwal et al., 2012;Bharat et al., 2006;Foucher et al., 2012;Gideon et al., 1992;Hwang and Inouye, 2001. ...
The development of new therapeutics against bacterial infections has aroused great interest over the last years in the context of drug resistance. The starting-point in the pursuit of new antibiotics for which bacterial resistance mechanisms do not exist is the identification of novel cellular targets. Genetics studies in the early 2000s have identified engA as a conserved bacterial gene whose product is a GTPase that could represent a potential drug target: it is conserved among bacteria, essential for cell survival, and absent in humans.Since EngA acts as an assembly factor for the bacterial ribosome, one of our aims was to develop an assay to screen inhibitors of the EngA-ribosome interactions. These interactions are modulated by EngA conformational changes that are in turn triggered by the binding of different nucleotides to the catalytic G-domain. As the interplay between all these events in bacteria is still not resolved, we have used a multi-technique approach to explore these questions in order to obtain useful information for the setting up of a robust screening assay.SAXS and limited proteolysis showed a conformational change occurring in solution upon addition of either di- or tri-phosphate nucleotides. While model validation analysis confirmed the GDP-bound conformation, the GTP-bound state does not match any known EngA structure. Binding studies have revealed modulation of interactions by different nucleotide-bound states. Furthermore, response to nucleotides occurs at high concentrations, suggesting that the role of EngA in promoting ribosome assembly could be monitored by the intracellular nucleotide concentration. Efforts on identifying the GTP-bound state 3D structure by crystallography have resulted in EngA structures in different crystal forms. Although all the obtained structures represent the GDP-bound state, packing analysis has revealed conserved crystal contacts that can potentially stabilise this conformation during nucleation. Specific mutations aiming at disrupting these contacts may help to promote crystallisation of alternative conformations. Cryo-EM investigation has been initiated in order to obtain the structure of the B. subtilis EngA:50S complex. So far, an electron density map at 6.4 Å resolution has been obtained and its interpretation is underway.
... In this study, the authors suggest that EngA might interact with both 50S and 30S ribosomal subunits at different stages of the assembly process [10]. The possibility that EngA might interact with 30S is also corroborated by the observation that it co-elutes with 16S rRNA [4,11,12]. In our previous studies, we argued that the role of EngA in ribosome biogenesis must arise from its ability to attain multiple guanine-nucleotide bound states, thereby permitting an intricate molecular mechanism. ...
EngA consists of two tandem GTPase-domains - GD1 and GD2 - followed by a KH-domain. EngA was considered to be a 50S assembly factor since it was shown to bind 50S and its deletion leads to the accumulation of immature 45S ribosomal subunits. Subsequently, we demonstrated an additional ribosome bound state of EngA bound to 50S, 30S, and 70S. While the former (50S binding) is achieved upon GTP binding at both GD1 and GD2, the latter is formed upon GTP hydrolysis at GD1, which is believed to trigger a large conformational change in the protein. The present study brings out two key aspects of EngA regulation: First, that distinctly stabilized GD1-KH interfaces allows EngA to exist in different ribosome bound states, and second is the importance of these states to ribosome assembly. Our analyses suggest that distinct inter-domain (GD-KH) interfaces are stabilized by interactions arising from unique sets of motifs, conserved across EngA homologues, and seem to be mechanistically linked to GTP/GDP binding. By experimentally measuring binding affinities for several interface mutants, we show that disrupting the interface interactions is necessary to realize EngA-ribosome binding. These findings are also supported by a recent cryo-EM structure of EngA bound to 50S, wherein the GD1-KH interface is completely disrupted leading to an ‘extended’ or ‘open state’ of the protein. Overall, it appears that the transition of EngA from a ‘closed state’ with GD1-KH forming a tight interface, to an ‘open state’ mediates interaction with ribosomal subunits.
... Obg is a highly conserved GTP hydrolysing protein and its GTP hydrolysing activity is known in several bacterial species. In this work, Obg enzyme assay were performed in the 30 reaction volume containing 1X assay buffer (50mM Tris pH 7.4, 100mM NaCl and 10mM, MgCl 2 ) using 500uM concentration of GTP and 2uM concentration of Obg (37). The reaction mixture is incubated for 30 minutes at 37 o C and to measure the released inorganic phosphate malachite green reagent was added and its absorbance is measured at 630nm in multimode plate reader. ...
... For the determination of GTPase activity of Obg, concentrated protein was incubated with the assay buffer (50mM Tris pH 7.4, 100mM NaCl and 10mM, MgCl 2 ) and GTP for 30 minutes as mentioned above. Active enzyme should have the ability to hydrolyse the GTP and to release the 3 or 4 fold more inorganic phosphate as compared to various controls which can be further detected by adding malachite green reagent (37). Unfortunately, purified Obg was found to be very less active and also high background was observed in the EngA. ...
... Pyrophosphatase (PPase) enzyme catalyze the formation of two inorganic phosphates so, three phosphate molecules are produced in a reaction. For the determination of in vitro activity, concentrated enzyme was incubated with assay buffer (50mM Tris pH 8.0, 25mM KCL, 10mM MgCl 2, 2mM DTT), L-methionine, ATP and Pyrophosphatase(37). Activity was measured by adding malachite green reagent after 30 minutes of incubation. ...
... This fragment showed 100% nucleic acid identity to PlasmoDB identifier PF3D7_ 0313500 encoding a part of the putative G-binding protein (Fig 2). Moreover, the PCR amplificate showed significant amino acid identity scores of 72% on the amino acid level to the EngA domain [34] of different prokaryotes. EngA proteins are a unique family of bacterial Ras GTPases with two tandem GTP binding domains and a KH-like domain [35] which is depicted in Fig 2. Next, two primers i.e. ...
... Similar results were obtained in an amino acid alignment with the 19 different human G-alpha subunits (data not shown). PfG shows different matches to the EngA2 domain [34] present in the Ras-like GTpase superfamily (amino acid position 800-875) comprising five G Box motifs for nucleotide binding.The different G Box motifs are depicted in Fig 2. Hence, it is worthy to note that the G box 3 motif DXXG contains asparagine (D) which forms a water-bridged contact with Mg 2+ while glycine (G) is hydrogen bonded to the gamma phosphate of GTP through the backbone amide. ...
During its development the malaria parasite P. falciparum has to adapt to various different environmental contexts. Key cellular mechanisms involving G-protein coupled signal transduction chains are assumed to act at these interfaces. Heterotrimeric G-proteins are absent in Plasmodium. We here describe the first cloning and expression of a putative, non-canonical Ras-like G protein (acronym PfG) from Plasmodium. PfG reveals an open reading frame of 2736 bp encoding a protein of 912 amino acids with a theoretical pI of 8.68 and a molecular weight of 108.57 kDa. Transcript levels and expression are significantly increased in the erythrocytic phase in particular during schizont and gametocyte formation. Most notably, PfG has GTP binding capacity and GTPase activity due to an EngA2 domain present in small Ras-like GTPases in a variety of Bacillus species and Mycobacteria. By contrast, plasmodial PfG is divergent from any human alpha-subunit. PfG was expressed in E. coli as a histidine-tagged fusion protein and was stable only for 3.5 hours. Purification was only possible under native conditions by Nickel-chelate chromatography and subsequent separation by Blue Native PAGE. Binding of a fluorescent GTP analogue BODIPY® FL guanosine 5'O-(thiotriphosphate) was determined by fluorescence emission. Mastoparan stimulated GTP binding in the presence of Mg2+. GTPase activity was determined colorimetrically. Activity expressed as absolute fluorescence was 50% higher for the human paralogue than the activity of the parasitic enzyme. The PfG protein is expressed in the erythrocytic stages and binds GTP after immunoprecipitation. Immunofluorescence using specific antiserum suggests that PfG localizes to the parasite cytosol. The current data suggest that the putitative, Ras-like G-protein might be involved in a non-canonical signaling pathway in Plasmodium. Research on the function of PfG with respect to pathogenesis and antimalarial chemotherapy is currently under way.
... In vitro growth was assessed by measuring OD600 of cultures at regular intervals, as well as by spotting serial dilutions of bacterial cultures on 7H11 agar plates supplemented with or without ATc, as described in Methods. Figure 4 demonstrates that repression of engA MS results in severely attenuated growth of Msm, which is similar to that of mmpL3 MS knockdown strain. Spotting of 10-fold serial dilutions of cultures on 7H11 plates revealed that growth of engA MS ( À ) strain is reduced by 41,000-fold in the presence of ATc, thus indicating the essential requirement of EngA in Msm as hypothesized earlier 28 (Fig. 4c,d). Conversely, repression of yidC does not affect growth of Msm in both 7H9 broth and on 7H11 agar media (Fig. 4e). ...
Recombination-based tools for introducing targeted genomic mutations in Mycobacterium tuberculosis are not efficient due to higher rate of illegitimate recombination compared with homologous DNA exchange. Moreover, involvement of multiple steps and specialized reagents make these tools cost ineffective. Here we introduce a novel clustered regularly interspaced short palindromic repeat (CRISPR) interference (CRISPRi) approach that efficiently represses expression of target genes in mycobacteria. CRISPRi system involves co-expression of the catalytically dead form of RNA-guided DNA endonuclease from the type II CRISPR system known as dCas9 and the small guide RNA specific to a target sequence, resulting in the DNA recognition complex that interferes with the transcription of corresponding DNA sequence. We show that co-expression of the codon-optimized dCas9 of S. pyogenes with sequence-specific guide RNA results in complete repression of individual or multiple targets in mycobacteria. CRISPRi thus offers a simple, rapid and cost-effective tool for selective control of gene expression in mycobacteria.
