In silico and experimental validation of protein–protein interactions between PknI and Rv2159c from Mycobacterium tuberculosis

Venkatesan, Arunkumar; Hassan, Sameer; Palaniyandi, Kannan; Narayanan, Sujatha

doi:10.1016/j.jmgm.2015.10.011

Cited by 15 publications

(12 citation statements)

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“…Displaying the prototypical kinase fold and sequence motifs characteristic of Hanks-type kinases, as already noticed [10,13], the crystal structure of PknI presents some striking features. The activation segment, defined as the stretch running from the DFG to the APE conserved motifs [14], lacks in PknI an activation loop and a P + 1 loop and is stabilized within the active site in a 'inward' conformation, incompatible with binding of peptide substrates ( Figs 4A,B and 5A).…”

Section: The Substrate Binding Cleft Of Pknimentioning

confidence: 62%

“…PknA and PknB, have been proven to be essential for the bacterial survival [4][5][6][7], thus, raising more general questions about the physiological roles of the ensemble of ePKs, especially in the course of infection, about interactions with other players in signalling like the two-component systems and their possible functional redundancy. Initially among the less studied mycobacterial ePKs, partly due to the nonessentiality of the corresponding gene in vitro [8], PknI (Rv2910c) has recently gained interest with the publication of a homology model [9], the identification of two possible interaction partners [10] and the description of the phenotype of a double pknI À /dacB2 À knockout in Mtb [11], confirming initial reports suggesting a role of PknI in infection [8]. In order to structurally characterize and compare PknI with other known ePKs, and eventually investigate the molecular basis of its role in the course of Mtb infection, we solved the crystal structure of the PknI catalytic domain.…”

Section: Introductionmentioning

confidence: 99%

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The crystal structure of PknI from Mycobacterium tuberculosis shows an inactive, pseudokinase‐like conformation

et al. 2017

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Section: The Substrate Binding Cleft Of Pknimentioning

confidence: 62%

Section: Introductionmentioning

confidence: 99%

The crystal structure of PknI from Mycobacterium tuberculosis shows an inactive, pseudokinase‐like conformation

et al. 2017

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“…In our previous study, we have shown the protein kinase PknI interacts with Ala-Gly-Trp motif residues of Rv2159c (Venkatesan et al, 2015 ). We expanded our previous observations and confirmed the results with pull down assay using recombinant His-PknI and GST-Rv2159c proteins.…”

Section: Resultsmentioning

confidence: 90%

“…Previous study from our lab identified the physical interaction between STPK, PknI and Rv2159c. The study also suggest Ala-Gly-Trp motif of Rv2159c is responsible for PknI interaction (Venkatesan et al, 2015 ).…”

Section: Introductionmentioning

confidence: 88%

Functional Characterization of PknI-Rv2159c Interaction in Redox Homeostasis of Mycobacterium tuberculosis

et al. 2016

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Mycobacterium tuberculosis adapts to stress conditions by responding to the signals from its external environment. M. tuberculosis genome encodes 11 eukaryotic like serine/threonine protein kinases (STPK) and their importance in regulating the physiology and virulence of the bacteria are being explored. Previous study from our lab identified the M. tuberculosis STPK, PknI interacts with two peroxidase proteins such as Rv2159c and Rv0148. In this study, we have characterized the biological function behind the PknI-Rv2159c interaction in M. tuberculosis. Point mutation of Ala-Gly-Trp motif identified that only Ala49 and Gly50 amino acids of Rv2159c are responsible for interaction and there is no phosphorylation involved in the PknI-Rv2159c interaction. Rv2159c is a member from the carboxymuconolactone decarboxylase family with peroxidase activity. Enzymatic assays with catalytic site point mutants showed that Cys84 of Rv2159c was responsible for its alkylhydroperoxidase activity. Interestingly, interaction with PknI increased its peroxidase activity by several folds. Gene knockdown of Rv2159c in M. tuberculosis showed increased sensitivity to peroxides such as cumene hydroperoxide and hydrogen peroxide. Proteomic analysis of differentially expressing Rv2159c strains by 2D gel electrophoresis and mass spectrometry revealed the differential abundance of 21 proteins. The total absence of oxidoreductase, GuaB1 suggests the essential role of Rv2159c in redox maintenance. Our findings provide new insights on signaling mechanisms of PknI in maintaining the redox homeostasis during oxidative stresses.

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“…Earlier studies reported that over expression of M. tuberculosis Rv0148 from multidrug-resistance isolates in Escherichia coli showed two-to three-folds of higher shift in MIC [18]. Previous studies from our lab identified that PknI, one of the 11 serine-threonine protein kinases (STPKs), interacts with two proteins Rv2159c and Rv0148 [19]. Rv2159c was characterized using gene knockdown studies, and its interaction with PknI increased its peroxidase activity several folds in the mutant strain [20].…”

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confidence: 96%

Protein–protein interaction of Rv0148 with Htdy and its predicted role towards drug resistance in Mycobacterium tuberculosis

et al. 2020

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Background: Mycobacterium tuberculosis resides inside host macrophages during infection and adapts to resilient stresses generated by the host immune system. As a response, M. tuberculosis codes for short-chain dehydrogenases/ reductases (SDRs). These SDRs are nicotinamide adenine dinucleotide-reliant oxidoreductases involved in cell homeostasis. The precise function of oxidoreductases in bacteria especially M. tuberculosis were not fully explored. This study aimed to know the detail functional role of one of the oxidoreductase Rv0148 in M. tuberculosis. Results: In silico analysis revealed that Rv0148 interacts with Htdy (Rv3389) and the protein interactions were confirmed using far western blot. Gene knockout mutant of Rv0148 in M. tuberculosis was constructed by specialized transduction. Macrophage cell line infection with this knockout mutant showed increased expression of pro-inflammatory cytokines. This knockout mutant is sensitive to oxidative, nitrogen, redox and electron transport inhibitor stress agents. Drug susceptibility testing of the deletion mutant showed resistance to first-line drugs such as streptomycin and ethambutol and second-line aminoglycosides such as amikacin and kanamycin. Based on interactorme analysis for Rv0148 using STRING database, we identified 220 most probable interacting partners for Htdy protein. In the Rv0148 knockout mutants, high expression of htdy was observed and we hypothesize that this would have perturbed the interactome thus resulting in drug resistance. Finally, we propose that Rv0148 and Htdy are functionally interconnected and involved in drug resistance and cell homeostasis of M. tuberculosis. Conclusions:Our study suggests that Rv0148 plays a significant role in various functional aspects such as intermediatory metabolism, stress, homeostasis and also in drug resistance.

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In silico and experimental validation of protein–protein interactions between PknI and Rv2159c from Mycobacterium tuberculosis

Cited by 15 publications

References 50 publications

The crystal structure of PknI from Mycobacterium tuberculosis shows an inactive, pseudokinase‐like conformation

The crystal structure of PknI from Mycobacterium tuberculosis shows an inactive, pseudokinase‐like conformation

Functional Characterization of PknI-Rv2159c Interaction in Redox Homeostasis of Mycobacterium tuberculosis

Protein–protein interaction of Rv0148 with Htdy and its predicted role towards drug resistance in Mycobacterium tuberculosis

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