Functional Characterization of the C-Terminus of YhaV in the Escherichia coli PrlF-YhaV Toxin-Antitoxin System
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Bacterial programmed cell death is regulated by the toxin-antitoxin (TA) system. YhaV (toxin) and Pr1F (antitoxin) have been recently identified as a type II TA system in Escherichia coli. YhaV homologs have conserved active residues within the C-terminus, and to characterize the function of this region, we purified native YhaV protein (without denaturing) and constructed YhaV proteins of varying lengths. Here, we report a new low-temperature method of purifying native YhaV, which is notable given the existing challenges of purifying this highly toxic protein. The secondary structures and thermostability of the purified native protein were characterized and no significant structural destruction was observed, suggesting that the observed inhibition of cell growth in vivo was not the result of structural protein damage. However, it has been reported that excessive levels of protein expression may result in protein misfolding and changes in cell growth and mRNA stability. To exclude this possibility, we used an [35S]-methionine prokaryotic cell-free protein synthesis system in vitro in the presence of purified YhaV, and two C-terminal truncated forms of this protein (YhaV-L and YhaV-S). Our results suggest that the YhaV C-terminal region is essential for mRNA interferase activity, and the W143 or H154 residues may play an analogous role to Y87 of RelE.
Abstract
Bacterial programmed cell death is regulated by the toxin-antitoxin (TA) system. YhaV (toxin) and Pr1F (antitoxin) have been recently identified as a type II TA system in Escherichia coli. YhaV homologs have conserved active residues within the C-terminus, and to characterize the function of this region, we purified native YhaV protein (without denaturing) and constructed YhaV proteins of varying lengths. Here, we report a new low-temperature method of purifying native YhaV, which is notable given the existing challenges of purifying this highly toxic protein. The secondary structures and thermostability of the purified native protein were characterized and no significant structural destruction was observed, suggesting that the observed inhibition of cell growth in vivo was not the result of structural protein damage. However, it has been reported that excessive levels of protein expression may result in protein misfolding and changes in cell growth and mRNA stability. To exclude this possibility, we used an [35S]-methionine prokaryotic cell-free protein synthesis system in vitro in the presence of purified YhaV, and two C-terminal truncated forms of this protein (YhaV-L and YhaV-S). Our results suggest that the YhaV C-terminal region is essential for mRNA interferase activity, and the W143 or H154 residues may play an analogous role to Y87 of RelE.
참고문헌 (39)
- Yamaguchi Y, "Toxin-antitoxin systems in bacteria and archaea", Annu. Rev. Genet., 45, pp.61~79 (2011)[DOI]
- Pandey DP, "Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes", Nucleic Acids Res., 33, pp.966~976 (2005)[DOI]
- Page R, "Toxin-antitoxin systems in bacterial growth arrest and persistence", Nat. Chem. Biol., 12, pp.208~214 (2016)[DOI]
- Thisted T, "Mechanism of post-segregational killing by the hok", sok system of plasmid R1. Sok antisense RNA regulates hok gene expression indirectly through the overlapping mok gene, 1992, pp.223, 41~54
- Kawano M, "Molecular characterization of long direct repeat (LDR) sequences expressing a stable mRNA encoding for a 35-amino-acid cell-killing peptide and a cis-encoded small antisense RNA in Escherichia coli", Mol. Microbiol., 45, pp.333~349 (2002)[DOI]
- Kawano M, "Divergently overlapping cis-encoded antisense RNA regulating toxin-antitoxin systems from E. coli: hok", sok, ldr, symR, pp.RNA Biol., 2012[DOI]
- Van Melderen L, "ATP-dependent degradation of CcdA by Lon protease. Effects of secondary structure and heterologous subunit interactions", J. Biol. Chem., 271, pp.27730~27738 (1996)[DOI]
- Buts L, "Toxinantitoxin modules as bacterial metabolic stress managers", Trends Biochem. Sci., 30, pp.672~679 (2005)[DOI]
- Brzozowska I, "Regulation of toxinantitoxin systems by proteolysis", Plasmid, 70, pp.33~41 (2013)[DOI]
- Muthuramalingam M, "Toxinantitoxin modules are pliable switches activated by multiple protease pathways", Toxins (Basel), 8, pp.E214~ (2016)[DOI]
- Zhang Y, "MazF cleaves cellular mRNAs specifically at ACA to block protein synthesis in Escherichia coli", Mol. Cell, 12, pp.913~923 (2003)[DOI]
- Kamada K, "Crystal structure of the MazE", MazF complex: molecular bases of antidote-toxin recognition, 2003, pp.11, 875~884
- Yamaguchi Y, "MqsR, a crucial regulator for quorum sensing and biofilm formation, is a GCU-specific mRNA interferase in Escherichia coli", J. Biol. Chem., 284, pp.28746~28753 (2009)[DOI]
- Hwang JY, "A ribosome profil ing study of mRNA cleavage by the endonuclease RelE", Nucleic Acids Res., 45, pp.327~336 (2016)
- Pedersen K, "The bacterial toxin RelE displays codonspecific cleavage of mRNAs in the ribosomal A site", Cell, 112, pp.131~140 (2003)[DOI]
- Galvani C, "Purification of the RelB and RelE proteins of Escherichia coli: RelE binds to RelB and to ribosomes", J. Bacteriol., 183, pp.2700~2703 (2001)[DOI]
- Neubauer C, "The structural basis for mRNA recognition and cleavage by the ribosome-dependent endonuclease RelE", Cell, 139, pp.1084~1095 (2009)[DOI]
- Zhang Y, "The inhibitory mechanism of protein synthesis by YoeB, an Escherichia coli toxin", J. Biol. Chem., 284, pp.6627~6638 (2009)[DOI]
- Keren I, "Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli", J. Bacteriol., 186, pp.8172~8180 (2004)[DOI]
- Zhang Y, "Characterization of YafO, an Escherichia coli toxin", J. Biol. Chem., 284, pp.25522~25531 (2009)[DOI]
- Feng S, "YoeB-ribosome structure: a canonical RNase that requires the ribosome for its specific activity", Nucleic Acids Res., 41, pp.9549~9556 (2013)[DOI]
- Schmidt O, "prlF and yhaV encode a new toxinantitoxin system in Escherichia coli", J. Mol. Biol., 372, pp.894~905 (2007)[DOI]
- Choi W, "Translation-dependent mRNA cleavage by YhaV in Escherichia coli", FEBS Lett., 591, pp.1853~1861 (2017)[DOI]
- Griffin MA, "Bacterial toxin RelE: a highly efficient ribonuclease with exquisite substrate specificity using atypical catalytic residues", Biochemistry, 52, pp.8633~8642 (2013)[DOI]
- Shinohara M, "The structural mechanism of the inhibition of archaeal RelE toxin by its cognate RelB antitoxin", Biochem. Biophys. Res. Commun., 400, pp.346~351 (2010)[DOI]
- Baker KE, "Ectopic RNase E sites promote bypass of 5'-end-dependent mRNA decay in Escherichia coli", Mol. Microbiol., 47, pp.75~88 (2003)
- Park JH, "ACA-specific RNA sequence recognition is acquired via the loop 2 region of MazF mRNA interferase", Proteins, 81, pp.874~883 (2013)[DOI]
- Simanshu DK, "Structural basis of mRNA recognition and cleavage by toxin MazF and its regulation by antitoxin MazE in Bacillus subtilis", Mol. Cell, 52, pp.447~458 (2013)[DOI]
- Kamphuis MB, "Model for RNA binding and the catalytic site of the RNase Kid of the bacterial parD toxin-antitoxin system", J. Mol. Biol., 357, pp.115~126 (2006)[DOI]
- Boggild A, "The crystal structure of the intact E. coli RelBE toxin-antitoxin complex provides the structural basis for conditional cooperativity", Structure, 20, pp.1641~1648 (2012)[DOI]
- Kamada K, "Conformational change in the catalytic site of the ribonuclease YoeB toxin by YefM antitoxin", Mol. Cell, 19, pp.497~509 (2005)[DOI]
- Schureck MA, "Defining the mRNA recognition signature of a bacterial toxin protein", Proc. Natl. Acad. Sci. USA, 112, pp.13862~13867 (2015)[DOI]
- Schureck MA, "Mechanism of endonuclease cleavage by the HigB toxin", Nucleic Acids Res., 44, pp.7944~7953 (2016)[DOI]
- Guex N, "SWISS-MODEL and the Swiss- PdbViewer: an environment for comparative protein modeling", Electrophoresis, 18, pp.2714~2723 (1997)[DOI]
- Herraez A, "Biomolecules in the computer: Jmol to the rescue", Biochem. Mol. Biol. Educ, 34, pp.255~261 (2006)[DOI]
- Brown BL, "Three dimensional structure of the MqsR:MqsA complex: a novel TA pair comprised of a toxin homologous to RelE and an antitoxin with unique properties", PLoS Pathog, 5, pp.e1000706~ (2009)[DOI]
- Selkoe DJ, "Folding proteins in fatal ways", Nature, 426, pp.900~904 (2003)[DOI]
- Park JH, "Intramolecular regulation of the sequence-specific mRNA interferase activity of MazF fused to a MazE fragment with a linker cleavable by specific proteases", Appl. Environ. Microbiol., 78, pp.3794~3799 (2012)[DOI]
- Chandrasegaran S, "Origins of programmable nucleases for genome engineering", J. Mol. Biol., 428, pp.963~989 (2016)[DOI]