Abstract
In Escherichia coli, RelE toxin participates in growth arrest and cell death by inducing mRNA degradation at the ribosomal A-site under stress conditions. The NMR structures of a mutant of E. coli RelE toxin, RelE(R81A/R83A), with reduced toxicity and its complex with an inhibitory peptide from RelB antitoxin, RelB(C) (Lys(47)-Leu(79)), have been determined. In the free RelE(R81A/R83A) structure, helix alpha4 at the C terminus adopts a closed conformation contacting with the beta-sheet core and adjacent loops. In the RelE(R81A/R83A)-RelB(C) complex, helix alpha3(*) of RelB(C) displaces alpha4 of RelE(R81A/R83A) from the binding site on the beta-sheet core. This helix replacement results in neutralization of a conserved positively charged cluster of RelE by acidic residues from alpha3(*) of RelB. The released helix alpha4 becomes unfolded, adopting an open conformation with increased mobility. The displacement of alpha4 disrupts the geometry of critical residues, including Arg(81) and Tyr(87), in a putative active site of RelE toxin. Our structures indicate that RelB counteracts the toxic activity of RelE by displacing alpha4 helix from the catalytically competent position found in the free RelE structure.
Highlights
To date, TA toxins are known to perturb one or more vital processes, such as DNA replication, RNA transcription, and protein translation, by targeting DNA gyrase [7], messenger RNA [8, 9], and/or ribosomes [5, 10]
A subgroup of toxins, including MazF, RelE, and YoeB, is named as mRNA interferase [11], because they perturb the stability of mRNA by sequencespecific cleavage
RelBC Abolishes the Residual Catalytic Activity of RelER81A/R83A—Overexpression of wild-type RelE alone in E. coli markedly hindered cell growth because of its cytotoxicity
Summary
Protein Sample Preparation—Recombinant expression and purification were carried out as described previously for RelBC (Lys47–Leu79) and wild-type RelE [16]. RelER81A/R83A mutant was obtained using QuikChange site-directed mutagenesis kit (Stratagene). Unlabeled or isotope-enriched (e.g. 15N or 15N,13C) protein was purified from crude lysate using nickelnitrilotriacetic acid resin (Qiagen) and further purified by size exclusion chromatography. For NMR spectroscopy, all samples were prepared in 25 mM sodium phosphate (pH 6.5) containing
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