Structural and Functional Characterization of Escherichia coli Toxin-Antitoxin Complex DinJ-YafQ

Yajing Liang,Zengqiang Gao,Fei Wang,Yangli Zhang,Yuhui Dong,Quansheng Liu

doi:10.1074/jbc.m114.559773

Abstract

Toxin YafQ functions as a ribonuclease in the dinJ-yafQ toxin-antitoxin system of Escherichia coli. Antitoxin DinJ neutralizes YafQ-mediated toxicity by forming a stable protein complex. Here, crystal structures of the (DinJ)2-(YafQ)2 complex and the isolated YafQ toxin have been determined. The structure of the heterotetrameric complex (DinJ)2-(YafQ)2 revealed that the N-terminal region of DinJ folds into a ribbon-helix-helix motif and dimerizes for DNA recognition, and the C-terminal portion of each DinJ exclusively wraps around a YafQ molecule. Upon incorporation into the heterotetrameric complex, a conformational change of YafQ in close proximity to the catalytic site of the typical microbial ribonuclease fold was observed and validated. Mutagenesis experiments revealed that a DinJ mutant restored YafQ RNase activity in a tetramer complex in vitro but not in vivo. An electrophoretic mobility shift assay showed that one of the palindromic sequences present in the upstream intergenic region of DinJ served as a binding sequences for both the DinJ-YafQ complex and the antitoxin DinJ alone. Based on structure-guided and site-directed mutagenesis of DinJ-YafQ, we showed that two pairs of amino acids in DinJ were important for DNA binding; the R8A and K16A substitutions and the S31A and R35A substitutions in DinJ abolished the DNA binding ability of the DinJ-YafQ complex.

Highlights

The type II TA dinJ-yafQ module autoregulates bacterial growth in response to environmental stimuli
The structure of the heterotetrameric complex (DinJ)2-(YafQ)2 revealed that the N-terminal region of DinJ folds into a ribbonhelix-helix motif and dimerizes for DNA recognition, and the C-terminal portion of each DinJ exclusively wraps around a YafQ molecule
A recent mutagenesis study on the ribonuclease activity of YafQ demonstrated that His-50, His-63, Asp-67, Arg-83, His-87, and Phe-91 substitutions of the predicted active site residues of YafQ abolished mRNA cleavage in vivo, whereas Asp-61 and Phe-91 mutations inhibited YafQ ribonuclease activity only moderately

Summary

Background

The type II TA dinJ-yafQ module autoregulates bacterial growth in response to environmental stimuli. In type IV TA systems, the protein antitoxin cannot form a complex with its cognate toxin but acts as an antagonist for its toxicity (e.g. the yeeU/yeeV module from E. coli) [15]. In type V TA systems, a protein antitoxin inhibits its cognate toxin by cleaving its mRNA (e.g. the ghoS/ ghoT module from E. coli) [16]. In the E. coli dinJ-yafQ TA system, YafQ toxin, which contains a microbial RNase fold, is an endoribonuclease that associates with the ribosome, and its overproduction causes growth inhibition or even cell death due to its RNA cleavage [7, 22]. Antitoxin DinJ forms a stable complex with toxin YafQ, sequestering its incorporation into ribosomes and neutralizing its toxicity. The R8A and K16A substitutions and the S31A and R35A substitutions of the predicted binding site residues of DinJ abolished DNA binding, as shown by an EMSA

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION