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J Biol Chem. 2014 Jul 25;289(30):21191-202. doi: 10.1074/jbc.M114.559773. Epub 2014 Jun 12.

Structural and functional characterization of Escherichia coli toxin-antitoxin complex DinJ-YafQ.

Author information

1
From the School of Life Sciences, University of Science and Technology of China, Hefei, Anhui Province 230027, China, the Multidiscipline Research Center, Institute of High Energy Physics of the Chinese Academy of Sciences, 19B Yuequan Road, Beijing 100049, China, and.
2
the Multidiscipline Research Center, Institute of High Energy Physics of the Chinese Academy of Sciences, 19B Yuequan Road, Beijing 100049, China, and.
3
the Key Laboratory of Molecular Biology on Infectious Disease, Chongqing Medical University, YiXueYuanlu-1, Chongqing 400016, China.
4
the Multidiscipline Research Center, Institute of High Energy Physics of the Chinese Academy of Sciences, 19B Yuequan Road, Beijing 100049, China, and dongyh@ihep.ac.cn.
5
the Multidiscipline Research Center, Institute of High Energy Physics of the Chinese Academy of Sciences, 19B Yuequan Road, Beijing 100049, China, and liuqsh@ihep.ac.cn.

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.

KEYWORDS:

Bacterial Toxin; Ribbon-Helix-Helix Transcription Repressor; Ribonuclease; Transcription Regulation; Translation Regulation; X-ray Crystallography

PMID:
24923448
PMCID:
PMC4110321
DOI:
10.1074/jbc.M114.559773
[Indexed for MEDLINE]
Free PMC Article

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