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Structural and functional characterization of Escherichia coli toxin-antitoxin complex DinJ-YafQ.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2014 Jul 25; Vol. 289 (30), pp. 21191-202. Date of Electronic Publication: 2014 Jun 12. - Publication Year :
- 2014
-
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.<br /> (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Subjects :
- Amino Acid Substitution
Bacterial Toxins genetics
Bacterial Toxins metabolism
Escherichia coli genetics
Escherichia coli metabolism
Escherichia coli Proteins genetics
Escherichia coli Proteins metabolism
Multiprotein Complexes genetics
Multiprotein Complexes metabolism
Mutagenesis, Site-Directed
Mutation, Missense
Protein Structure, Quaternary
Structure-Activity Relationship
Bacterial Toxins chemistry
Escherichia coli chemistry
Escherichia coli Proteins chemistry
Multiprotein Complexes chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 289
- Issue :
- 30
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 24923448
- Full Text :
- https://doi.org/10.1074/jbc.M114.559773