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

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.