Molecular Basis of Operator Recognition by the DNA-Binding Domain of Escherichia coli HigA Antitoxin

Abstract

Bacterial toxin-antitoxin (TA) systems are genetic modules consisting of two genes, one of which codes for a toxin (usually a protein) that is toxic to the host cell in its free form and the other an antidote of toxin, i.e., antitoxin, which may be an RNA or a protein. Under normal growth conditions, the antitoxin keeps the toxin inactive. During phage infection or stress (such as antibiotic stress), the toxin is liberated from antitoxin inhibition, which causes bacterial growth inhibition. In type II TA systems, both the toxin and antitoxin are proteins. Under favorable growth conditions, bacteria employ several additional strategies to keep toxin protein production in check. One of the strategies is transcriptional repression of the TA operon by the antitoxin or the antitoxin-toxin complex in a feedback manner. Here, we have studied the repressor activity of Escherichia coli HigA antitoxin by studying its binding to the operator DNA of the HigBA TA operon. We purified the DNA-binding domain (DBD) of HigA and studied its binding to the specific operon DNA sequences using NMR spectroscopy and isothermal titration calorimetry (ITC). The results showed that the isolated HigA DBD is well-folded in solution and binds specifically to the palindromic operator DNA sequences from the promoter region of the HigBA operon. NMR chemical shift perturbation (CSP) experiments have revealed the residues of the HigA DBD involved in DNA recognition. High-confidence AlphaFold 3 models of the HigA-DNA complexes matched well with the NMR CSP-derived HADDOCK models, revealing the DNA recognition mode of HigA.