RESUMEN
The toxin-antitoxin (TA) systems are small operon systems that are involved in important physiological processes in bacteria such as stress response and persister cell formation. Escherichia coli HigBA complex belongs to the type II TA systems and consists of a protein toxin called HigB and a protein antitoxin called HigA. The toxin HigB is a ribosome-dependent endoribonuclease that cleaves the translating mRNAs at the ribosome A site. The antitoxin HigA directly binds the toxin HigB, rendering the HigBA complex catalytically inactive. The existing biochemical and structural studies had revealed that the HigBA complex forms a heterotetrameric assembly via dimerization of HigA antitoxin. Here, we report a high-resolution crystal structure of E. coli HigBA complex that revealed a well-ordered DNA binding domain in HigA antitoxin. Using SEC-MALS and ITC methods, we have determined the stoichiometry of complex formation between HigBA and a 33â bp DNA and report that HigBA complex as well as HigA homodimer bind to the palindromic DNA sequence with nano molar affinity. Using E. coli growth assays, we have probed the roles of key, putative active site residues in HigB. Spectroscopic methods (CD and NMR) and molecular dynamics simulations study revealed intrinsic dynamic in antitoxin in HigBA complex, which may explain the large conformational changes in HigA homodimer in free and HigBA complexes observed previously. We also report a truncated, heterodimeric form of HigBA complex that revealed possible cleavage sites in HigBA complex, which can have implications for its cellular functions.