2.09 Å Resolution structure of E. coli HigBA toxin–antitoxin complex reveals an ordered DNA-binding domain and intrinsic dynamics in antitoxin

Abstract

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. E. 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 33 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 MD 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.

Identification, functional characterization, assembly and structure of ToxIN type III toxin–antitoxin complex from E. coli

“…To prevent any accidental activation of the TA system, the antitoxin in type II TA systems usually acts as an auto-repressor, which is a characteristic feature of type II TA systems ( 36 ). Almost all the antitoxins in type II TA systems contain a DNA binding domain via which the antitoxin specifically binds to DNA sequences in the promoter/operator region of the TA operon ( 1 , 37 ). Whether type III TA systems are further regulated directly by ToxI or ToxN or other host or viral factors remains to be seen.…”

Dynamics-Based Regulatory Switches of Type II Antitoxins: Insights into New Antimicrobial Discovery

“…For instance, mqsRA promoters located in the preceding sequence for the MqsR toxin drive the constitutive production of the MqsA antitoxin [ 47 ]. There are type II TA operons that contain a single operator recognized by the antitoxin dimer, and the antitoxin and the cognate TA complex bind to the operator with a similar affinity, as exemplified by the E. coli higBA module; however, HigA undergoes a large conformational change upon binding to HigB [ 48 ]. As with the higBA module, the E. coli dinJ-yafQ system exhibited that both DinJ and the DinJ-YafQ complex repress the transcription to a similar extent [ 49 ].…”

Prokaryote toxin–antitoxin modules: Complex regulation of an unclear function

“…One such example is the E. coli higBA module, where operator binding by HigA does not seem to be influenced by the presence of HigB, as both HigA and the HigBA complex bind the operator with a similar nanomolar affinity. 58 This may be somewhat surprising given that HigB binding induces a large conformational change to HigA.…”