Structural insights into assembly and regulation of HigBA toxin-antitoxin system from Escherichia coli
In the last few decades, bacterial Toxin-antitoxin (TA) systems have been identified to play crucial roles in bacterial survival under stressful conditions and virulence. TA systems are pair of genetic elements where one of the genes codes for a protein (toxin), which is toxic to the host cell, and the other gene code for its antidote (antitoxin), which can be an RNA or a protein. Under favourable growth conditions, the antitoxin inhibits the toxin activity; however, when the bacterial cell encounters stressful conditions such as antibiotic exposure, starvation, phage infection, etc., the toxin is released from antitoxin inhibition resulting in cell growth arrest or cell death. The TA systems have been mainly implicated in plasmid maintenance, inhibition of bacteriophage propagation (abortive infection), and survival against antibiotic exposure (persister cell formation). The current thesis work is focused on understanding the structural basis of toxin inhibition and autoregulation of operon expression in the HigBA type II TA system from E. coli. This study reports a high-resolution 2.09 Å crystal structure of the HigBA complex from E. coli K-12. This structure reveals the overall organization and mechanism of antitoxin HigA binding to toxin HigB. We also report a 2.3 Å resolution crystal structure of a truncated heterodimeric HigBA complex. This structure signifies the role of helices 𝛼1 and 𝛼2 in the dimerization of HigA. Also, we propose that the dimeric structure may indicate the possible proteolytic cleavage sites in toxin HigB and antitoxin HigA, which may have implications in HigBA complex disassembly and regulation in bacteria under proteolytic stress. Further using CD spectroscopy, NMR spectroscopy, and MD simulation studies, we suggest that E. coli HigA antitoxin is well-folded and stable in solution; however, it shows an intrinsic dynamic behavior. Using EMSA, SEC-MALS, and ITC experiments we establish that HigBA binds to its 33bp promoter DNA (Pal-1 DNA) in a 2:1 (HigA: Pal-1) ratio, and through ITC experiments we report that both the HigBA complex and HigA have comparable high binding affinity towards 33bp Pal-1 DNA. Therefore, we suggest that the toxin HigB has little or no effect on the antitoxin’s DNA binding activity. Further, the C-terminal DBD of HigA (HigA_DBD) was cloned and purified for the NMR-based titration experiments to identify the DNA binding residues. The sequential backbone assignments of HigA_DBD were achieved and using NMR CSP data from the titration experiments with different Pal-1 DNA sequences, we reveal that residues from helix 𝛼7, 𝛼8, loop L1 and loop L2 of the DNA binding domain of HigA interact with Pal-1 promoter DNA sequence. Further, we report the NMR CSP data-driven HADDOCK model of Pal-1 DNA bound HigBA complex. Finally, we report a low-resolution cryoEM structure of the HigBA and 27bp Pal-1 DNA complexes, confirming that two HigBA complexes bind the Pal-1 DNA sequence.