The molecular basis of protein toxin HicA–dependent binding of the protein antitoxin HicB to DNA

Abstract

Toxin-antitoxin (TA) systems are present in many bacteria and play important roles in bacterial growth, physiology, and pathogenicity. Those that are best studied are the type II TA systems, in which both toxins and antitoxins are proteins. The HicAB system is one of the prototypic TA systems, found in many bacterial species. Complex interactions between the protein toxin (HicA), the protein antitoxin (HicB), and the DNA upstream of the encoding genes regulate the activity of this system, but few structural details are available about how HicA destabilizes the HicB-DNA complex. Here, we determined the X-ray structures of HicB and the HicAB complex to 1.8 and 2.5 Å resolution, respectively, and characterized their DNA interactions. This revealed that HicB forms a tetramer and HicA and HicB form a heterooctameric complex that involves structural reorganization of the C-terminal (DNA-binding) region of HicB. Our observations indicated that HicA has a profound impact on binding of HicB to DNA sequences upstream of hicAB in a stoichiometric-dependent way. At low ratios of HicA:HicB, there was no effect on DNA binding, but at higher ratios, the affinity for DNA declined cooperatively, driving dissociation of the HicA:HicB:DNA complex. These results reveal the structural mechanisms by which HicA de-represses the HicB-DNA complex. This work was supported by the Biotechnology and Biological Sciences Research Council-funded South West Biosciences Doctoral Training Partnership Training Grant BB/J014400/1. The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S10 and Tables S1-S12. The atomic coordinates and structure factors (codes 6G1C, 6G1N, and 6G26) have been deposited in the Protein Data Bank (http://wwpdb.org/). Experimental SAXS data and derived models of both HicB 4 and HicAB 4 have been deposited in the Small Angle Scattering Biological Data Bank (SASBDB) under the accession codes SASDD45 and SASDD55.

Entropic pressure controls oligomerization ofVibrio choleraeParD2 antitoxin

“…Higher toxin to antitoxin ratios lead to de-repression via the formation of toxin-antitoxin complexes with altered stoichiometry. For antitoxins that bind only to a single site, regulation is less complex and the toxin only serves to weaken operator binding (Brown et al, 2013; Turnbull & Gerdes, 2018; Winter et al, 2018; Manav et al, 2019). For parDE modules, the mechanism of transcription regulation is not known.…”

Pseudomonas aeruginosa antitoxin HigA functions as a diverse regulatory factor by recognizing specific pseudopalindromic DNA motifs

“…1A), it displays different concentration‐dependent binding profiles and different dissociation rates to various operators. It is well known that the antitoxin level is transcriptionally and post‐transcriptionally controlled within bacterial cells by the concentration‐dependent DNA binding mode, which could be modified by forming complexes with cognate toxins (Fivianhughes and Davis, 2010; De et al ., 2013; Winter et al ., 2018). We speculated that PaHigA regulates diverse cellular pathways through a complex and finely tuned mechanism due to the distinct binding affinity of PaHigA for various operators.…”

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

“…These typically interact with the toxin by covering the active site (Figure 1c). [27][28][29][30][31] In the case of Proteus vulgaris higBA and the closely related Pseudomonas putida graTA, the folded antitoxin does not cover the active site, but still inhibits its ribonuclease activity via an allosteric mechanism (Figure 1d). 32,33 Also HigB from E. coli and Shigella flexneri are inhibited in a similar way, although the corresponding antitoxin structurally diverges from those in the P. vulgaris and P. putida systems.…”

“…42 Higher order association of toxin-antitoxin complexes outside the context of transcription regulation has since then been reported for hicAB, vapXD, and parDE. 28,30,31,38,43 In the case of hicAB, these higher order complexes differ between different species with heterotetramers, heterohexamers as well as hetero-octamers being observed. 28,30,31 It is tempting to speculate that such higher order structures carry some functionality, for example, further harnessing the activity of the toxin, as was recently suggested.…”

“…28,30,31,38,43 In the case of hicAB, these higher order complexes differ between different species with heterotetramers, heterohexamers as well as hetero-octamers being observed. 28,30,31 It is tempting to speculate that such higher order structures carry some functionality, for example, further harnessing the activity of the toxin, as was recently suggested. 28 Nevertheless, in the case of the E. coli PaaA2-ParE2 pair, disruption of the higher order assembly does not affect antitoxin activity in vivo.…”