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. 2019 Jun;111(6):1449-1462.
doi: 10.1111/mmi.14229. Epub 2019 Apr 1.

Structural basis of transcriptional regulation by the HigA antitoxin

Marc A Schureck  1 , Jeffrey Meisner  1 , Eric D Hoffer  1 , Dongxue Wang  1 , Nina Onuoha  1 , Shein Ei Cho  1 , Pete Lollar 3rd  2   3 , Christine M Dunham  1
Affiliations

Structural basis of transcriptional regulation by the HigA antitoxin

Marc A Schureck et al. Mol Microbiol. 2019 Jun.

Abstract

Bacterial toxin-antitoxin systems are important factors implicated in growth inhibition and plasmid maintenance. Type II toxin-antitoxin pairs are regulated at the transcriptional level by the antitoxin itself. Here, we examined how the HigA antitoxin regulates the expression of the Proteus vulgaris higBA toxin-antitoxin operon from the Rts1 plasmid. The HigBA complex adopts a unique architecture suggesting differences in its regulation as compared to classical type II toxin-antitoxin systems. We find that the C-terminus of the HigA antitoxin is required for dimerization and transcriptional repression. Further, the HigA structure reveals that the C terminus is ordered and does not transition between disorder-to-order states upon toxin binding. HigA residue Arg40 recognizes a TpG dinucleotide in higO2, an evolutionary conserved mode of recognition among prokaryotic and eukaryotic transcription factors. Comparison of the HigBA and HigA-higO2 structures reveals the distance between helix-turn-helix motifs of each HigA monomer increases by ~4 Å in order to bind to higO2. Consistent with these data, HigBA binding to each operator is twofold less tight than HigA alone. Together, these data show the HigB toxin does not act as a co-repressor suggesting potential novel regulation in this toxin-antitoxin system.

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Figures

Figure 1.
Figure 1.. HigA dimerization is dispensable for toxin inhibition but necessary for transcriptional repression.
A. Organization of the hig operon with the regions HigA recognizes shown in grey shading and the −35 and −10 promoter regions boxed. B. Spot dilution assay of E. coli BW25113 transformed with indicated plasmids, overexpressed and the indicated amounts were plated on LB and LB in the presence of 0.2% arabinose. C. β-gal assays of E. coli BW25113 transformed with indicated plasmids in LB medium. D. β-gal assays of E. coli BW25113 transformed with indicated plasmids in M9 maltose media and 0.2% arabinose.
Figure 2.
Figure 2.. HigA is an obligate dimer.
A. 1.9 Å X-ray crystal structure of HigA reveals the maintenance of the dimer interface in the absence of the HigB toxin. The DNA binding helix-turn-helix (HTH) motif and the dimer interface are indicated. B. Analytical ultracentrifugation of HigA produced a signal-average s20,w peak at 2.15 S corresponding to an estimated molecular weight of 25.9 kDa. C. Comparison of the HigA dimer (green) and HigA in the context of the HigBA complex (blue; PDB code 4MCT) reveals a 5° move away from the DNA binding surface involving α1, α2, and α3.
Figure 3.
Figure 3.. Structural basis of HigA-DNA operator recognition.
A. 2.9 Å X-ray crystal structure of HigA bound to higO2. One helix-turn-helix (HTH) motif of a HigA monomer is boxed. The higO2 is shown with the blue arrows indicating the inverted repeats HigA recognizes and the specific nucleotides contacted are shown in bold. B. Zoomed in view of HigA α2 and α3 of the HTH motif that interact directly with nucleotides T+6, G+7 and T+8. HigA residue Arg40 hydrogen bonds to G+7. The phosphate of G+7 is contacted by Thr34 and Thr37 that may serve to stabilize the interaction between Arg40 and the nucleobase of G+7 (right panel). C. Schematic representation of interactions between HigA residues with higO2. Van der waals interactions between Ala36 in both monomers is shaded grey.
Figure 4.
Figure 4.. HigA Arg40 and G+7 are necessary for HigA recognition of higO2.
A. Spot dilution assay of E. coli BW25113 transformed with indicated plasmids, overexpressed with the indicated amounts were plated on LB and LB in the presence of 0.2% arabinose. B. β-gal assays of E. coli BW25113 transformed with indicated plasmids. C. Electrophoretic mobility shift assay of HigA R40A and higO2. D. Size exclusion chromatography analysis of HigA wild-type and the R40A variant. E. Electrophoretic mobility shift assay of the wild-type HigA and higO2 containing a G+7 to A+7 mutation (along with a C+7 to U+7 to maintain Watson-Crick base-pairing).
Figure 5.
Figure 5.. Structural rearrangements of HigA during operator recognition.
A. Superposition of free HigA (this study), the HigB-HigA complex (PDB code 4MCT), and DNA operator-bound HigA (this study) using the second HigA monomer (grey) as an anchor point. The HigA dimer hinges ~12° away from the DNA surface upon DNA recognition as compared to HigA bound to HigB. In the context of both apo HigA and in the HigB-HigA complex, clashes between α2 and α3 with DNA would occur. Rearrangement of the one monomer of HigA is required to allow the HTH motif to fully engage DNA. B. The HigA N terminus packs against the toxin HigB and forms interactions with α5 that may restrict its conformation (top panel). In the absence of HigB, the interactions between loop 1 and α5 are disrupted (bottom panel).
Figure 6.
Figure 6.. HigA and HigBA recognition of higO1 and higO2.
A. The band intensities from EMSAs as plotted as percent HigA bound versus HigA concentration for binding to Phig with scrambled higO1 and of HigA and Phig with scrambled higO2. In each assay, the HigA concentration increases from 0–1000 nM (gel shown in Fig. S2). B. The band intensities from EMSAs as plotted as percent HigBA bound versus HigBA concentration for binding to Phig with scrambled higO1 and of HigA and Phig with scrambled higO2. In each assay, the HigBA concentration increases from 0–600 nM (gel shown in Fig. S2). Curves represent the data from which binding affinities given in the main text were derived.
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