Structure-function analyses reveal the molecular architecture and neutralization mechanism of a bacterial HEPN-MNT toxin-antitoxin system

Abstract

Toxin-antitoxin (TA) loci in bacteria are small genetic modules that regulate various cellular activities, including cell growth and death. The two-gene module encoding a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domain and a cognate MNT (minimal nucleotidyltransferase) domain have been predicted to represent a novel type II TA system prevalent in archaea and bacteria. However, the neutralization mechanism and cellular targets of the TA family remain unclear. The toxin SO_3166 having a HEPN domain and its cognate antitoxin SO_3165 with an MNT domain constitute a typical type II TA system that regulates cell motility and confers plasmid stability in the bacterium Shewanella oneidensis Here, we report the crystal structure and solution conformation of the SO_3166-SO_3165 pair, representing the first complex structures in this TA family. The structures revealed that SO_3165 and SO_3166 form a tight heterooctamer (at a 2:6 ratio), an organization that is very rare in other TA systems. We also observed that SO_3166 dimerization enables the formation of a deep cleft at the HEPN-domain interface harboring a composite RX4-6H active site that functions as an RNA-cleaving RNase. SO_3165 bound SO_3166 mainly through its two α-helices (α2 and α4), functioning as molecular recognition elements. Moreover, their insertion into the SO_3166 cleft sterically blocked the RX4-6H site or narrowed the cleft to inhibit RNA substrate binding. Structure-based mutagenesis confirmed the important roles of these α-helices in SO_3166 binding and inhibition. Our structure-function analysis provides first insights into the neutralization mechanism of the HEPN-MNT TA family.

Keywords: RNA binding protein; RNA ribonuclease; RNA-protein interaction; crystal structure; small-angle X-ray scattering (SAXS); toxin; toxin-antitoxin system.

Figure 1.

RNA cleavage activity of HEPN toxin SO_3166. A, the ompA (1–306 nt) mRNA cleavage activity of SO_3166 and the SO_3166–SO_3165 complex at different time points (in vitro). B, E. coli total tRNAs treated with SO_3166 at different time points (in vitro). C, total RNAs isolated from MR-1/pHGE and MR-1/pHGE-SO_3166 after a 40-min induction with 0.5 mm IPTG added at a turbidity of 0.7 at 600 nm (in vivo). HI indicates heat-inactivated SO_3166 or SO_3166–SO_3165 complex. M indicates ssRNA ladder in A and B, and DNA ladder in C.

Figure 2.

Overview of HEPN-MNT SO_3166–SO_3165 complex structure. A, overall crystal structure of the SO_3166–SO_3165 complex shown as a schematic. The heterotetramer structure is composed of one SO_3165 (in green and labeled using the subscript A) binding SO_3166 molecules (in cyan/pink/yellow and labeled using the subscripts A–C, respectively) simultaneously. B, heterooctamer structure of the SO_3166–SO_3165 complex generated by crystallographic symmetry. It is composed of two SO_3165 (in green/dark green and labeled using the subscripts A and A′, respectively) binding six SO_3166 molecules (in cyan/yellow/pink/blue/magenta/wheat and labeled using subscripts A–C, A′–C′, respectively). C, purified SO_3165 (blue) and SO_3166–SO_3165 complex (red) eluted from gel filtration chromatogram (Superdex^TM 200 10/300 GL) at 16.3 and 12.9 ml, respectively. D, solution conformation of the SO_3166–SO_3165 complex by SAXS analysis. Left: Curve 1, experimental data; Curve 2, scattering patterns computed from the DAMMIN model; Curve 3, scattering patterns computed from the GASBOR model; insertion in right above, P(r) function. Right, DAMMIN models overlap with the tight heterooctamer crystal structure. The experimental data compare well with the theoretical curves of the heterooctamer crystal structure of the complex.

Figure 3.

Structural characteristics of HEPN toxin SO_3166. A, structure-based sequence alignment of SO_3166 with its representative homologs performed using Clustal X (version 1.81) and ESPript 3. They include SO_3166 from S. oneidensis and the homologs from Pectobacterium carotovorum (P. carotovorum), Oleibacter marinus (O. marinus), Marinomonas aquimarina (M. aquimarina), Clostridium butyricum (C. butyricum), and Desulfotomaculum gibsoniae (D. gibsoniae). The residues that were previously identified important for SO_3166 toxicity are highlighted using black arrows. The conserved RX4–6H motif are labeled using a red box. B, homodimer structure of SO_3166 shown as a schematic from the side view. The two subunits SO_3166_A and SO_3166_B are shown in cyan and pink, respectively. The RX4–6H motifs from both subunits are highlighted in red. C, the molecular surface representation of the SO_3166 dimer from the same view as B (blue, +7.1KT; red, −7.1KT), colored by its local electrostatic potential. The deep cleft formed by HEPN-domain dimerization of both subunits is highlighted using an ellipse. D, surface representation of the SO_3166 dimer from the top view. The key residues (except Cys-15 and Leu-107) for SO_3166 toxicity are highlighted in red. E, structural superimposition of SO_3166_A (dark gray) and the homolog HI0074 (light gray, PDB ID 1JOG). The regions containing RX4–6H motif in SO_3166 (Val⁹⁴–Asp¹⁰³) and HI0074 (Asp¹⁰⁴–Tyr¹¹³) are highlighted in cyan and orange, respectively. The conformations of the regions (especially the catalytic histidine) are remarkably different.

Figure 4.

Structural characteristics of MNT antitoxin SO_3165. A, structure-based sequence alignment of SO_3165 with its representative homologs as described in the legend to Fig. 3A. They include SO_3165 from S. oneidensis (S. oneidensis) and the homologs from P. carotovorum (P. carotovorum), O. marinus (O. marinus), P. syringae (P. syringae), P. caricapapayae (P. caricapapayae), and Halomonas ilicicola (H. ilicicola). The conserved residues are boxed in blue, identical conserved and low conserved residues are highlighted in red background and red letters, respectively. B, the molecular surface representation of SO_3165 (blue, +7.4KT; red, −7.4KT), colored by its local electrostatic potential. The helix α1 is covered with dominantly positive charges, whereas helices α2- and α4-mediated SO_3166 binding are covered with dominantly negative charges. C, structural superimposition of SO_3165 (light gray) and the homolog HI0073 (heavy gray, PDB ID 1NO5). The β1–β2 loop in HI0073 and the corresponding region in SO_3165 (also the helix α4) are highlighted in orange and green, respectively. They have significant differences in a β1–β2 loop conformation and the extra helix α4 in SO_3165, although they share overall similar folds. The binding zinc ion in HI0073 is shown as a yellow sphere.

Figure 5.

Contacts analysis (H-bonds and salt bridges) between SO_3165 and SO_3166. A, direct interactions between SO_3166_A and SO3165_A. B, direct interactions between SO_3166_B and SO3165_A. C, direct interactions between SO_3166_C and SO3165_A. SO_3166 and SO_3165 are shown in dark gray and light gray, respectively. The interacting residues are shown as sticks and their colorings are the same as described in the legend to Fig. 2A. The RX4–6H motif is highlighted in red. The residues that are previously identified important for SO_3166 toxicity are highlighted using ellipses.

Figure 6.

Structural basis of SO_3166 toxicity suppression by SO_3165. A and B, heterooctamer structure of the SO_3166–SO_3165 complex from side view (A) and top view (B), respectively. SO_3166 and SO_3165 are shown as surface and schematics, respectively, and their colorings are the same as described in the legend to Fig. 2B. C and D, close-up views show the contacts between SO_3166 and SO_3165. The residues important for SO_3166 toxicity are highlighted in red. The helix α4 of SO_3165 can bind into the cleft formed by the SO_3166 dimer (C) and sterically block the two active sites of SO_3166 simultaneously. The helix α2 and α2–α3 loops of the symmetry-related SO_3165 dimer can bind to the edge of the cleft (D), and the narrowed cleft will probably cause the unfavorable binding of the substrate by SO_3166.

Figure 7.

The roles of helices α2 and α4 of SO_3165 in the inhibition of SO_3166 toxicity by functional studies. A, three truncations: Δ(98–113) and Δ(114–125) in the helix α4 and Δ(52–65) in the helix α2 of SO_3165 are constructed for SO_3166 toxicity inhibition studies. The regions important for SO_3166 toxicity are highlighted in red as described in the legend to Fig. 6. B, the viability (CFUs/ml) of BL21 hosts carrying the pET28a-based plasmids were induced with 0.3 mm IPTG added at A₆₀₀ ∼ 0.1. Three independent cultures were conducted, error bars indicate mean ± S.E. (n = 3). C, viabilities were tested after induced for 4 h. Three independent cultures of each strain were tested and only representative images are shown.