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Auxiliary interfaces support the evolution of specific toxin–antitoxin pairing

Nature Chemical Biology volume 17, pages 1296–1304 (2021)Cite this article

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Abstract

Toxin–antitoxin (TA) systems are a large family of genes implicated in the regulation of bacterial growth and its arrest in response to attacks. These systems encode nonsecreted toxins and antitoxins that specifically pair, even when present in several paralogous copies per genome. Salmonella enterica serovar Typhimurium contains three paralogous TacAT systems that block bacterial translation. We determined the crystal structures of the three TacAT complexes to understand the structural basis of specific TA neutralization and the evolution of such specific pairing. In the present study, we show that alteration of a discrete structural add-on element on the toxin drives specific recognition by their cognate antitoxin underpinning insulation of the three pairs. Similar to other TA families, the region supporting TA-specific pairing is key to neutralization. Our work reveals that additional TA interfaces beside the main neutralization interface increase the safe space for evolution of pairing specificity.

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Fig. 1: S. Typhimurium TacAT TA modules are specific.
Fig. 2: The NDs of all TacA antitoxins bind and neutralize their cognate toxins in a similar manner.
Fig. 3: The specificity of TacA antitoxins for their cognate toxin is conferred by residues in their α4 helix region.
Fig. 4: Full-length TacA2 forms multiple interfaces with TacT2.
Fig. 5: RHH domains extend the neutralization scope of TacA antitoxins.

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Data availability

Atomic models of TacA2–TacT2Y137F, TacA152–88–TacT1Y140F and TacTA358–93–TacT3Y143F protein complexes were deposited at the PDB under accession nos. 7AK7, 7AK8 and 7AK9, respectively. Source data are provided with this paper.

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Acknowledgements

We thank members of S.H.’s lab, V. Pelicic and D. Mavridou, for comments on the manuscript. We thank G. Frankel for sharing S. enterica serovar Typhi genomic DNA. This work was supported by an MRC Career Development Award (no. MR/M009629/1) from the UK Medical Research Council and a starting grant from the European Research Council (grant no. 757369) to S.H. The crystallization facility at Imperial College London was funded by BBSRC (BB/D524840/1) and the Wellcome Trust (202926/Z/16/Z).

Author information

Authors and Affiliations

  1. Department of Microbiology, Harvard Medical School, Boston, MA, USA

    Grzegorz J. Grabe, Rachel T. Giorgio, Alexander M. J. Hall, Laurent Dubois, Tyler A. Sisley, Julian A. Rycroft & Sophie Helaine

  2. Department of Life Sciences, Imperial College London, London, UK

    Rhodri M. L. Morgan

  3. School of Life Sciences, University of Sussex, Brighton, UK

    Stephen A. Hare

  4. MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK

    Sophie Helaine

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Contributions

G.J.G. purified and crystallized all protein samples. G.J.G., R.T.G., A.M.J.H., L.D., T.A.S. and J.A.R. performed neutralization and toxicity experiments, and constructed plasmids used in the present study. A.M.J.H. and G.J.G. performed co-precipitation experiments. G.J.G., R.M.L.M. and S.A.H. determined the crystal structures of the three protein complexes. S.H. and G.J.G. wrote the manuscript. All authors contributed to data interpretation and the writing of the manuscript. S.H. and G.J.G. initiated and orchestrated the project.

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Correspondence to Sophie Helaine.

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The authors declare no competing interests.

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Peer review information Nature Chemical Biology thanks Michael Laub, Ditlev Brodersen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Identification of the neutralization domain of TacA1-3 antitoxins.

Toxin neutralization assays for TacT1, TacT2 or TacT3 each co-expressed with either full-length or gradual N-terminal truncations of cognate antitoxin. Neutralization efficiencies were measured by OD600 as the ability to counteract toxin-induced bacterial growth inhibition and normalized to that conferred by cognate full-length antitoxin. Data represent the mean percentage ±SEM (n= 3).

Source data

Extended Data Fig. 2 Details of the neutralization interfaces.

a, Cartoon representation of a 180° vertical rotation view of the structure of the neutralization interface of the three TacAT complexes shown in Fig. 2a: TacAT1 (orange and light yellow), TacAT2 (navy and light blue) and TacAT3 (burgundy and light pink). Dashed lines represent a disordered loop 3 region observed in each toxin structure (PDB IDs: 7AK8; 7AK7; 7AK9). N termini of each molecule are indicated on structure. b, Secondary structure elements and amino acid sequence alignment of the three TacT toxins with the residues interacting with the N- and C-terminal portions of the antitoxin neutralization domain (min. 10 Å2 surface) highlighted in green and violet, respectively. The dashed line and grey amino acids represent the disordered loop 3 region. Asterisks, colons and dots show identity, high and low similarity of aligned amino acids, respectively. c, Secondary structure elements and sequence alignment of the neutralization domain of the three TacA antitoxins with white and black gradient representing the surface contribution in Å2 to the toxin-antitoxin interface. The surface contribution of each residue was calculated using the PISA software. The green box in the TacA1 sequence highlights the region involved in intertwining of the TacA1 peptide fragments. Asterisks, colons and dots show identity, high and low similarity of aligned amino acids, respectively. The two TacA2 C-terminal amino acids (E96 and K97) not observed in the structure are shown in red. d, TacA1 peptides intertwine and bind two TacT1 toxins. Density modification (2Fo-Fc) map (green mesh; 0.8 σ) of the intertwining TacA1 linker regions (sticks) connecting α3 and α4 helices of two TacA1 molecules colored in orange and violet. TacT1 toxins are shown as grey and orange surfaces.

Extended Data Fig. 3 The neutralization domain of TacA antitoxins targets TBS and ACP sites in TacT toxins.

a, Toxicity assays for overexpressed TacT3, TacT3K80E and TacT3R84E variants. Toxicity was measured by OD600 as the ability to inhibit bacterial growth and normalized to that of wild-type TacT3 toxin. Data represent the mean percentage ±SEM (n= 3). b, Co-precipitation of purified Salmonella aminoacyl-tRNA (lane 1) with purified His-tagged catalytic mutants TacT3Y143F (YF) (lane 2), TacT3Y143F, K80E (YF KE) (lane 3) or TacT3Y143F, R84E (YF RE) (lane 4). Proteins were separated by SDS-PAGE and detected with Coomassie staining, whereas tRNA molecules were separated on acid-urea PAGE and revealed by methylene blue staining. Representative experiment (left panel) and average tRNA signal intensities normalized to that of TacT3Y143F (YF) (right panel) ±SEM (n=4). c, Co-precipitation of purified Salmonella aminoacyl-tRNA (lane 1) with purified His-tagged TacT3Y143F toxin in absence (lane 2) or presence (lane 3) of co-purified TacA3ND peptide. Proteins were separated by SDS-PAGE followed by Coomassie (TacT3Y143F) or silver (TacA3ND) staining, whereas tRNA molecules were separated on acid-urea PAGE and revealed by methylene blue staining. Representative experiment (left panel) and average tRNA signal intensities normalized to that of TacT3Y143F (T3YF) (right panel) ±SEM (n=4). d, Toxin neutralization assays for TacT1, TacT2 or TacT3 each co-expressed with either wild-type or K78A, K85A and K80A mutants tested either in neutralization domain (ND; TacA152–88, TacA258–97, and TacA358–93; top) or a shorter (Pep; TacA155–88, TacA262–97, and TacA360–93; bottom) peptide variant of cognate antitoxin. Grey stars indicate residues K78, K85 and K80 in TacT1, TacT2 and TacT3, respectively. Neutralization efficiencies were measured by OD600 as the ability to counteract toxin-induced bacterial growth inhibition and normalized to that conferred by wild-type antitoxin peptide. Data represent the mean percentage ±SEM (n= 3).

Source data

Extended Data Fig. 4 Evolutionary intermediates of TacAT systems in Salmonella genus.

a, Comparison of genomic locations of S. Typhimurium 14028 S tacA1-tacT1 (orange and light yellow) and S. bongori NCTC12419 tacA-tacTSb (dark brown and brown-grey) modules. b, Secondary structure elements and sequence alignment of the two S. Typhimurium TacT1 and TacT2 and S. bongori TacTSb toxins. Grey highlights indicate the α4 helix region of the toxins. Asterisks, colons and dots show identity, high and low similarity of aligned amino acids, respectively. c, Toxin neutralization assays for S. Typhimurium TacT1, TacT2, TacT3 or S. bongori TacTSb each co-expressed with TacA1, TacA2, TacA3 or TacA1Sb in S. Typhimurium tacAT triple deletion mutant. d, Comparison of genomic locations of tacA2-tacT2 (blue) and tacA2b-tacT2b (green) modules in S. Typhimurium 14028 S, and S. Typhi Ty2 serovars. e, Secondary structure elements and sequence alignment of the two S. Typhi TacT2Ty and TacT2bTy toxins. Grey highlights indicate the α4 helix region of the toxins. Asterisks, colons and dots show identity, high and low similarity of aligned amino acids, respectively. f, Toxin neutralization assays for S. Typhi TacT2bTy co-expressed with S. Typhi TacA2Ty or TacA2bTy in S. Typhimurium tacAT triple deletion mutant. Neutralization efficiencies in c and f were measured by OD600 as the ability to counteract toxin-induced bacterial growth inhibition and normalized to that conferred by cognate antitoxin. Data represent the mean percentage ±SEM (n= 3). g, Phylogenetic tree based on amino acid sequence alignment of Salmonella TacT1, TacTSb, TacT2, TacT2STy, TacT2bSTy, and TacT3, Escherichia AtaT and ItaT, and Klebsiella KacT.

Source data

Extended Data Fig. 5 TacA and TacT C-terminal regions and their role in toxicity and neutralization.

a, Secondary structure elements and sequence of TacT3 C-terminal region (top). Red dashed lines point to the sites of C-terminal truncations of the α4 helix overexpressed in the toxicity assay (bottom). Toxicity was measured by OD600 as the ability to inhibit bacterial growth and normalized to that of wild-type TacT3 toxin. Data represent the mean percentage ±SEM (n= 3). b, Cartoon representation of the interface between α4 helices of TacA3 (burgundy) and TacT3 (violet; black arrow) with the main residues contributing to the interface represented as sticks. Black dashed lines represent hydrogen bonds. c, Toxin neutralization assays for TacT3 and TacT3 C-terminal truncations each co-expressed with full-length TacA1, TacA2 or TacA3. d, Amino acid identity between TacA1, TacA2 and TacA3 of the α3 or α4 helix regions each twelve amino acids long. Identity is reported pairwise. e, Secondary structure elements of TacA1, TacA2 or TacA3 where the grey element represents the truncation of the α4 helix region, and the dashed line stands for the N-terminal part of the antitoxin (top). Toxin neutralization assays for TacT1, TacT2 or TacT3 each co-expressed with TacA1, TacA2 or TacA3 truncated of their α4 helix region. Neutralization efficiencies in c and e were measured by OD600 as the ability to counteract toxin-induced bacterial growth inhibition and normalized to that conferred by cognate full-length antitoxin. Data represent the mean percentage ±SEM (n= 3).

Source data

Extended Data Fig. 6 Basis of specificity between TacA1-TacT1 and TacA2-TacT2.

a, Cartoon representation of the α4 helix regions of TacA1 (orange) and TacA2 (navy) interaction with their respective toxin. Pink sticks represent residues of TacA2 that form contact with the TacT2 α4 helix or their equivalent residues in TacA1 (top). Violet sticks represent antitoxin residues that have significantly co-evolved with the cognate toxin as identified by GREMLIN (bottom). Black dashed lines represent hydrogen bonds. b, Table listing co-varying residue pairs in TacA1-TacT1 or TacA2-TacT2 as identified by GREMLIN. A higher score indicates higher co-variance. Yellow boxes show the residues depicted in panel A (bottom) that were selected for mutagenesis in the specificity switch experiment in Fig. 3b. c, Secondary structure elements of the C-terminal region of the TacA antitoxins (top) and sequence alignment of TacA1, TacA2 and TacA1 variants tested in the specificity switch experiment in Fig. 3b (bottom). Orange letters represent TacA1 residues, navy letters represent TacA2 residues. d, Secondary structure elements of the C-terminal region of the TacA antitoxins (top) and sequence alignment of the neutralization domain of S. bongori TacASb and S. Typhimurium TacA1 and TacA2 antitoxins (bottom). Dots, colons, and asterisks show identities shared with TacA1, TacA2, or both TacA1 and TacA2, respectively. e, Secondary structure elements of the C-terminal region of the TacA antitoxins (top) and sequence alignment of the neutralization domains of S. Typhi TacA2Ty and TacA2bTy antitoxins (bottom). Asterisks show identity. Pink and violet highlights in c-e indicate TacA2 amino acids interacting with TacT2 α4 helix or co-varying amino acids identified by GREMLIN, respectively.

Extended Data Fig. 7 Neutralization domains do not disrupt toxin dimer.

a-b, Cartoon representation of the structure of TacAND-TacT complexes. One toxin chain and its bound antitoxin are colored while other chains are in light grey. Ac-CoA and CoA molecules are shown as stick representations. a, TacT1 (light yellow) bound by TacA152–70 and TacA171–88 antitoxin peptides (orange). b, TacT3 (light pink) bound by TacA358–93 antitoxin peptide (burgundy). c, Size-exclusion chromatography-multiangle light scattering (SEC-MALS) chromatograms for each TacAT complexes (TacAT1 orange, TacAT2 navy, and TacAT3 burgundy). Theoretical molecular weights of various complex stoichiometries and experimentally calculated molecular weights for each peak are shown on the right.

Extended Data Fig. 8 TacA2 antitoxin is flexible and forms multiple toxin interfaces.

a, Cartoon representation of the two TacA2 dimers present in the TacA2-TacT2 complex. Four antitoxin chains engage each toxin monomer either with their P, S, T, and Q region (left) or P’, S’, T’, and Q’ (right). TacA2 chain color code used is dictated by interfaces formed on the left (non-prime letters). b, Cartoon representation of the TacT2 loop 3 disordered site (left) with the main contributing amino acids represented as sticks (right). Light-blue dashed lines represent the disrupted TBS surrounded by three TacA2 antitoxin molecules forming P (navy), S (marine), and T (violet) interfaces. Black dashed lines represent hydrogen bonds. c, Cartoon representation of the TacT2 Ac-CoA site masked by TacA2 (left) with the main contributing amino acid represented as sticks (right). TacA2 Q interface (green) with its helix α1 (left) together with P interface (navy) jointly engage in masking the front of the acetyl group of the Ac-CoA in TacT2 (light blue) ACP. d, Cartoon representation of part of the S interface (left) formed between the α3 helix of TacA2 (marine) and the α2 helix of TacT2 (light blue) with main contributing amino acids represented as sticks. Black dashed lines represent hydrogen bonds. Sequence alignment of the region of TacT2 participating in the S interface (right), highlighting conservation of three key amino acids (yellow highlight) across the three toxins. e, Table showing a pair of co-evolving residues in the antitoxin and toxin in the three TacAT systems as identified by GREMLIN in the S interface (left). Amino acid sequence alignment of the α3 helix region in the three TacA antitoxins (right). Yellow highlights indicate amino acid identity across the three antitoxins. f, Toxicity assay in trans-complementation between two variants of TacT3 expressed from two different plasmids in S. Typhimurium. Toxicity efficiencies were measured by OD600 as the ability to inhibit bacterial growth and normalized to that of wild-type TacT3 expressed from vector 1 co-transformed with an empty vector (EV) 2. Data represent the mean percentage ±SEM (n= 4). g, Surface representation of a partially reconstituted toxic state through trans-complementation of TacT3ACP (salmon) by TacT3TBS (pink) heterodimer combination in the co-expression experiment in Figure S8f. Grey disks indicate the mutated sites. Grey dashed line shows a non-functional site, whereas green highlight indicates a reconstituted functional site. h, Secondary structure elements and amino acid sequence of each of the four regions of TacA2 antitoxin engaged in the P, S, T, and Q interfaces with TacT2. Brown highlights indicate residues dually involved in P and S or T and Q interfaces. Dashed line indicates a disordered region. i, Differential conformation of the α2 helix of TacA2 as adopted in P (navy) and S (marine) interfaces. Only the region 48-63 is shown for both molecules. Black dashed lines represent hydrogen bonds.

Source data

Extended Data Fig. 9 Promiscuity of neutralization of the TacA full-length chimeras.

a, Secondary structure elements of the chimeric TacA1-3 antitoxin (top) tested in toxin neutralization assays against TacT1, TacT2 and TacT3 (bottom). Neutralization efficiencies were measured by OD600 as the ability to counteract toxin-induced bacterial growth inhibition and normalized to that of conferred by full-length cognate antitoxin. Data represent the mean percentage ±SEM (n= 3). b, Sequence alignment of the region (helix α1) involved in the T and Q interfaces across the three TacA antitoxins. Yellow highlight indicates the critical residue of the T and Q interfaces as identified in the TacT2-TacA2 complex structure. c, Table summarizing the area in Å2 of each P, S, T, or Q interfaces, as calculated using the PISA software obtained for different structures of GNAT toxin-antitoxin complexes. The top row indicates PDB identification codes. The sum of all toxin-antitoxin interfaces, total available toxin area, and a percentage of toxin area engaged in interface with the antitoxin are also indicated.

Source data

Extended Data Fig. 10 Additional interfaces in other TA complexes.

a, Comparison of the structure of three GNAT toxin-antitoxin complexes (KacAT, AtaRT, and TacAT2). Toxins are in wheat, antitoxin chains in grey and dark grey, with regions involved in P and S interfaces shown in magenta and cyan, respectively (top). Decomposition of the structure of each antitoxin chains involved in P (teal and grey) and S (pink and dark grey) interfaces (bottom). Whereas KacA and AtaR contribute to the S interface through extension of their α2 helix, TacA2 forms an α3 helix. Green color in TacA2 shows additional amino acids involved in the S interface whose counterparts where not observed for KacA or AtaR. b, Primary (P) and secondary (S) interfaces present in other TA complex structures. Toxins are represented as surface while antitoxins as cartoon. Dark grey coloring indicates an additional copy of a molecule (toxin or antitoxin). Teal (antitoxin) and cyan (toxin) coloring indicate the P interface. Dark (antitoxin) and light (toxin) pink colors highlight the S interface. Only amino acids contributing a minimum of 15 Å2 to the interface (as calculated by PISA) are colored. TA complexes were grouped according to the number of interfaces present in a single antitoxin. In RES-Xre (PDB: 6GW6) and YoeB-YefM (PDB: 6L8E) complexes (left), each antitoxin forms a single P or S interface, while in VapD-VapX (PDB: 6ZN8) and MbcT-MbcA (PDB: 6FKG) complexes (right), each antitoxin forms both P and S interfaces. Surface areas in Å2 of the different interfaces are indicated underneath each structure and were obtained using the PISA software.

Supplementary information

Supplementary Information

Supplementary Tables 1–5.

Reporting Summary

Supplementary Data

Representative growth kinetics.

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Grabe, G.J., Giorgio, R.T., Hall, A.M.J. et al. Auxiliary interfaces support the evolution of specific toxin–antitoxin pairing. Nat Chem Biol 17, 1296–1304 (2021). https://doi.org/10.1038/s41589-021-00862-y

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