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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Jaffe, A., Ogura, T. & Hiraga, S. Effects of the CCD function of the F plasmid on bacterial growth. J. Bacteriol. 163, 841–849 (1985).
Fineran, P. C. et al. The phage abortive infection system, ToxIN, functions as a protein–RNA toxin–antitoxin pair. Proc. Natl Acad. Sci. USA 106, 894–899 (2009).
Helaine, S. et al. Internalization of salmonella by macrophages induces formation of nonreplicating persisters. Science 343, 204–208 (2014).
Ross, B. N., Micheva-Viteva, S., Hong-Geller, E. & Torres, A. G. Evaluating the role of Burkholderia pseudomallei K96243 toxins BPSS0390, BPSS0395, and BPSS1584 in persistent infection. Cell. Microbiol. 21, e13096 (2019).
Yamaguchi, Y., Park, J. H. & Inouye, M. Toxin–antitoxin systems in bacteria and archaea. Annu. Rev. Genet. 45, 61–79 (2011).
Page, R. & Peti, W. Toxin–antitoxin systems in bacterial growth arrest and persistence. Nat. Chem. Biol. 12, 208–214 (2016).
Huang, C. Y., Gonzalez-Lopez, C., Henry, C., Mijakovic, I. & Ryan, K. R. hipBA toxin–antitoxin systems mediate persistence in Caulobacter crescentus. Sci. Rep. 10, 2865 (2020).
Goulard, C., Langrand, S., Carniel, E. & Chauvaux, S. The Yersinia pestis chromosome encodes active addiction toxins. J. Bacteriol. 192, 3669–3677 (2010).
Wilbaux, M., Mine, N., Guérout, A. M., Mazel, D. & Van Melderen, L. Functional interactions between coexisting toxin-antitoxin systems of the ccd family in Escherichia coli O157:H7. J. Bacteriol. 189, 2712–2719 (2007).
Aakre, C. D. et al. Evolving new protein–protein interaction specificity through promiscuous intermediates. Cell 163, 594–606 (2015).
Fiebig, A., Castro Rojas, C. M., Siegal-Gaskins, D. & Crosson, S. Interaction specificity, toxicity and regulation of a paralogous set of ParE/RelE-family toxin–antitoxin systems. Mol. Microbiol. 77, 236–251 (2010).
Ramage, H. R., Connolly, L. E. & Cox, J. S. Comprehensive functional analysis of Mycobacterium tuberculosis toxin–antitoxin systems: implications for pathogenesis, stress responses, and evolution. PLoS Genet 5, e1000767 (2009).
Hallez, R. et al. New toxins homologous to ParE belonging to three-component toxin-antitoxin systems in Escherichia coli O157:H7. Mol. Microbiol. 76, 719–732 (2010).
Cheverton, A. M. et al. A salmonella toxin promotes persister formation through acetylation of tRNA. Mol. Cell 63, 86–96 (2016).
Rycroft, J. A. et al. Activity of acetyltransferase toxins involved in Salmonella persister formation during macrophage infection. Nat. Commun. 9, 1993 (2018).
Jurenas, D. et al. AtaT blocks translation initiation by N-acetylation of the initiator tRNAfMet. Nat. Chem. Biol. 13, 640–646 (2017).
Ovchinnikov, S. V. et al. Mechanism of translation inhibition by type II GNAT toxin AtaT2. Nucleic Acids Res. 48, 8617–8625 (2020).
Wilcox, B. et al. Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle. Nucleic Acids Res. 46, 7873–7885 (2018).
Qian, H. et al. Identification and characterization of acetyltransferase-type toxin–antitoxin locus in Klebsiella pneumoniae. Mol. Microbiol. 108, 336–349 (2018).
McVicker, G. & Tang, C. M. Deletion of toxin–antitoxin systems in the evolution of Shigella sonnei as a host-adapted pathogen. Nat. Microbiol. 2, 16204 (2016).
Van Acker, H., Sass, A., Dhondt, I., Nelis, H. J. & Coenye, T. Involvement of toxin–antitoxin modules in Burkholderia cenocepacia biofilm persistence. Pathog. Dis. 71, 326–335 (2014).
Narimisa, N., Sadeghi Kalani, B., Mohammadzadeh, R. & Masjedian Jazi, F. Combination of antibiotics—nisin reduces the formation of persister cell in Listeria monocytogenes. Microb. Drug Resist. 27, 137–144 (2021).
Iqbal, N., Guérout, A. M., Krin, E., Le Roux, F. & Mazel, D. Comprehensive functional analysis of the 18 Vibrio cholerae N16961 toxin–antitoxin systems substantiates their role in stabilizing the superintegron. J. Bacteriol. 197, 2150–2159 (2015).
Zhang, C., Yashiro, Y., Sakaguchi, Y., Suzuki, T. & Tomita, K. Substrate specificities of Escherichia coli ItaT that acetylates aminoacyl-tRNAs. Nucleic Acids Res. 48, 7532–7544 (2020).
Burckhardt, R. M. & Escalante-Semerena, J. C. Small-molecule acetylation by GCN5-related N-acetyltransferases in bacteria. Microbiol. Mol. Biol. Rev. 84, e00090-19 (2020).
Jurėnas, D., Garcia-Pino, A. & Van Melderen, L. Novel toxins from type II toxin–antitoxin systems with acetyltransferase activity. Plasmid 93, 30–35 (2017).
Yashiro, Y., Yamashita, S. & Tomita, K. Crystal structure of the enterohemorrhagic Escherichia coli AtaT–AtaR toxin–antitoxin complex. Structure 27, 476–484.e3 (2019).
Jurėnas, D., Van Melderen, L. & Garcia-Pino, A. Mechanism of regulation and neutralization of the AtaR–AtaT toxin–antitoxin system. Nat. Chem. Biol. 15, 285–294 (2019).
Qian, H. et al. Toxin–antitoxin operon kacAT of Klebsiella pneumoniae is regulated by conditional cooperativity via a W-shaped KacA-KacT complex. Nucleic Acids Res. 47, 7690–7702 (2019).
Walling, L. R. & Butler, J. S. Structural determinants for antitoxin identity and insulation of cross talk between homologous toxin–antitoxin systems. J. Bacteriol. 198, 3287–3295 (2016).
Plach, M. G. et al. Evolutionary diversification of protein–protein interactions by interface add-ons. Proc. Natl Acad. Sci. USA 114, E8333–E8342 (2017).
Lite, T.-L. V. et al. Uncovering the basis of protein-protein interaction specificity with a combinatorially complete library. eLife 9, e60924 (2020).
Balakrishnan, S., Kamisetty, H., Carbonell, J. G., Lee, S. I. & Langmead, C. J. Learning generative models for protein fold families. Proteins Struct. Funct. Bioinform. 79, 1061–1078 (2011).
Schreiter, E. R. & Drennan, C. L. Ribbon–helix–helix transcription factors: variations on a theme. Nat. Rev. Microbiol. 5, 710–720 (2007).
Yashiro, Y., Sakaguchi, Y., Suzuki, T. & Tomita, K. Mechanism of aminoacyl-tRNA acetylation by an aminoacyl-tRNA acetyltransferase AtaT from enterohemorrhagic E. coli. Nat. Commun. 11, 5438 (2020).
Xue, L. et al. Distinct oligomeric structures of the YoeB–YefM complex provide insights into the conditional cooperativity of type II toxin–antitoxin system. Nucleic Acids Res. 48, 10527–10541 (2020).
Skjerning, R. B., Senissar, M., Winther, K. S., Gerdes, K. & Brodersen, D. E. The RES domain toxins of RES-Xre toxin–antitoxin modules induce cell stasis by degrading NAD+. Mol. Microbiol. 111, 221–236 (2019).
Bertelsen, M. B. et al. Structural basis for toxin inhibition in the VapXD toxin–antitoxin system. Structure 29, 139–150.e3 (2021).
Freire, D. M. et al. An NAD+ phosphorylase toxin triggers Mycobacterium tuberculosis cell death. Mol. Cell 73, 1282–1291.e8 (2019).
Ahidjo, B. A. et al. VapC toxins from Mycobacterium tuberculosis are ribonucleases that differentially inhibit growth and are neutralized by cognate VapB antitoxins. PLoS ONE 6, e21738 (2011).
Nolle, N., Schuster, C. F. & Bertram, R. Two paralogous yefM–yoeB loci from Staphylococcus equorum encode functional toxin–antitoxin systems. Microbiol. (U. Kingd.) 159, 1575–1585 (2013).
Guérout, A. M. et al. Characterization of the phd-doc and ccd toxin–antitoxin cassettes from Vibrio superintegrons. J. Bacteriol. 195, 2270–2283 (2013).
Połom, D., Boss, L., Węgrzyn, G., Hayes, F. & Kędzierska, B. Amino acid residues crucial for specificity of toxin–antitoxin interactions in the homologous Axe–Txe and YefM–YoeB complexes. FEBS J. 280, 5906–5918 (2013).
Minor, D. L. & Kim, P. S. Context-dependent secondary structure formation of a designed protein sequence. Nature 380, 730–734 (1996).
Garcia-Pino, A. et al. Allostery and intrinsic disorder mediate transcription regulation by conditional cooperativity. Cell 142, 101–111 (2010).
Kumar, P., Issac, B., Dodson, E. J., Turkenburg, J. P. & Mande, S. C. Crystal structure of Mycobacterium tuberculosis YefM antitoxin reveals that it is not an intrinsically unstructured protein. J. Mol. Biol. 383, 482–493 (2008).
Sterckx, Y. G. J. et al. Small-angle X-ray scattering—and nuclear magnetic resonance-derived conformational ensemble of the highly flexible antitoxin PaaA2. Structure 22, 854–865 (2014).
Chan, W. T., Espinosa, M. & Yeo, C. C. Keeping the wolves at bay: antitoxins of prokaryotic type II toxin–antitoxin systems. Front. Mol. Biosci. 3, https://doi.org/10.3389/fmolb.2016.00009 (2016).
De Jonge, N. et al. Rejuvenation of CcdB-poisoned gyrase by an intrinsically disordered protein domain. Mol. Cell 35, 154–163 (2009).
Harms, A., Brodersen, D. E., Mitarai, N. & Gerdes, K. Toxins, targets, and triggers: an overview of toxin–antitoxin biology. Mol. Cell 70, 768–784 (2018).
Scheich, C., Kümmel, D., Soumailakakis, D., Heinemann, U. & Büssow, K. Vectors for co-expression of an unrestricted number of proteins. Nucleic Acids Res. 35, e43 (2007).
Clabbers, M. T. B., Gruene, T., Parkhurst, J. M., Abrahams, J. P. & Waterman, D. G. Electron diffraction data processing with DIALS. Acta Crystallogr. D Struct. Biol. 74, 506–518 (2018).
Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D Struct. Biol. 75, 861–877 (2019).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).
Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).
Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. E. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015).
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).
The authors declare no competing interests.
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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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).
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).
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.
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).
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.
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.
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.
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.
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.
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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
Nature Chemical Biology (2021)