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The structural basis of hyperpromiscuity in a core combinatorial network of type II toxin-antitoxin and related phage defense systems

Ernits, Karin LU ; Saha, Chayan Kumar LU orcid ; Brodiazhenko, Tetiana ; Chouhan, Bhanu LU ; Shenoy, Aditi ; Buttress, Jessica A ; Duque-Pedraza, Julián J LU ; Bojar, Veda LU ; Nakamoto, Jose A LU orcid and Kurata, Tatsuaki LU , et al. (2023) In Proceedings of the National Academy of Sciences of the United States of America 120(33). p.1-12
Abstract

Toxin-antitoxin (TA) systems are a large group of small genetic modules found in prokaryotes and their mobile genetic elements. Type II TAs are encoded as bicistronic (two-gene) operons that encode two proteins: a toxin and a neutralizing antitoxin. Using our tool NetFlax (standing for Network-FlaGs for toxins and antitoxins), we have performed a large-scale bioinformatic analysis of proteinaceous TAs, revealing interconnected clusters constituting a core network of TA-like gene pairs. To understand the structural basis of toxin neutralization by antitoxins, we have predicted the structures of 3,419 complexes with AlphaFold2. Together with mutagenesis and functional assays, our structural predictions provide insights into the... (More)

Toxin-antitoxin (TA) systems are a large group of small genetic modules found in prokaryotes and their mobile genetic elements. Type II TAs are encoded as bicistronic (two-gene) operons that encode two proteins: a toxin and a neutralizing antitoxin. Using our tool NetFlax (standing for Network-FlaGs for toxins and antitoxins), we have performed a large-scale bioinformatic analysis of proteinaceous TAs, revealing interconnected clusters constituting a core network of TA-like gene pairs. To understand the structural basis of toxin neutralization by antitoxins, we have predicted the structures of 3,419 complexes with AlphaFold2. Together with mutagenesis and functional assays, our structural predictions provide insights into the neutralizing mechanism of the hyperpromiscuous Panacea antitoxin domain. In antitoxins composed of standalone Panacea, the domain mediates direct toxin neutralization, while in multidomain antitoxins the neutralization is mediated by other domains, such as PAD1, Phd-C, and ZFD. We hypothesize that Panacea acts as a sensor that regulates TA activation. We have experimentally validated 16 NetFlax TA systems and used domain annotations and metabolic labeling assays to predict their potential mechanisms of toxicity (such as membrane disruption, and inhibition of cell division or protein synthesis) as well as biological functions (such as antiphage defense). We have validated the antiphage activity of a RosmerTA system encoded by
Gordonia phage Kita, and used fluorescence microscopy to confirm its predicted membrane-depolarizing activity. The interactive version of the NetFlax TA network that includes structural predictions can be accessed at http://netflax.webflags.se/.

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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Antitoxins/genetics, Bacterial Toxins/metabolism, Prokaryotic Cells/metabolism, Operon/genetics, Computational Biology, Bacterial Proteins/genetics
in
Proceedings of the National Academy of Sciences of the United States of America
volume
120
issue
33
article number
e2305393120
pages
1 - 12
publisher
National Academy of Sciences
external identifiers
  • pmid:37556498
  • scopus:85167528527
ISSN
1091-6490
DOI
10.1073/pnas.2305393120
language
English
LU publication?
yes
id
f7ce0a32-349d-402b-b343-9589c103546a
date added to LUP
2023-08-16 17:45:22
date last changed
2024-06-16 08:32:23
@article{f7ce0a32-349d-402b-b343-9589c103546a,
  abstract     = {{<p>Toxin-antitoxin (TA) systems are a large group of small genetic modules found in prokaryotes and their mobile genetic elements. Type II TAs are encoded as bicistronic (two-gene) operons that encode two proteins: a toxin and a neutralizing antitoxin. Using our tool NetFlax (standing for Network-FlaGs for toxins and antitoxins), we have performed a large-scale bioinformatic analysis of proteinaceous TAs, revealing interconnected clusters constituting a core network of TA-like gene pairs. To understand the structural basis of toxin neutralization by antitoxins, we have predicted the structures of 3,419 complexes with AlphaFold2. Together with mutagenesis and functional assays, our structural predictions provide insights into the neutralizing mechanism of the hyperpromiscuous Panacea antitoxin domain. In antitoxins composed of standalone Panacea, the domain mediates direct toxin neutralization, while in multidomain antitoxins the neutralization is mediated by other domains, such as PAD1, Phd-C, and ZFD. We hypothesize that Panacea acts as a sensor that regulates TA activation. We have experimentally validated 16 NetFlax TA systems and used domain annotations and metabolic labeling assays to predict their potential mechanisms of toxicity (such as membrane disruption, and inhibition of cell division or protein synthesis) as well as biological functions (such as antiphage defense). We have validated the antiphage activity of a RosmerTA system encoded by<br>
 Gordonia phage Kita, and used fluorescence microscopy to confirm its predicted membrane-depolarizing activity. The interactive version of the NetFlax TA network that includes structural predictions can be accessed at http://netflax.webflags.se/.<br>
 </p>}},
  author       = {{Ernits, Karin and Saha, Chayan Kumar and Brodiazhenko, Tetiana and Chouhan, Bhanu and Shenoy, Aditi and Buttress, Jessica A and Duque-Pedraza, Julián J and Bojar, Veda and Nakamoto, Jose A and Kurata, Tatsuaki and Egorov, Artyom A and Shyrokova, Lena and Johansson, Marcus J O and Mets, Toomas and Rustamova, Aytan and Džigurski, Jelisaveta and Tenson, Tanel and Garcia-Pino, Abel and Strahl, Henrik and Elofsson, Arne and Hauryliuk, Vasili and Atkinson, Gemma C}},
  issn         = {{1091-6490}},
  keywords     = {{Antitoxins/genetics; Bacterial Toxins/metabolism; Prokaryotic Cells/metabolism; Operon/genetics; Computational Biology; Bacterial Proteins/genetics}},
  language     = {{eng}},
  month        = {{08}},
  number       = {{33}},
  pages        = {{1--12}},
  publisher    = {{National Academy of Sciences}},
  series       = {{Proceedings of the National Academy of Sciences of the United States of America}},
  title        = {{The structural basis of hyperpromiscuity in a core combinatorial network of type II toxin-antitoxin and related phage defense systems}},
  url          = {{http://dx.doi.org/10.1073/pnas.2305393120}},
  doi          = {{10.1073/pnas.2305393120}},
  volume       = {{120}},
  year         = {{2023}},
}