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

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...

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Published in:	Proceedings of the National Academy of Sciences - PNAS Vol. 120; no. 33; p. e2305393120
Main Authors:	Ernits, Karin, Saha, Chayan Kumar, Brodiazhenko, Tetiana, Chouhan, Bhanu, Shenoy, Aditi, Buttress, Jessica A, Duque-Pedraza, Julián J, Bojar, Veda, Nakamoto, Jose A, Kurata, Tatsuaki, Egorov, Artyom A, Shyrokova, Lena, Johansson, Marcus J O, Mets, Toomas, Rustamova, Aytan, Džigurski, Jelisaveta, Tenson, Tanel, Garcia-Pino, Abel, Strahl, Henrik, Elofsson, Arne, Hauryliuk, Vasili, Atkinson, Gemma C
Format:	Journal Article
Language:	English
Published:	United States National Academy of Sciences 15-08-2023
Subjects:	AlphaFold Annotations antitoxin Antitoxins Antitoxins/genetics Bacterial Proteins/genetics Bacterial Toxins/metabolism Bioinformatics and Systems Biology Bioinformatik och systembiologi Biologi Biological Sciences Cell division Combinatorial analysis Computational Biology Depolarization Fluorescence Fluorescence microscopy Membranes Mutagenesis Natural Sciences Naturvetenskap Neutralization Neutralizing Operon/genetics Operons Panacea phage Phages Prokaryotes Prokaryotic Cells/metabolism Protein biosynthesis Protein synthesis Proteins Structure-function relationships Toxicity toxin Toxins Toxins and antitoxins toxin phage antitoxin AlphaFold Panacea
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Summary:	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 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/.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Edited by Marlene Belfort, University at Albany, State University of New York, Albany, NY; received April 4, 2023; accepted July 11, 2023 1K.E. and C.K.S. contributed equally to this work.
ISSN:	0027-8424 1091-6490 1091-6490
DOI:	10.1073/pnas.2305393120