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Mechanism of regulation and neutralization of the AtaR–AtaT toxin–antitoxin system

Abstract

GCN5-related N-acetyl-transferase (GNAT)-like enzymes from toxin–antitoxin modules are strong inhibitors of protein synthesis. Here, we present the bases of the regulatory mechanisms of ataRT, a model GNAT-toxin–antitoxin module, from toxin synthesis to its action as a transcriptional de-repressor. We show the antitoxin (AtaR) traps the toxin (AtaT) in a pre-catalytic monomeric state and precludes the effective binding of ac-CoA and its target Met-transfer RNAfMet. In the repressor complex, AtaR intrinsically disordered region interacts with AtaT at two different sites, folding into different structures, that are involved in two separate functional roles, toxin neutralization and placing the DNA-binding domains of AtaR in a binding-compatible orientation. Our data suggests AtaR neutralizes AtaT as a monomer, right after its synthesis and only the toxin–antitoxin complex formed in this way is an active repressor. Once activated by dimerization, later neutralization of the toxin results in a toxin–antitoxin complex that is not able to repress transcription.

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Fig. 1: Structure and Met-tRNAfMet binding site mapping of AtaT.
Fig. 2: Structure of free AtaR and mapping of the toxin neutralization region.
Fig. 3: Mechanism of recognition and neutralization of AtaT by AtaR.
Fig. 4: Transcription repression by AtaR and AtaR–AtaT.
Fig. 5: Structure of AtaT–AtaR2–Opr22–AtaR2–AtaT.
Fig. 6: Cartoon representation of the molecular model for the mechanism of regulation of the ataRT operon.

Data availability

All the structures have been deposited in the PDB database with the following accession numbers: 6GTO, 6GTQ, 6GTP, 6GTR and 6GTS. All data needed to evaluate the conclusions in the paper are present in the paper and/or the Methods. Additional data related to this paper may be requested from the authors.

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Acknowledgements

We acknowledge the use of the synchrotron-radiation facility at the Soleil synchrotron Gif-sur-Yvette, France, under proposals 20150717, 20160750 and 20170756; Diamond Light Source, Didcot, UK, under proposal MX9426; and access support from the European Community’s Seventh Framework Program (FP7/2007–2013) under BioStruct-X (projects 1673 and 6131). We also thank the staff from Swing, PROXIMA-1 and PROXIMA-2A beamlines at Soleil for assistance with data collection. F. Goormaghtigh and A. Talavera are thanked for technical assistance with the Flow Cytometry and SEC-multiangle light scattering measurements. This work was supported by grants from the Fonds National de Recherche Scientifique nos. FNRS-MIS F.4505.16, FNRS-EQP U.N043.17F, FRFS-WELBIO CR-2017S-03 and FNRS-PDR T.0066.18 to A.G.-P. and FNRS-PDR T.0147.15F and FNRS-CDR J.0061.16F to L.V.M.; the Program ‘Actions de Recherche Concertée’ 2016–2021 from the ULB, the Fonds d’Encouragement à la Recherche (FER) ULB to A.G.-P.; and the Fonds Jean Brachet and the Fondation Van Buuren to A.G.-P. and L.V.M. D.J. was supported by a PhD grant from the Fonds National de Recherche Scientifique FNRS-ASPIRANT.

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D.J., L.V.M. and A.G.-P. designed research. D.J. performed the research. D.J. and A.G.-P. analyzed the data. D.J., L.V.M. and A.G.-P. wrote the paper.

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Correspondence to Abel Garcia-Pino.

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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). https://doi.org/10.1038/s41589-018-0216-z

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