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Structural basis of substrate specificity and neutralization mechanism of the FaRel2/ATfaRel2 toxin-antitoxin system from Coprobacillus sp. D7

par Dominguez Molina, Lucia
Président du jury Vanhamme, Luc
Promoteur Garcia-Pino, Abel
Publication Non publié, 2023-11-17

Thèse de doctorat

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Résumé :

Toxin-antitoxin (TA) systems are widespread in bacterial and archaeal genomes. These TA modulesgenerally encode a toxic component and its cognate antidote in the same operon. In the case of type IITA systems, the antitoxin binds to the toxin to form a nontoxic complex via protein-protein interactionand thus, the toxin is retained in an inactive state protecting the bacterial cells. Under unfavourableconditions in the cell, the TA systems are activated, and the labile antitoxin is degraded by proteases;therefore, the toxin can find its target in the bacterial cell leading to growth inhibition or cell death. Arecent discovery reveals multiple families of small alarmone synthetases (SASs) in which they areencoded in conserved bicistronic architectures that recall toxin-antitoxin modules. ToxSAS (toxicSASs) enzymes act through translation inhibition mediated by pyrophosphorylation of tRNA CCAends, such as FaRel2, or through synthesis of the toxic alarmone (p)ppApp and ATP depletion (such asFaRel). Both enzymatic reactions use ATP as a pyrophosphate donor. In this thesis, we have studiedthe FaRel2 toxSAS effector from Coprobacillus sp. D7 (Coprobacillus) promotes growth arrest in E.coli by inhibiting protein synthesis. FaRel2 transfers a pyrophosphate moiety from ATP to the 3’CCAend of the tRNA. Indeed, this modification abrogates the aminoacylation of tRNA and the starvationsignal of cellular amino acids by the ribosome-RelA enzyme. Virtually nothing is known about theneutralisation mechanism that ATfaRel2 antitoxin exerts to counteract FaRel2 toxicity and diversitytoxicity activity in the new toxSAS subfamilies. In this study, we show that the pseudo-Zn2+ fingerdomain (pZFD) of the ATfaRel2 antitoxin prevents the access of ATP to the pyrophosphate donor siteof the FaRel2 toxin, without affecting the recruitment of the tRNA pyrophosphate acceptor. On thecontrary, toxSASs producing (p)ppApp are inhibited via Tis1 antitoxin domains by occlusion of thepyrophosphate acceptor binding site. Consequently, the auxiliary pZFD of AT2faRel is dispensable forthe neutralisation of FaRel. We have unravelled the crystal structures of FaRel2 bound to APCPP, anon-hydrolysable analogue of ATP and the ATfaRel2:FaRel2 complex. Furthermore, we haveelucidated the neutralisation mechanism in the FaRel2-ATfaRel2 TA system, in which oligomerizationof the C-terminal domain plays an important role in complex stabilisation and neutralisation comparedto the closed inactive conformation state observed in the fused CapRelSJ46 toxSAS TA module.Together, our study establishes the general principles of inhibition of toxSAS by structured antitoxindomains directly coupled to substrate specificity.