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A nucleotide-switch mechanism mediates opposing catalytic activities of Rel enzymes


Bifunctional Rel stringent factors, the most abundant class of RelA/SpoT homologs, are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3′ of guanosine tri-/diphosphate (GTP/GDP) to synthesize the bacterial alarmone (p)ppGpp, and also catalyze the 3′ pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide-switch mechanism controls catalysis by Thermus thermophilus Rel (RelTt). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of RelTt (RelTtNTD) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation.

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Fig. 1: Structure of RelTtNTD in a resting and closed state.
Fig. 2: Structure of RelTtNTD in the open active SYN state, molecular bases of the interdomain allosteric interplay.
Fig. 3: Nucleotide binding controls RelTt allosteric switch, off the ribosome.
Fig. 4: RelTtNTD catalytic domain conformational dynamics in the presence of nucleotides assessed by smFRET.
Fig. 5: Conformational dynamics of the α6–α7 motif is coupled to nucleotide binding.
Fig. 6: Regulation of Rel catalytic activities by substrate nucleotides.

Data availability

All the structures have been deposited in the PDB database with the following accession numbers; 6S2V, 6S2T and 6S2U. 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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We acknowledge the use of the synchrotron-radiation facility at the SOLEIL synchrotron Gif-sur-Yvette, France, under proposals 20150717, 20160750 and 20170756. We also thank the staff from Swing, PROXIMA-1 and PROXIMA-2A beamlines at SOLEIL for assistance with data collection. This work was supported by grants from the Fonds National de Recherche Scientifique, nos. FNRS-EQP U.N043.17F, FRFS-WELBIO CR-2017S-03 and FNRS-PDR T.0066.18, and the Joint Programming Initiative on Antimicrobial Resistance (grant no. JPI-EC-AMR-R.8004.18-) to A.G.-P. The Program ‘Actions de Recherche Concertée’ 2016-2021 and Fonds d’Encouragement à la Recherche from the ULB, Fonds Jean Brachet and the Fondation Van Buren to A.G.-P.; the Molecular Infection Medicine Sweden, Swedish Research council (grant no. 2017-03783), and Ragnar Söderberg foundation fellowship to V.H.; J. Hendrix and J. Hofkens are grateful to the Research Foundation Flanders (FWO Vlaanderen, grant no. G0B4915N) and large infrastructure grant (no. ZW15_09 GOH6316N) and the KU Leuven Research Fund (no. C14/16/053); J.Hofkens thanks financial support of the Flemish government through long-term structural funding Methusalem (CASAS2, Meth/15/04). K.V.N. was supported by a PhD grant from the Fonds National de Recherche Scientifique FNRS-FRIA. N.V. acknowledges the Agency for Innovation by Science and Technology in Flanders for a PhD grant. H. Tamman was supported by a Chargé de Recherches fellowship from the FNRS (no. CR/DM-392). H. Takada was supported by the postdoctoral grant from the Umeå Centre for Microbial Research (UCMR).

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H. Tamman, K.V.N., N.V., D.S. and A.T. performed biophysical, structural biology and smFRET experiments. H. Takada performed biochemical assays. Y.P. was involved in the initial steps of the preparation of T. thermophilus ribosomes. J. Hendrix and J. Hofkens supervised the smFRET data analysis. V.H., J. Hendrix and A.G.-P. designed research and wrote the paper.

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Correspondence to Vasili Hauryliuk, Jelle Hendrix or Abel Garcia-Pino.

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Tamman, H., Van Nerom, K., Takada, H. et al. A nucleotide-switch mechanism mediates opposing catalytic activities of Rel enzymes. Nat Chem Biol 16, 834–840 (2020).

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