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

Abstract

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.

References

  1. 1.

    Laffler, T. & Gallant, J. A. Stringent control of protein synthesis in E. coli. Cell 3, 47–49 (1974).

    CAS  Article  Google Scholar 

  2. 2.

    Cashel, M. & Gallant, J. Two compounds implicated in the function of the RC gene of Escherichia coli. Nature 221, 838–841 (1969).

    CAS  Article  Google Scholar 

  3. 3.

    Hauryliuk, V., Atkinson, G. C., Murakami, K. S., Tenson, T. & Gerdes, K. Recent functional insights into the role of (p)ppGpp in bacterial physiology. Nat. Rev. Microbiol. 13, 298–309 (2015).

    CAS  Article  Google Scholar 

  4. 4.

    Atkinson, G. C., Tenson, T. & Hauryliuk, V. The RelA/SpoT homolog (RSH) superfamily: distribution and functional evolution of ppGpp synthetases and hydrolases across the tree of life. PLoS ONE 6, e23479 (2011).

    CAS  Article  Google Scholar 

  5. 5.

    Stent, G. S. & Brenner, S. A genetic locus for the regulation of ribonucleic acid synthesis. Proc. Natl Acad. Sci. USA 47, 2005–2014 (1961).

    CAS  Article  Google Scholar 

  6. 6.

    Hogg, T., Mechold, U., Malke, H., Cashel, M. & Hilgenfeld, R. Conformational antagonism between opposing active sites in a bifunctional RelA/SpoT homolog modulates (p)ppGpp metabolism during the stringent response [corrected]. Cell 117, 57–68 (2004).

    CAS  Article  Google Scholar 

  7. 7.

    Avarbock, D., Salem, J., Li, L. S., Wang, Z. M. & Rubin, H. Cloning and characterization of a bifunctional RelA/SpoT homologue from Mycobacterium tuberculosis. Gene 233, 261–269 (1999).

    CAS  Article  Google Scholar 

  8. 8.

    Mechold, U., Murphy, H., Brown, L. & Cashel, M. Intramolecular regulation of the opposing (p)ppGpp catalytic activities of Rel(Seq), the Rel/Spo enzyme from Streptococcus equisimilis. J. Bacteriol. 184, 2878–2888 (2002).

    CAS  Article  Google Scholar 

  9. 9.

    Van Nerom, K., Tamman, H., Takada, H., Hauryliuk, V. & Garcia-Pino, A. The Rel stringent factor from Thermus thermophilus: crystallization and X-ray analysis. Acta Crystallogr. F 75, 561–569 (2019).

    CAS  Article  Google Scholar 

  10. 10.

    Singal, B. et al. Crystallographic and solution structure of the N-terminal domain of the Rel protein from Mycobacterium tuberculosis. FEBS Lett. 591, 2323–2337 (2017).

    CAS  Article  Google Scholar 

  11. 11.

    Brown, A., Fernandez, I. S., Gordiyenko, Y. & Ramakrishnan, V. Ribosome-dependent activation of stringent control. Nature 534, 277–280 (2016).

    CAS  Article  Google Scholar 

  12. 12.

    Arenz, S. et al. The stringent factor RelA adopts an open conformation on the ribosome to stimulate ppGpp synthesis. Nucleic Acids Res. 44, 6471–6481 (2016).

  13. 13.

    Loveland, A. B. et al. Ribosome*RelA structures reveal the mechanism of stringent response activation. eLife 5, e17029 (2016).

  14. 14.

    Sun, D. et al. A metazoan ortholog of SpoT hydrolyzes ppGpp and functions in starvation responses. Nat. Struct. Mol. Biol. 17, 1188–1194 (2010).

    CAS  Article  Google Scholar 

  15. 15.

    Aravind, L. & Koonin, E. V. The HD domain defines a new superfamily of metal-dependent phosphohydrolases. Trends Biochem. Sci. 23, 469–472 (1998).

    CAS  Article  Google Scholar 

  16. 16.

    Manav, M. C. et al. Structural basis for (p)ppGpp synthesis by the Staphylococcus aureus small alarmone synthetase RelP. J. Biol. Chem. 293, 3254–3264 (2018).

    CAS  Article  Google Scholar 

  17. 17.

    Steinchen, W. et al. Structural and mechanistic divergence of the small (p)ppGpp synthetases RelP and RelQ. Sci. Rep. 8, 2195 (2018).

    Article  Google Scholar 

  18. 18.

    Takada, H. et al. Ribosome association primes the stringent factor Rel for recruitment of deacylated tRNA to ribosomal A-site. Preprint at bioRxiv https://doi.org/10.1101/2020.01.17.910273 (2020).

  19. 19.

    Takada, H. et al. The C-terminal RRM/ACT domain is crucial for fine-tuning the activation of ‘Long’ RelA-SpoT homolog enzymes by ribosomal complexes. Front. Microbiol. 11, 277–307 (2020).

  20. 20.

    Steinchen, W. et al. Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone. Proc. Natl Acad. Sci. USA 112, 13348–13353 (2015).

    CAS  Article  Google Scholar 

  21. 21.

    Okar, D. A. et al. PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6-bisphosphate. Trends Biochem. Sci. 26, 30–35 (2001).

    CAS  Article  Google Scholar 

  22. 22.

    Gratani, F. L. et al. Regulation of the opposing (p)ppGpp synthetase and hydrolase activities in a bifunctional RelA/SpoT homologue from Staphylococcus aureus. PLoS Genet. 14, e1007514 (2018).

    Article  Google Scholar 

  23. 23.

    Ronneau, S. et al. Regulation of (p)ppGpp hydrolysis by a conserved archetypal regulatory domain. Nucleic Acids Res. 47, 843–854 (2019).

    CAS  Article  Google Scholar 

  24. 24.

    Kabsch, W. XDS. Acta Crystallogr D 66, 125–132 (2010).

    CAS  Article  Google Scholar 

  25. 25.

    Collaborative Computational Project, N. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

  26. 26.

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  Article  Google Scholar 

  27. 27.

    Terwilliger, T. C. et al. phenix.mr_rosetta: molecular replacement and model rebuilding with Phenix and Rosetta. J. Struct. Funct. Genomics 13, 81–90 (2012).

    CAS  Article  Google Scholar 

  28. 28.

    Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012).

    CAS  Article  Google Scholar 

  29. 29.

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  Google Scholar 

  30. 30.

    Smart, O. S. et al. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr. D 68, 368–380 (2012).

    CAS  Article  Google Scholar 

  31. 31.

    Talavera, A. et al. Phosphorylation decelerates conformational dynamics in bacterial translation elongation factors. Sci. Adv. 4, eaap9714 (2018).

    Article  Google Scholar 

  32. 32.

    Otosu, T., Ishii, K. & Tahara, T. Note: simple calibration of the counting-rate dependence of the timing shift of single photon avalanche diodes by photon interval analysis. Rev. Sci. Instrum. 84, 036105 (2013).

    Article  Google Scholar 

  33. 33.

    Schrimpf, W., Barth, A., Hendrix, J. & Lamb, D. C. PAM: a framework for integrated analysis of imaging, single-molecule, and ensemble fluorescence data. Biophys. J. 114, 1518–1528 (2018).

    CAS  Article  Google Scholar 

  34. 34.

    Hellenkamp, B. et al. Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study. Nat. Methods 15, 669 (2018).

    CAS  Article  Google Scholar 

  35. 35.

    Kudryavtsev, V. et al. Combining MFD and PIE for accurate single-pair Forster resonance energy transfer measurements. Chem. Phys. Chem. 13, 1060–1078 (2012).

    CAS  Article  Google Scholar 

  36. 36.

    Tomov, T. E. et al. Disentangling subpopulations in single-molecule FRET and ALEX experiments with photon distribution analysis. Biophys. J. 102, 1163–1173 (2012).

    CAS  Article  Google Scholar 

  37. 37.

    Antonik, M., Felekyan, S., Gaiduk, A. & Seidel, C. A. Separating structural heterogeneities from stochastic variations in fluorescence resonance energy transfer distributions via photon distribution analysis. J. Phys. Chem. B 110, 6970–6978 (2006).

    CAS  Article  Google Scholar 

  38. 38.

    Kalinin, S. et al. A toolkit and benchmark study for FRET-restrained high-precision structural modeling. Nat. Methods 9, 1218–1225 (2012).

    CAS  Article  Google Scholar 

  39. 39.

    Polikanov, Y. S., Steitz, T. A. & Innis, C. A. A proton wire to couple aminoacyl-tRNA accommodation and peptide-bond formation on the ribosome. Nat. Struct. Mol. Biol. 21, 787–793 (2014).

    CAS  Article  Google Scholar 

  40. 40.

    Antoun, A., Pavlov, M. Y., Tenson, T. & Ehrenberg, M. M. Ribosome formation from subunits studied by stopped-flow and Rayleigh light scattering. Biol. Proced. Online 6, 35–54 (2004).

    CAS  Article  Google Scholar 

  41. 41.

    Murina, V., Kasari, M., Hauryliuk, V. & Atkinson, G. C. Antibiotic resistance ABCF proteins reset the peptidyl transferase centre of the ribosome to counter translational arrest. Nucleic Acids Res. 46, 3753–3763 (2018).

    CAS  Article  Google Scholar 

  42. 42.

    Kudrin, P. et al. The ribosomal A-site finger is crucial for binding and activation of the stringent factor RelA. Nucleic Acids Res. 46, 1973–1983 (2018).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

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 or 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 (2020). https://doi.org/10.1038/s41589-020-0520-2

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