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Affinity-based capture and identification of protein effectors of the growth regulator ppGpp

An Author Correction to this article was published on 10 May 2019

Abstract

The nucleotide ppGpp is a highly conserved regulatory molecule in bacteria that helps tune growth rate to nutrient availability. Despite decades of study, how ppGpp regulates growth remains poorly understood. Here, we developed and validated a capture-compound mass spectrometry approach that identified >50 putative ppGpp targets in Escherichia coli. These targets control many key cellular processes and include 13 enzymes required for nucleotide synthesis. We demonstrated that ppGpp inhibits the de novo synthesis of all purine nucleotides by directly targeting the enzyme PurF. By solving a structure of PurF bound to ppGpp, we designed a mutation that ablates ppGpp-based regulation, leading to dysregulation of purine-nucleotide synthesis following ppGpp accumulation. Collectively, our results provide new insights into ppGpp-based growth control and a nearly comprehensive set of targets for future exploration. The capture compounds developed should also enable the rapid identification of ppGpp targets in any species, including pathogens.

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Fig. 1: ppGpp inhibits growth independently of its effects on RNA polymerase.
Fig. 2: Overview of the capture-compound mass spectrometry approach for identifying ppGpp targets.
Fig. 3: ppGpp inhibits the de novo synthesis of purine nucleotides.
Fig. 4: ppGpp directly inhibits de novo synthesis of purine nucleotides by binding PurF.
Fig. 5: Preventing the regulation of PurF by ppGpp leads to a dysregulation of purine nucleotides.

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Data availability

Structural data for the PurF–ppGpp complex used to generate Figs. 4d–f, 5a and Supplementary Fig. 4b–d have been deposited in the Protein Data Bank under accession number PDB 6CZF. Raw proteomic LC–MS2 data as sources of Table 1, Fig. 2c and the Supplementary Dataset have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository under dataset identifier PXD010402. All other data generated or analyzed during this study are included in this published article (and its Supplementary Information files) or are available from the corresponding author on reasonable request.

References

  1. 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).

    Article  CAS  Google Scholar 

  2. Potrykus, K., Murphy, H., Philippe, N. & Cashel, M. ppGpp is the major source of growth rate control in E. coli. Environ. Microbiol. 13, 563–575 (2011).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  4. Liu, K., Bittner, A. N. & Wang, J. D. Diversity in (p)ppGpp metabolism and effectors. Curr. Opin. Microbiol. 24, 72–79 (2015).

    Article  Google Scholar 

  5. Haseltine, W. A. & Block, R. Synthesis of guanosine tetra- and pentaphosphate requires the presence of a codon-specific, uncharged transfer ribonucleic acid in the acceptor site of ribosomes. Proc. Natl Acad. Sci. USA 70, 1564–1568 (1973).

    Article  CAS  Google Scholar 

  6. Sarubbi, E. et al. Characterization of the spoT gene of Escherichia coli. J. Biol. Chem. 264, 15074–15082 (1989).

    CAS  PubMed  Google Scholar 

  7. Mechold, U., Potrykus, K., Murphy, H., Murakami, K. S. & Cashel, M. Differential regulation by ppGpp versus pppGpp in Escherichia coli. Nucleic Acids Res. 41, 6175–6189 (2013).

    Article  CAS  Google Scholar 

  8. Kanjee, U., Ogata, K. & Houry, W. A. Direct binding targets of the stringent response alarmone (p)ppGpp. Mol. Microbiol. 85, 1029–1043 (2012).

    Article  CAS  Google Scholar 

  9. Potrykus, K. & Cashel, M. (p)ppGpp: still magical? Annu. Rev. Microbiol. 62, 35–51 (2008).

    Article  CAS  Google Scholar 

  10. Dalebroux, Z. D. & Swanson, M. S. ppGpp: magic beyond RNA polymerase. Nat. Rev. Microbiol. 10, 203–212 (2012).

    Article  CAS  Google Scholar 

  11. Ross, W. et al. ppGpp binding to a site at the RNAP-DksA interface accounts for its dramatic effects on transcription initiation during the stringent response. Mol. Cell 62, 811–823 (2016).

    Article  CAS  Google Scholar 

  12. Traxler, M. F. et al. The global, ppGpp-mediated stringent response to amino acid starvation in Escherichia coli. Mol. Microbiol. 68, 1128–1148 (2008).

    Article  CAS  Google Scholar 

  13. Zhang, Y., Zbornikova, E., Rejman, D. & Gerdes, K. Novel (p)ppGpp binding and metabolizing proteins of Escherichia coli. mBio 9, e02188–17 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Corrigan, R. M., Bellows, L. E., Wood, A. & Gründling, A. ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria. Proc. Natl Acad. Sci. USA 113, E1710–E1719 (2016).

    Article  CAS  Google Scholar 

  15. Schreiber, G. et al. Overexpression of the relA gene in Escherichia coli. J. Biol. Chem. 266, 3760–3767 (1991).

    CAS  PubMed  Google Scholar 

  16. Köster, H. et al. Capture compound mass spectrometry: a technology for the investigation of small molecule protein interactions. Assay. Drug. Dev. Technol. 5, 381–390 (2007).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Mann, M. Functional and quantitative proteomics using SILAC. Nat. Rev. Mol. Cell Biol. 7, 952–958 (2006).

    Article  CAS  Google Scholar 

  19. Li, G. W., Burkhardt, D., Gross, C. & Weissman, J. S. Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources. Cell 157, 624–635 (2014).

    Article  CAS  Google Scholar 

  20. Verstraeten, N., Fauvart, M., Versées, W. & Michiels, J. The universally conserved prokaryotic GTPases. Microbiol. Mol. Biol. Rev. 75, 507–542 (2011).

    Article  CAS  Google Scholar 

  21. Bennett, B. D. et al. Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat. Chem. Biol. 5, 593–599 (2009).

    Article  CAS  Google Scholar 

  22. Cashel, M. Regulation of bacterial ppGpp and pppGpp. Annu. Rev. Microbiol. 29, 301–318 (1975).

    Article  CAS  Google Scholar 

  23. Messenger, L. J. & Zalkin, H. Glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli: purification and properties. J. Biol. Chem. 254, 3382–3392 (1979).

    CAS  PubMed  Google Scholar 

  24. Krahn, J. M. et al. Coupled formation of an amidotransferase interdomain ammonia channel and a phosphoribosyltransferase active site. Biochemistry 36, 11061–11068 (1997).

    Article  CAS  Google Scholar 

  25. Jensen, K. F., Dandanell, G., Hove-Jensen, B. & WillemoËs, M. Nucleotides, nucleosides, and nucleobases. Ecosal Plus 3, (2008).

  26. Hove-Jensen, B., Harlow, K. W., King, C. J. & Switzer, R. L. Phosphoribosylpyrophosphate synthetase of Escherichia coli: properties of the purified enzyme and primary structure of the prs gene. J. Biol. Chem. 261, 6765–6771 (1986).

    CAS  PubMed  Google Scholar 

  27. Milon, P. et al. The nucleotide-binding site of bacterial translation initiation factor 2 (IF2) as a metabolic sensor. Proc. Natl Acad. Sci. USA 103, 13962–13967 (2006).

    Article  CAS  Google Scholar 

  28. Rojas, A. M., Ehrenberg, M., Andersson, S. G. E. & Kurland, C. G. ppGpp inhibition of elongation factors Tu, G and Ts during polypeptide synthesis. Mol. Gen. Genet. 197, 36–45 (1984).

    Article  CAS  Google Scholar 

  29. Kriel, A. et al. Direct regulation of GTP homeostasis by (p)ppGpp: a critical component of viability and stress resistance. Mol. Cell 48, 231–241 (2012).

    Article  CAS  Google Scholar 

  30. Liu, K. et al. Molecular mechanism and evolution of guanylate kinase regulation by (p)ppGpp. Mol. Cell 57, 735–749 (2015).

    Article  CAS  Google Scholar 

  31. Krásný, L. & Gourse, R.L. An alternative strategy for bacterial ribosome synthesis: Bacillus subtilis rRNA transcription regulation. EMBO J. 23, 4473–4483 (2004).

    Article  Google Scholar 

  32. Hochstadt-Ozer, J. & Cashel, M. The regulation of purine utilization in bacteria. V. Inhibition of purine phosphoribosyltransferase activities and purine uptake in isolated membrane vesicles by guanosine tetraphosphate. J. Biol. Chem. 247, 7067–7072 (1972).

    CAS  PubMed  Google Scholar 

  33. Stevens, A. J. et al. Design of a split intein with exceptional protein splicing activity. J. Am. Chem. Soc. 138, 2162–2165 (2016).

    Article  CAS  Google Scholar 

  34. Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).

    Article  CAS  Google Scholar 

  35. Dai, P. et al. Salt effect accelerates site-selective cysteine bioconjugation. ACS Cent. Sci. 2, 637–646 (2016).

    Article  CAS  Google Scholar 

  36. Park, J. O. et al. Metabolite concentrations, fluxes and free energies imply efficient enzyme usage. Nat. Chem. Biol. 12, 482–489 (2016).

    Article  CAS  Google Scholar 

  37. Wang, B., Zhao, A., Novick, R. P. & Muir, T. W. Key driving forces in the biosynthesis of autoinducing peptides required for staphylococcal virulence. Proc. Natl Acad. Sci. USA 112, 10679–10684 (2015).

    Article  CAS  Google Scholar 

  38. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  40. Muchmore, C. R., Krahn, J. M., Kim, J. H., Zalkin, H. & Smith, J. L. Crystal structure of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli. Protein Sci. 7, 39–51 (1998).

    Article  CAS  Google Scholar 

  41. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D. Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  42. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Guo, J. Kraemer, and P. Culviner for comments on the manuscript. We thank T. Muir (Princeton University) and S. Lovett (Brandeis University) for providing protein-expression vectors. This research made use of the Pilatus detector (RR029205) at NE-CAT beamline 24-IDC (GM103403) of the Advanced Photon Source (DE-AC02-06CH11357). We thank members of the Drennan laboratory for collecting the diffraction data at APS. We thank the Koch Institute Swanson Biotechnology Center (Biopolymer and Proteomic Core Facility) for help with quantitative mass spectrometry and the Whitehead Institute Metabolite Profiling Core Facility for measuring metabolite levels. Instrumentation resources from the Biophysical Instrumentation Facility for the Study of Complex Macromolecular Systems (NSF-0070319), the Structural Biology Core Facility, and the BioMicro Center in the Department of Biology at MIT are gratefully acknowledged. This work was supported by a fellowship from the Jane Coffin Childs Memorial Fund for Medical Research to B.W. and an NIH grant to M.T.L. (R01GM082899), who is also supported as an Investigator of the Howard Hughes Medical Institute.

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B.W., P.D., and B.L.P. designed and synthesized capture compounds. D.D. performed phylogenetic analyses. A.D.R. analyzed proteomics data. R.A.G. helped with X-ray structure determination. B.W. performed all other experiments. B.W. and M.T.L. designed experiments, analyzed data, prepared figures, and wrote the manuscript.

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Correspondence to Michael T. Laub.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Tables 1–9

Reporting Summary

Supplementary Note

Synthetic procedures

Supplementary Dataset

SILAC mass spectrometry results

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Wang, B., Dai, P., Ding, D. et al. Affinity-based capture and identification of protein effectors of the growth regulator ppGpp. Nat Chem Biol 15, 141–150 (2019). https://doi.org/10.1038/s41589-018-0183-4

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