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

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.

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.

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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.

Author information

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.

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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