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Arginine phosphorylation marks proteins for degradation by a Clp protease

Abstract

Protein turnover is a tightly controlled process that is crucial for the removal of aberrant polypeptides and for cellular signalling. Whereas ubiquitin marks eukaryotic proteins for proteasomal degradation, a general tagging system for the equivalent bacterial Clp proteases is not known. Here we describe the targeting mechanism of the ClpC–ClpP proteolytic complex from Bacillus subtilis. Quantitative affinity proteomics using a ClpP-trapping mutant show that proteins phosphorylated on arginine residues are selectively targeted to ClpC–ClpP. In vitro reconstitution experiments demonstrate that arginine phosphorylation by the McsB kinase is required and sufficient for the degradation of substrate proteins. The docking site for phosphoarginine is located in the amino-terminal domain of the ClpC ATPase, as resolved at high resolution in a co-crystal structure. Together, our data demonstrate that phosphoarginine functions as a bona fide degradation tag for the ClpC–ClpP protease. This system, which is widely distributed across Gram-positive bacteria, is functionally analogous to the eukaryotic ubiquitin–proteasome system.

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Figure 1: Pull-down of ClpP trapping mutants.
Figure 2: Effect of McsB on the activity of ClpCP in vitro.
Figure 3: Binding of pArgAA to ClpC.
Figure 4: ClpCP protease activity towards a pArg-containing substrate protein.
Figure 5: Crystal structure of ClpCNTD in complex with pArg.
Figure 6: The pArg–ClpCP degradation system.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) under accession code 5HBN. The mass spectrometry data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) with the dataset identifier PXD003305.

References

  1. Goldberg, A. L. The mechanism and functions of ATP-dependent proteases in bacterial and animal cells. Eur. J. Biochem. 203, 9–23 (1992)

    Article  CAS  PubMed  Google Scholar 

  2. Löwe, J. et al. Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution. Science 268, 533–539 (1995)

    Article  ADS  PubMed  Google Scholar 

  3. Glickman, M. H. et al. A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and eIF3. Cell 94, 615–623 (1998)

    Article  CAS  PubMed  Google Scholar 

  4. Elsasser, S. & Finley, D. Delivery of ubiquitinated substrates to protein-unfolding machines. Nat. Cell Biol. 7, 742–749 (2005)

    Article  CAS  PubMed  Google Scholar 

  5. Kerscher, O., Felberbaum, R. & Hochstrasser, M. Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu. Rev. Cell Dev. Biol. 22, 159–180 (2006)

    Article  CAS  PubMed  Google Scholar 

  6. Finley, D. Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu. Rev. Biochem. 78, 477–513 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Burns, K. E., Liu, W. T., Boshoff, H. I., Dorrestein, P. C. & Barry, C. E., III . Proteasomal protein degradation in Mycobacteria is dependent upon a prokaryotic ubiquitin-like protein. J. Biol. Chem. 284, 3069–3075 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Pearce, M. J., Mintseris, J., Ferreyra, J., Gygi, S. P. & Darwin, K. H. Ubiquitin-like protein involved in the proteasome pathway of Mycobacterium tuberculosis. Science 322, 1104–1107 (2008)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Battesti, A. & Gottesman, S. Roles of adaptor proteins in regulation of bacterial proteolysis. Curr. Opin. Microbiol. 16, 140–147 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Flynn, J. M., Neher, S. B., Kim, Y. I., Sauer, R. T. & Baker, T. A. Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals. Mol. Cell 11, 671–683 (2003)

    Article  CAS  PubMed  Google Scholar 

  11. Sauer, R. T. & Baker, T. A. AAA+ proteases: ATP-fueled machines of protein destruction. Annu. Rev. Biochem. 80, 587–612 (2011)

    Article  CAS  PubMed  Google Scholar 

  12. Keiler, K. C., Waller, P. R. & Sauer, R. T. Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science 271, 990–993 (1996)

    Article  ADS  CAS  PubMed  Google Scholar 

  13. Fuhrmann, J. et al. McsB is a protein arginine kinase that phosphorylates and inhibits the heat-shock regulator CtsR. Science 324, 1323–1327 (2009)

    Article  ADS  CAS  PubMed  Google Scholar 

  14. Krüger, E., Zühlke, D., Witt, E., Ludwig, H. & Hecker, M. Clp-mediated proteolysis in Gram-positive bacteria is autoregulated by the stability of a repressor. EMBO J. 20, 852–863 (2001)

    Article  PubMed  PubMed Central  Google Scholar 

  15. Schmidt, A. et al. Quantitative phosphoproteomics reveals the role of protein arginine phosphorylation in the bacterial stress response. Mol. Cell. Proteomics 13, 537–550 (2014)

    Article  CAS  PubMed  Google Scholar 

  16. Derré, I., Rapoport, G. & Msadek, T. CtsR, a novel regulator of stress and heat shock response, controls clp and molecular chaperone gene expression in gram-positive bacteria. Mol. Microbiol. 31, 117–131 (1999)

    Article  PubMed  Google Scholar 

  17. Kirstein, J., Dougan, D. A., Gerth, U., Hecker, M. & Turgay, K. The tyrosine kinase McsB is a regulated adaptor protein for ClpCP. EMBO J. 26, 2061–2070 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Elsholz, A. K., Michalik, S., Zühlke, D., Hecker, M. & Gerth, U. CtsR, the Gram-positive master regulator of protein quality control, feels the heat. EMBO J. 29, 3621–3629 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Elsholz, A. K. et al. Global impact of protein arginine phosphorylation on the physiology of Bacillus subtilis. Proc. Natl Acad. Sci. USA 109, 7451–7456 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  20. Boisvert, F. M., Chénard, C. A. & Richard, S. Protein interfaces in signaling regulated by arginine methylation. Sci. STKE 2005, re2 (2005)

    PubMed  Google Scholar 

  21. Feng, J. et al. Trapping and proteomic identification of cellular substrates of the ClpP protease in Staphylococcus aureus. J. Proteome Res. 12, 547–558 (2013)

    Article  CAS  PubMed  Google Scholar 

  22. Miethke, M., Hecker, M. & Gerth, U. Involvement of Bacillus subtilis ClpE in CtsR degradation and protein quality control. J. Bacteriol. 188, 4610–4619 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Derré, I., Rapoport, G. & Msadek, T. The CtsR regulator of stress response is active as a dimer and specifically degraded in vivo at 37 degrees C. Mol. Microbiol. 38, 335–347 (2000)

    Article  PubMed  Google Scholar 

  24. Fuhrmann, J. et al. Structural basis for recognizing phosphoarginine and evolving residue-specific protein phosphatases in Gram-positive bacteria. Cell Reports 3, 1832–1839 (2013)

    Article  CAS  PubMed  Google Scholar 

  25. Trentini, D. B., Fuhrmann, J., Mechtler, K. & Clausen, T. Chasing phosphoarginine proteins: development of a selective enrichment method using a phosphatase trap. Mol. Cell. Proteomics 13, 1953–1964 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Schlothauer, T., Mogk, A., Dougan, D. A., Bukau, B. & Turgay, K. MecA, an adaptor protein necessary for ClpC chaperone activity. Proc. Natl Acad. Sci. USA 100, 2306–2311 (2003)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kirstein, J., Zühlke, D., Gerth, U., Turgay, K. & Hecker, M. A tyrosine kinase and its activator control the activity of the CtsR heat shock repressor in B. subtilis. EMBO J. 24, 3435–3445 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kojetin, D. J. et al. Structural and motional contributions of the Bacillus subtilis ClpC N-domain to adaptor protein interactions. J. Mol. Biol. 387, 639–652 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Wang, F. et al. Structure and mechanism of the hexameric MecA-ClpC molecular machine. Nature 471, 331–335 (2011)

    Article  ADS  CAS  PubMed  Google Scholar 

  30. Woo, K. M., Kim, K. I., Goldberg, A. L., Ha, D. B. & Chung, C. H. The heat-shock protein ClpB in Escherichia coli is a protein-activated ATPase. J. Biol. Chem. 267, 20429–20434 (1992)

    Article  CAS  PubMed  Google Scholar 

  31. Hagai, T. & Levy, Y. Ubiquitin not only serves as a tag but also assists degradation by inducing protein unfolding. Proc. Natl Acad. Sci. USA 107, 2001–2006 (2010)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kirstein, J. et al. Adaptor protein controlled oligomerization activates the AAA+ protein ClpC. EMBO J. 25, 1481–1491 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Stannek, L., Gunka, K., Care, R. A., Gerth, U. & Commichau, F. M. Factors that mediate and prevent degradation of the inactive and unstable GudB protein in Bacillus subtilis. Front. Microbiol. 5, 758 (2015)

    Article  PubMed  PubMed Central  Google Scholar 

  34. Chan, P., Curtis, R. A. & Warwicker, J. Soluble expression of proteins correlates with a lack of positively-charged surface. Sci. Rep. 3, 3333 (2013)

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  35. Nguyen, H. D. et al. Construction of plasmid-based expression vectors for Bacillus subtilis exhibiting full structural stability. Plasmid 54, 241–248 (2005)

    Article  CAS  PubMed  Google Scholar 

  36. Gerth, U. et al. Fine-tuning in regulation of Clp protein content in Bacillus subtilis. J. Bacteriol. 186, 179–191 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Msadek, T. et al. ClpP of Bacillus subtilis is required for competence development, motility, degradative enzyme synthesis, growth at high temperature and sporulation. Mol. Microbiol. 27, 899–914 (1998)

    Article  CAS  PubMed  Google Scholar 

  38. Wis´niewski, J. R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–362 (2009)

    Article  CAS  Google Scholar 

  39. Käll, L., Canterbury, J. D., Weston, J., Noble, W. S. & MacCoss, M. J. Semi-supervised learning for peptide identification from shotgun proteomics datasets. Nat. Methods 4, 923–925 (2007)

    Article  PubMed  CAS  Google Scholar 

  40. Taus, T. et al. Universal and confident phosphorylation site localization using phosphoRS. J. Proteome Res. 10, 5354–5362 (2011)

    Article  CAS  PubMed  Google Scholar 

  41. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008)

    Article  CAS  PubMed  Google Scholar 

  42. Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011)

    Article  ADS  CAS  PubMed  Google Scholar 

  43. Cox, J. et al. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell. Proteomics 13, 2513–2526 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Vizcaíno, J. A. et al. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013. Nucleic Acids Res. 41, D1063–D1069 (2013)

    Article  PubMed  CAS  Google Scholar 

  45. Suzuki, Y., Takeda, Y. & Ikuta, T. Immunoblotting conditions for human hemoglobin chains. Anal. Biochem. 378, 218–220 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Nørby, J. G. Coupled assay of Na+,K+-ATPase activity. Methods Enzymol. 156, 116–119 (1988)

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Brünger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  PubMed  Google Scholar 

  51. Terwilliger, T. C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr. D 64, 61–69 (2008)

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Debreczeni, J. E. & Emsley, P. Handling ligands with Coot. Acta Crystallogr. D 68, 425–430 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Moriarty, N. W., Grosse-Kunstleve, R. W. & Adams, P. D. electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation. Acta Crystallogr. D 65, 1074–1080 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Schrodinger, LLC. The PyMOL Molecular Graphics System v.1.3r1 (2010)

  58. Lee, B. G. et al. Structures of ClpP in complex with acyldepsipeptide antibiotics reveal its activation mechanism. Nat. Struct. Mol. Biol. 17, 471–478 (2010)

    Article  CAS  PubMed  Google Scholar 

  59. Waksman, G., Shoelson, S. E., Pant, N., Cowburn, D. & Kuriyan, J. Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms. Cell 72, 779–790 (1993)

    Article  CAS  PubMed  Google Scholar 

  60. Rittinger, K. et al. Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding. Mol. Cell 4, 153–166 (1999)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank E. Charpentier for providing B. subtilis strains and plasmids, S. Spiess for advice on B. subtilis genetic manipulation, G. Dürnberger for assistance with statistical analysis, A. Schmidt for help with mass spectrometry interpretation, R. Beveridge for performing native mass spectrometry analysis, J. Kley for help with ITC experiments and G. Bourenkov and T. Schneider at DESY for assistance during data collection. This work was supported by the Austrian Research Promotion Agency (FFG). The IMP is funded by Boehringer Ingelheim.

Author information

Authors and Affiliations

Authors

Contributions

D.B.T. performed mass spectrometry and biochemical experiments; M.J.S. performed the structural analysis and biochemical experiments; A.H. and R.K. performed in vivo complementation assays and biochemical experiments; L.D. assisted with biochemical experiments; T.C. designed the study with expert assistance from K.M. for the mass spectrometry part; D.B.T., M.J.S. and T.C. wrote the manuscript, with contributions from all authors.

Corresponding author

Correspondence to Tim Clausen.

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The authors declare no competing financial interests.

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Nature thanks S. Gygi, Y. Shi and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Development and validation of the ClpPX-TRAP mutant.

a, Co-NTA purification of wild-type and TRAP mutant ClpP(6His) expressed in heat-stressed B. subtilis wild-type cells. SDS–PAGE (left) reveals co-purification of two ClpP protein species. MALDI–MS analysis (bottom) estimates that the mass difference between the two ClpP species was 1,036 and 1,042 Da for wild-type and TRAP ClpP, respectively. These values fit to the expected mass of the 6His tag (1,065 Da), indicating that the purified proteins correspond to recombinant (tagged) and endogenous ClpP. Native-PAGE (right) shows that the two proteins are present predominantly as a composite, heptameric complex. Therefore, ClpP(6His)TRAP expression in B. subtilis under heat-stress conditions is complicated by the formation of mixed complexes with endogenous (active) ClpP (cartoon). As the untagged endogenous ClpP was observed at similar levels as ClpP(6His), it is unlikely that efficient substrate-trapping complexes—built up exclusively by inactive ClpPTRAP—are formed in vivo. b, Engineering of the ClpP cross mutant. Crystal structure of the ClpP heptamer (PDB code 3KTI; ref. 58) shown in top (left) and side (right) view. Zoomed-in picture shows interaction between Arg142 and Glu119 of two neighbouring subunits. To avoid the interaction of the recombinant ClpP(6His) trapping mutant with endogenous ClpP, these two residues were inter-exchanged (Glu119Arg/Arg142Glu), thus leading to an electrostatic repulsion between the cross-mutant ClpP(6His)X-TRAP and wild-type ClpP, while allowing formation of the respective homo-heptamers, as schematically indicated. c, Co-NTA purification of ClpP(6His)X and ClpP(6His)X-TRAP expressed in heat-stressed B. subtilis wild-type cells. SDS–PAGE (left) shows that the X variants do not co-purify with endogenous ClpP, demonstrating that the Glu119Arg/Arg142Glu mutation prevents the formation of hetero-oligomers (see cartoon). Native-PAGE analysis (middle) suggests that the ClpP(6His)X protein has a reduced heptamerization propensity. However, the inactive version (Ser98Ala, ‘TRAP’) of the ClpP(6His)X mutant is present predominantly as a heptameric complex, probably owing to the stabilization by trapped substrates. ClpP(6His)X-TRAP thus represents a tool to trap substrates in the wild-type background of B. subtilis. Our experimental approach has the advantage of avoiding the use of the ΔclpP B. subtilis strain, which has an extremely pleiotropic phenotype (including increased levels of McsB and McsA14) that would largely bias the characterization of ClpP substrates. The ClpPX protein represents the ideal negative control: it has reduced ability to heptamerize and therefore to degrade proteins, and its overexpression is expected to have little effect on the overall levels of protein degradation and consequently on the abundance of substrate proteins.

Extended Data Figure 2 In vivo characterization of ClpPX-TRAP variants used in pull-down experiments.

a, SDS–PAGE (left) and native-PAGE (right) analysis of co-NTA purifications of experiment 1: pull-down of His-tagged ClpP variants in wild-type B. subtilis. Numbers denote biological replicates. Each sample represents approximately 10% of the total purification. b, SDS–PAGE (top) and native-PAGE (bottom) analysis of co-NTA purifications of experiment 2: pull-down of His-tagged ClpP variants in the wild-type (left) and ΔclpC (right) B. subtilis strain. Each sample represents approximately 5% of the total purification.

Extended Data Figure 3 ITC binding data.

For each binding study, the analysed interactions are indicated on the top, and the respective Kd values, when detected, are shown below. For reference, a structural alignment of the ClpC (grey) and ClpA (purple; PDB code 1K6K) NTDs, showing a high degree of structural similarity, is presented.

Extended Data Figure 4 Preparation and validation of caseinpArg as a model substrate of ClpCP.

a, Top, size exclusion chromatography (SEC) separation of caseinpArg from the B. subtilis McsBA complex after in vitro phosphorylation. The red markings indicate the fractions that were pooled. Bottom, SDS–PAGE analysis of the fractions indicates that the SEC procedure could separate at least 95% of the McsB protein from the pooled β-casein fractions. b, Evaluating the effect of the McsB contamination on the degradation of caseinpArg. Top, ClpCP in vitro degradation assay towards untreated β-casein. Middle, ClpCP in vitro degradation of caseinpArg, with and without additional McsB kinase. Bottom, addition of either inactive or active McsB did not have an effect on the initial degradation rate of caseinpArg. Of note, the degradation of untreated casein in the presence of McsBA is slightly delayed compared to the degradation of pre-modified caseinpArg. c, An alternative, improved protocol for purifying caseinpArg. After in vitro phosphorylation of β-casein by G. stearothermophilus McsB(6His), a Ni-NTA column was used to capture the tagged kinase. The flow-through fraction was then applied to a SEC column to further separate remaining McsB from β-casein. Two different fractions of the β-casein peak were collected, the later-eluting one (yellow) having a higher degree of purity in relation to the earlier one (orange). d, Activation of the ClpC ATPase activity by the different caseinpArg preparations (2 μM concentration each). Each fraction contains a different (substoichiometric) amount of the McsB contamination. ATPase measurements reveal an almost identical activation of ClpC for all fractions, indicating that the residual amounts of McsB present in the caseinpArg sample do not contribute to ClpC activation. Error bars denote standard deviation of technical triplicates. e, To obtain substrate samples varying in the amount of arginine phosphorylation, casein was pre-incubated with McsBA for increasing times. The YwlE arginine phosphatase was added to the sample phosphorylated most strongly (120 min incubation with McsBA) as a negative control. The resultant caseinpArg samples were subjected to pArg immunoblots using a pArg-specific antibody24 (right, top) and to ClpCP degradation assays (right, bottom).

Extended Data Figure 5 Binding pockets of pArg, pTyr and pSer/Thr.

The binding pockets are shown as surface representation and coloured according to their electrostatics as calculated with PyMol (blue: positive, red: negative). Bound phosphoamino acids are presented as sticks with nitrogens and oxygens coloured blue and red, respectively. a, b, pArg-binding sites 1 and 2, respectively, of the ClpCNTD domain. The sites are characterized by a ‘bipolar’ architecture with both a positive and a negative area, jointly required to recognize a pArg side chain. c, d, pTyr-binding site of the Src SH2 domain (PDB code 1SPS; ref. 59) and pSer/Thr-binding site of the 14-3-3 domain (PDB code 1QJB; ref. 60). Both pTyr and pSer were part of a peptide but are shown in isolation for clarity. In contrast to the pArg-binding site, pTyr- and pSer/Thr-specific pockets are uniformly positively charged.

Extended Data Figure 6 Sequence alignment of the pArg-binding site of ClpC from different species and of the homologous regions of other Clp ATPases.

The two symmetrical regions (comprising residues 6–68 and 80–142, approximately) of each protein are aligned. Residues interacting with the pArg molecule (the Glu residue binding to the guanidinium group and the Arg/Thr residues interacting with the phosphate) are circled in black and marked by an arrow. Each of the two pArg-binding sites comprises Glu and Thr from one symmetrical region and Arg and Thr from the other. The alignment shows high conservation of the critical residues of ClpC proteins from different McsB-containing bacteria (B. subtilis, Listeria monocytogenes, Staphylococcus aureus, Bacillus anthracis and Peptoclostridium difficile). Conversely, the residues are not conserved in related Clp proteins (ClpA and ClpB) from McsB-deficient, Gram-negative bacteria.

Extended Data Figure 7 Intact mass analysis of McsB-treated and untreated (control) β-casein.

a, Unprocessed MS spectra. Zoomed view of the 13+ charge state species shows two predominant arginine phosphorylation states in the McsB-treated sample (top): 1 phopshorylation (diamond, m/z 1,852) and 2 phosphorylations (triangle, m/z 1,852), while only the non-pArg form (circle, m/z 1,848) can be visualized in the untreated control (bottom). b, Deconvoluted MS spectra, showing the average proportion of unmodified (circle, 23,982 Da), 1 pArg-containing (diamond, 24,063 Da) and 2 pArg-containing (triange, 24,142 Da) β-casein.

Extended Data Table 1 Summary of identified phospho-sites in various ClpP-trapping experiments
Extended Data Table 2 Data collection and refinement statistics
Extended Data Table 3 Summary of protein expression and purification conditions

Supplementary information

Supplementary Information

The file contains the Supplementary Discussion, Supplementary References and Supplementary Figures (Supplementary Figure 1: selected MS-MS spectra of identified pArg peptides; Supplementary Figure 2: uncropped images of the SDS-PAGE gels). (PDF 6908 kb)

Supplementary Table 1

Quantitative MS analysis of ClpP trapping pull-downs. MaxQuant protein identification, label free quantification (LFQ) and statistical analysis (ClpPX vs. ClpPX-TRAP comparison) results are reported. Results for Experiment 1 (B. subtilis wt), Experiment 2 B. subtilis wt, and Experiment 2 B. subtilis ΔclpC are separated in different tabs. (XLSX 4484 kb)

Supplementary Table 2

Phosphorylation analysis of ClpP trapping pull-downs. Mascot identification of phosphopeptides with respective Percolator false discovery rate analysis and PhosphoRS evaluation of phosphosite localization are reported. Unmodified peptides are not shown. (XLSX 93 kb)

Supplementary Table 3

Phosphoproteomic analysis of total cell extracts from ΔclpC B. subtilis. Mascot identification of phosphopeptides with respective Percolator false discovery rate analysis and PhosphoRS evaluation of phosphosite localization are reported. Unmodified peptides are not shown. (XLSX 6747 kb)

Supplementary Table 4

Phosphoproteomic analysis of total cell extracts and protein aggregates from ΔclpP B. subtilis. Mascot identification of phosphopeptides with respective Percolator false discovery rate analysis and PhosphoRS evaluation of phosphosite localization are reported. Unmodified peptides are not shown. (XLSX 223 kb)

Supplementary Table 5

Phosphoproteomic analysis of protein aggregates extracted from heat-shocked B. subtilis wt. Mascot identification of phosphopeptides with respective Percolator false discovery rate analysis and PhosphoRS evaluation of phosphosite localization are reported. Unmodified peptides are not shown. (XLSX 6374 kb)

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Trentini, D., Suskiewicz, M., Heuck, A. et al. Arginine phosphorylation marks proteins for degradation by a Clp protease. Nature 539, 48–53 (2016). https://doi.org/10.1038/nature20122

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