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Arginine phosphorylation marks proteins for degradation by a Clp protease

Nature volume 539, pages 48–53 (2016)Cite this article

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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.

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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.

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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

Author notes

  1. Débora Broch Trentini and Marcin Józef Suskiewicz: These authors contributed equally to this work.

Authors and Affiliations

  1. Research Institute of Molecular Pathology (IMP), Dr-Bohr-Gasse 7, Vienna, 1030, Austria

    Débora Broch Trentini, Marcin Józef Suskiewicz, Alexander Heuck, Robert Kurzbauer, Luiza Deszcz, Karl Mechtler & Tim Clausen

  2. Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Dr-Bohr-Gasse 3, Vienna, 1030, Austria

    Karl Mechtler

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  1. Débora Broch Trentini
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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.

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Correspondence to Tim Clausen.

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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
Full size table
Extended Data Table 2 Data collection and refinement statistics
Full size table
Extended Data Table 3 Summary of protein expression and purification conditions
Full size table

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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