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. 2016 Nov 3;539(7627):48-53.
doi: 10.1038/nature20122. Epub 2016 Oct 6.

Arginine Phosphorylation Marks Proteins for Degradation by a Clp Protease

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Arginine Phosphorylation Marks Proteins for Degradation by a Clp Protease

Débora Broch Trentini et al. Nature. .
Free PMC article

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.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Extended Data Figure 1
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 (as shown in the 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 McsA) 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
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
Extended Data Figure 3. 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 antibody (right, top) and to ClpCP degradation assays (right, bottom).
Extended Data Figure 4
Extended Data Figure 4. 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
Extended Data Figure 4. 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 5
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
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
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.
Figure 1
Figure 1. Pull-down of ClpP trapping mutants.
a, Cartoon illustrating the experimental workflow. As indicated, all pull-down experiments were done in triplicates. LC–MS/MS, liquid chromatography tandem mass spectrometry. b, Volcano plot illustrating proteins identified in ClpPX (control) and ClpPX-TRAP pull-downs after expression in a B. subtilis wild-type (WT) strain. Proteins were considered as ClpP substrates (shaded area) when the X-TRAP/control relative protein intensity (x axis) was >2 and the corresponding limma P value (y axis) was <0.05. Phosphorylated proteins are shown as filled squares (red: pArg, blue: pSer/Thr/Tyr). In a few cases, the phosphorylated residue could not be unambiguously localized. As the same phosphopeptides have been observed to contain a pArg in previous experiments, they are labelled as probable pArg (open red squares). Identified pArg proteins are listed on the left. c, Volcano plots of the ClpP pull-downs performed in B. subtilis wild-type and ΔclpC strains in parallel. For comparison, pArg proteins identified in the B. subtilis wild-type pull-downs are marked in yellow/orange in the ΔclpC plot.
Figure 2
Figure 2. Effect of McsB on the activity of ClpCP in vitro.
a, ClpCP-mediated degradation of β-casein in the presence of different effector proteins. Here and in the following, a quantification of the β-casein band is presented (original SDS–PAGE gels in Supplementary Fig. 2). Bottom panel shows the kinase activity of assayed McsB variants in an autoradiography plot. b, In contrast to active McsB, the inactive McsBE212A kinase cannot stimulate casein degradation by ClpCP. c, Effect of the YwlE arginine phosphatase on ClpCP activation by McsBA. The inactive YwlED118N mutant was used as a negative control.
Figure 3
Figure 3. Binding of pArgAA to ClpC.
a, b, Inhibitory effect of pArgAA on McsBA-activated (a) and MecA-activated (b) ClpCP. c, Pull-down experiment monitoring the interaction of the NTD (wild type and E32A/E106A mutant) with MecA (10 μM each) in the presence pArgAA. d, ITC profile of pArgAA binding to full-length double Walker B mutant ClpC (ClpCDWB(FL); left) and the NTD of ClpC (ClpCNTD(1–150); right). Determined Kd values are indicated.
Figure 4
Figure 4. ClpCP protease activity towards a pArg-containing substrate protein.
a, Preparation of the caseinpArg model substrate. SEC, size exclusion chromatography. b, Binding of caseinpArg (35 μM) to ClpC NTD (10 μM) at increasing amounts of pArgAA. c, Degradation of caseinpArg by ClpCP without adaptor proteins and the inhibitory effect of pArgAA on this activity. d, Pull-down experiment monitoring ClpCP complex formation in the presence of MecA, casein and caseinpArg. e, Preparation of substrate samples that contain increasing amounts of caseinpArg after prolonged incubation with McsBA. ClpCP degradation of resultant caseinpArg samples is directly correlated to the degree of substrate phosphorylation seen in pArg immunoblots (Extended Data Fig. 3e). AU, arbitrary units.
Figure 5
Figure 5. Crystal structure of the ClpC NTD in complex with pArg.
a, Overall structure of the ClpC NTD domain bound to two pArgAA molecules. The cartoon representation is coloured in light grey (residues 70–148) and dark grey (4–69) to highlight the two symmetrical halves of the NTD. b, c, Zoomed view of pArg-binding sites 1 (b) and 2 (c), with labelled interacting residues. The 2FoFc omit electron densities of the pArgAA ligands, calculated at 1.6 Å resolution, are contoured at 1σ. d, Overlap of MecA- and pArg-binding sites. Shown is the hexameric organization of the ClpC1–485 (grey) complex with MecA121–218 (blue) (PDB code 3PXG; ref. 29) superimposed with pArgAA (orange). Bottom left, zoomed-in view shows two adjacent ClpC NTDs with pArgAA and MecA interactors. Bottom right, zoomed-in view illustrates that the pArg phosphoryl group and Glu184 (Glu198 in second binding site) of MecA compete for the same ClpC binding pocket. e, Scheme representing the distinct substrate (S) preferences of the wild-type ClpCP and ClpCPEA protease complexes. f, Degradation assays comparing the activity of wild-type ClpCP and ClpCPEA towards MecA-delivered (left) and pArg-labelled (middle, right) casein. YwlE was used as a control for the pArg-dependent degradation. g, ATPase activity of ClpC and ClpCEA in the presence of putative substrate proteins. Levels are normalized to the induced ATPase activity of the ClpC–caseinpArg complex. Error bars show the s.d. of three independent experiments.
Figure 6
Figure 6. The pArg–ClpCP degradation system.
a, Thermotolerance assay analysing the in vivo complementation of ΔclpC by expressing ClpC and ClpCEA. Levels are normalized to the values before heat shock (time 0), and numbers above bars represent the fraction of cells surviving after 2 h heat shock. CFU, colony-forming units. Error bars show the s.d. of three independent experiments. b, The pArg–ClpCP system. Left, cartoon representation shows that after phosphorylation by the McsB arginine kinase, pArg-tagged proteins are targeted to the ClpCP protease. Binding of pArg proteins to one of the 12 NTD binding pockets stimulates the ATPase activity of ClpC, leading to the translocation of the captured substrate into the ClpP protease cage and to protein degradation. Right, a model of the respective ClpCP complex (ClpC NTD in light grey, ClpC AAA1/2 in white, ClpP in dark grey, substrate in black).

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