Dóra
Balogh‡
a,
Maria
Dahmen‡
a,
Matthias
Stahl
a,
Marcin
Poreba
b,
Malte
Gersch§
a,
Marcin
Drag
b and
Stephan A.
Sieber
*a
aCenter for Integrated Protein Science at the Department of Chemistry, Technische Universität München, Lichtenbergstraße 4, Garching bei München, D-85747, Germany. E-mail: stephan.sieber@mytum.de
bDepartment of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland
First published on 28th October 2016
Caseinolytic proteases (ClpP) are important for recognition and controlled degradation of damaged proteins. While the majority of bacterial organisms utilize only a single ClpP, Listeria monocytogenes expresses two isoforms (LmClpP1 and LmClpP2). LmClpPs assemble into either a LmClpP2 homocomplex or a LmClpP1/2 heterooligomeric complex. The heterocomplex in association with the chaperone ClpX, exhibits a boost in proteolytic activity for unknown reasons. Here, we use a combined chemical and biochemical strategy to unravel two activation principles of LmClpPs. First, determination of apparent affinity constants revealed a 7-fold elevated binding affinity between the LmClpP1/2 heterocomplex and ClpX, compared to homooligomeric LmClpP2. This tighter interaction favors the formation of the proteolytically active complex between LmClpX and LmClpP1/2 and thereby accelerating the overall turnover. Second, screening a diverse library of fluorescent labeled peptides and proteins with various ClpP mutants allowed the individual analysis of substrate preferences for both isoforms within the heterocomplex. In addition to Leu and Met, LmClpP2 preferred a long aliphatic chain (2-Aoc) in the P1 position for cleavage. Strikingly, design and synthesis of a corresponding 2-Aoc chloromethyl ketone inhibitor resulted in stimulation of proteolysis by 160% when LmClpP2 was partially alkylated on 20% of the active sites. Determination of apparent affinity constants also revealed an elevated complex stability between partially modified LmClpP2 and the cognate chaperone LmClpX. Thus, the stimulation of proteolysis through enhanced binding to the chaperone seems to be a characteristic feature of LmClpPs.
Interestingly, some bacterial strains such as Mycobacterium tuberculosis and Listeria monocytogenes encode two ClpP isoforms (ClpP1 and ClpP2).14–18 While L. monocytogenes ClpP2 (LmClpP2) resembles related enzymes in other bacteria such as Escherichia coli and Staphylococcus aureus, LmClpP1 shares only 44% sequence identity with E. coli ClpP, forms predominantly inactive heptamers, lacks a conserved N-terminal chaperone binding motif and exhibits a truncated catalytic triad in which Asp172 is replaced by an Asn residue.15 Mutational studies and in-depth X-ray crystallographic analysis revealed that this Asn residue is responsible for a conformational selection of the inactive heptameric state.15 Accordingly, mutation of this Asn to an Asp induced tetradecamer formation and increased catalytic activity of LmClpP1.15
Negative stain EM images of mixed LmClpP1 and LmClpP2 indicated the formation of heterocomplexes composed of two homoheptameric rings.14 Importantly, it was found that LmClpP1 is only active when complexed with LmClpP2, which forces LmClpP1 into an active conformational state.15 The molecular reason for this surprising finding could be explained by a crystal structure of the LmClpP1/2 heterocomplex.16 All active sites within the heterocomplex, including Asn of LmClpP1, were aligned in an active conformation, which demonstrated that heterocomplex formation regulates LmClpP1 activity. While the heterocomplex was less active in peptidase assays, a 10-fold increase in proteolytic activity over the homocomplex was observed when in the presence of LmClpX. From a functional perspective, this implies that the cell produces a hyperactive enzyme during stress conditions, when misfolded proteins must be removed efficiently. Indeed, quantitative real-time PCR showed increased clpP1 and clpP2 expression under heat stress.16 So far, no systematic analysis of LmClpP1 and LmClpP2 peptidolytic and proteolytic cleavage specificities has been performed, leaving their role in substrate recognition unresolved. Structural studies and beta-lactone inhibitor screenings showed subtle differences in the P1 binding pockets and revealed a preference of LmClpP2 for medium to long aliphatic and aromatic side chains. LmClpP1, on the other hand, did not bind inhibitors specifically and only bound the natural product vibralactone.14,15
Interestingly, ClpP1 and ClpP2 from M. tuberculosis (MtClpP1 and MtClpP2) also assemble into a similar heterocomplex.17–19 However, many mechanistic and functional differences compared to the L. monocytogenes heterocomplex have been reported. For example, MtClpP1 and MtClpP2 are both tetradecamers, which show only weak proteolytic activity on their own in the presence of chaperones.17 Peptidolytic activity is only enabled through MtClpP1/2 heterocomplex formation and requires the presence of activating peptides.17,19,20 Proteolytic substrate turnover is facilitated in association with the chaperones MtClpX or MtClpC1.17,21 Moreover, peptide substrate screenings with MtClpP1 revealed a cleavage preference after Met, Leu, Phe and Ala residues, while MtClpP2 was largely inactive.22 Although structural information remains elusive, mutational studies within the hydrophobic clefts of MtClpPs showed that chaperones only bind the heterocomplex via MtClpP2.23 The stoichiometry of the L. monocytogenes complexes remains unknown. However, as tetradecameric LmClpP1(N172D) is inactive in proteolysis assays with LmClpX, the LmClpP1/2 heterocomplex does not seem to bind LmClpX via the ClpP1 ring.16
Here, we utilize peptide libraries to identify LmClpP1 and LmClpP2 cleavage specificities. While these preferences were largely abrogated in the proteolytic complexes, both isoforms retained a certain degree of individual specificity in protein digests. Biochemical analyses were used to dissect the multiple steps of proteolysis and revealed that the elevated activity of the heterocomplex stems from a 7-fold increased binding affinity for the LmClpX chaperone. Surprisingly, stimulation of proteolysis was also observed when a customized chloromethyl ketone-based (CMK) inhibitor partially modified the active sites of homotetradecameric LmClpP2. A closer mechanistic inspection of this inhibition mode revealed an increased affinity for the chaperone LmClpX to be the fundamental activation principle.
Fig. 1 Peptide library screenings. (a) Principle of ClpP peptidase assay: after cleavage of an ACC-tagged tripeptide by ClpP an increase in fluorescence is measured (please refer to ESI† for the structures of the R1, R2 and R3 residues). (b) Cleavage specificity of LmClpP variants in peptidase assays represented as a heatmap. Each row is normalized to the lowest (0) and to the highest (1) activity. Substrates in gray and peptides containing Cys could not be obtained. Please refer to the ESI† for the structures of the substrates, for the nomenclature of non-natural amino acids and for the absolute values and errors of the peptidase activity. Peptidase activities were measured in triplicates. |
Comparison of P2 and P3 libraries revealed a less stringent specificity and identified some additional preferences and similarities of cleavage sites (Fig. 1a). For example, homoarginine (hArg) was well tolerated at the P2 position and 3,4-dichlorophenylalanine at the P3 position by both LmClpP variants. D-Amino acids were not processed, suggesting a high degree of stereochemical discrimination by LmClpP. For example, Leu at the P1 position was readily turned over in its L-configuration, but the corresponding D-stereoisomer could not be processed by any of the constructs. As the substrate specificity extends beyond the scope of natural amino acids, it is possible that chaperone-independent hydrolysis of peptidic, cellular metabolites could be an additional function of LmClpP1/2.
Fig. 2 Cleavage specificity in a protease assay. (a) Principle of ClpP protease assay: SsrA-tagged substrate proteins are digested by the ClpXP complex and the produced peptides are analyzed by MS/MS. (b) Cleavage specificity of LmClpP variants in protease assays represented as a heatmap. ClpXP digests of substrate proteins were analyzed by Protein|Clpper.25 log2(S) > 0 means that cleavage occurred more often than expected from the amino acid composition of the substrate proteins, log2(S) < 0 represents less frequent cleavage than random cleavage. Data represent two independent experiments with five different substrate proteins. |
The peptidolytic activity of LmClpP2, LmClpP1wt/2wt, LmClpP1wt/2m, LmClpP1m/2wt and LmClpP1(N172D) was measured by the hydrolysis of Ac-Ala-hArg-Leu-ACC, a substrate that showed optimal turnover by both isomers. Inhibitors were used in three concentrations, 100, 10 and 1 μM to estimate their potency (Fig. 3b and S2†). As all complexes deviate in their peptidolytic rates, substrate turnover in the absence of inhibitor was normalized to 100% activity. AV170 and both CMK inhibitors were the most effective against LmClpP2, followed by D3 and E2. A similar profile was observed for heterooligomeric LmClpP1wt/2wt and LmClpP1m/2wt, suggesting that inhibition of LmClpP2 alone is sufficient to affect the overall complex activity. In contrast, the inhibition pattern for LmClpP1 in LmClpP1wt/2m and LmClpP1(N172D) complexes were quite different. Here, only the CMKs showed any inhibitory effect, and Leu-CMK exhibited significantly higher potency compared to the 2-Aoc analog. Leu-CMK completely abolished LmClpP1 activity in the heterocomplex at 1 μM, reflecting its preference for small residues at the P1 site. With the first potent LmClpP1 inhibitors in hand, we commenced with mechanistic studies of LmClpXP proteolysis.
Fig. 4 Activation of the ClpXP proteolysis by small molecules. (a) Protease activity of LmClpP variants with Aoc-CMK and Leu-CMK. Pie diagrams illustrate the degree of modification of LmClpP2 with Aoc-CMK, determined by intact protein mass spectrometry (means from triplicate experiments are shown). (b) Protease activity of the LmClpP2 homocomplex with lactones D3 and E2. (c) ATPase activity of LmClpX6 (0.2 μM) in the presence of LmClpP214 (0.1 μM) and Aoc-CMK (4.25 μM) with 20 mM ATP. Data represent three independent experiments which were measured in quadruplicate (mean ± sd). (d) Kinetic scheme for protein degradation by ClpXP (parts of this figure have been adapted from Kim et al.27). (e) Protease activity of LmClpP (varying concentrations of LmClpP14) with and without Aoc-CMK (in 25-fold excess to LmClpP214). Curves were fit to the Hill equation (y = Vmin + (Vmax − Vmin)xn/(Kappn + xn)). Protease activity data represent at least two independent experiments which were measured in triplicate (mean ± sd). |
Importantly, partial binding alone cannot explain the proteolytic enhancement since aromatic lactone E2 inhibited LmClpXP2 without significant activation, while D3 showed 41% activation (Fig. 4b). In addition, all three heterocomplex constructs revealed only marginal to no enhancement of turnover with Aoc-CMK, suggesting that homotetradecameric LmClpP2 is required to trigger this effect. Similarly, Leu-CMK stimulated LmClpXP2 to a much lesser extent than Aoc-CMK, highlighting that both the intrinsic reactivity as well as the compound structure are crucial for the activity. We thus commenced with in-depth analysis of the mechanistic requirements responsible for these effects.
First, we focused on the homocomplex and addressed the nature of its activation by studying its conformational stability. Select lactones, including E2 and phenyl esters such as AV170, are known to destabilize tetradecameric ClpP and to induce its dissociation into inactive heptamers.10,11 In fact, deoligomerization of LmClpP2 could be observed upon E2 binding, while the tetradecameric complex was retained with both the CMKs and D3 (Fig. S4†). This demonstrates a principle difference in the binding mode, in which those molecules that disrupt the oligomeric state thereby inactivate LmClpP, while those that only partially modify LmClpP and retain the tetradecameric state stimulate LmClpP.
Second, as partial inhibition does not trigger peptidolysis but proteolysis, we investigated the role of the associated LmClpX chaperone. ClpX recognizes tagged substrates and catalyzes their ATP-dependent unfolding, which represents the rate-determining step of proteolysis.27 Thus, LmClpX ATPase activity was determined in the presence of partially Aoc-CMK-inhibited LmClpP2. No increase in ATP turnover could be observed, suggesting that ATP hydrolysis occurs independently of the association with Aoc-CMK-modified LmClpP (Fig. 4c).
Third, LmClpXP2 assembly is in equilibrium with individual LmClpP2 and LmClpX complexes. This equilibrium precedes all subsequent steps required for protein turnover, including ClpX-mediated substrate binding, unfolding and translocation into the proteolytic chamber (Fig. 4d). A shift in equilibrium towards the proteolytically-active LmClpXP2 complex could therefore significantly enhance overall activity. We thus elucidated the direct interaction between LmClpP2 and LmClpX in proteolytic assays, in which the concentration of LmClpP2 was systematically varied and an apparent affinity constant was calculated. Importantly, while the Kapp of LmClpXP2 with unmodified LmClpP2 was 443 nM, the Kapp for partially Aoc-CMK-modified LmClpP2 dropped to 162 nM. This increase in affinity through partial inhibitor binding to ClpP reveals an intriguing mechanism of activation (Fig. 4e). A similar enhancement in ClpX affinity was previously observed for E. coli ClpP fully inactivated by diisopropyl fluorophosphonate (DFP) which contributed to a model of functional communication between ClpX and ClpP during substrate processing.28 Here, we show that the mechanism of proteolytic stimulation extends beyond complete active site binding and is largely affected by the degree of modification, inhibitor structure as well as the ClpP isoform investigated.
Inspired by the results of partial Aoc-CMK inhibition, we focused on the interaction between LmClpP1/2 and LmClpX, and determined their affinity. Proteolytic assays were performed with varying LmClpP1/2 concentrations, and apparent affinity constants were calculated as outlined above (Fig. 5a). Importantly, the Kapp for LmClpXP1/2 was 85 nM and thus about 7-fold lower compared to the LmClpXP2 homocomplex. These results suggest a strong shift in the equilibrium of complex formation to the LmClpXP1/2 form, thereby enhancing substrate turnover.
To further prove this theory, we determined the Kapp for protein unfolding, which is the rate-limiting step before proteolysis.27 To focus on LmClpX activity, we utilized LmClpP2 and LmClpP1/2 containing Ser98 to Ala mutations. While catalytically inactive, these mutant proteins are still able to bind LmClpX and facilitate the translocation of the linear peptide chain into the barrel. Systematic variation of LmClpP concentrations revealed Kapp values of 2362 nM and 224 nM for homo- and heterocomplexes, respectively (Fig. 5b). In agreement with the proteolysis data, these results indicate that LmClpX and LmClpP1/2 form the functional complex with higher affinity at the rate-limiting, substrate-unfolding step, thereby enhancing the overall activity.
Although these studies provide a mechanistic basis for understanding the elevated proteolytic activity, the nature of this high-affinity interaction, as well as how the heterocomplex or partially-inhibited homocomplex facilitate tighter LmClpX binding, remain to be explored. No significant structural differences between the LmClpP2 homocomplex and heterocomplex with respect to LmClpP1 were observed.16 However, the N-terminal regions, which are crucial for chaperone interaction, could not be characterized with high resolution so far. In order to unravel more general aspects of heterocomplex assembly, analysis of its quaternary structural organization would be required.
We initiated our studies by inspecting both LmClpP isoforms and discovered that the LmClpP2 peptidase, similar to its S. aureus homolog,25 prefers substrates with a long aliphatic side chain at the P1 site. As ClpP exhibits hydrolytic activity of small substrates that access the proteolytic chamber by diffusion, it is possible that it may have specialized functions beyond the cleavage of proteins. Although the preference for aliphatic amino acids such as Leu and Met, similar to other ClpPs, was retained in proteolytic studies, the overall selectivity observed for peptidase activity was largely abrogated.22,25 However, a striking feature of LmClpP1 is its additional preference for Gln at the P1 site. An additional feature of LmClpP1/2 is the accelerated proteolytic rate when associated with LmClpX. We show that the reason for this second principle of ClpP activation is a higher affinity between LmClpP1/2 and LmClpX, which shifts the equilibrium to the active LmClpXP1/2 heterocomplex. As the apical sites of LmClpP1 and LmClpP2 do not significantly change in the homo- and heterocomplexes, structural differences in the loop regions, which could not be resolved so far, may be responsible for a tighter interaction. Importantly, LmClpP2 is prone to a third mechanism of activation based on partial active site binding by irreversible inhibitors. While modification of 20% of all catalytic serines by a CMK inhibitor increased proteolysis by 160%, alkylation of approximately 50% of the sites resulted in full inhibition. Again, the reason for this proteolytic stimulation was an increase in binding affinity of the chaperone for partially CMK-modified LmClpP2, which shifted the equilibrium to the active proteolytic complex. Thus, modulating the affinity between ClpP and ClpX seems to be a unifying principle for proteolytic fine-tuning. Interestingly, the activation heavily depended on the inhibitor used. While Aoc-CMK induced the strongest effect, a CMK inhibitor containing Leu at the P1 site was only marginally stimulatory, suggesting that binding of long aliphatic chains into the S1 pocket could be a crucial parameter. Similarly, the long aliphatic beta-lactone inhibitor D3 also stimulated proteolysis, although to a much lesser extent, which suggests that this effect is more general and can also be observed by lactone active-site acylation in addition to CMK alkylation. On the contrary, aromatically-decorated lactone E2, a known disruptor of the ClpP tetradecamer,10 inhibited turnover, which emphasizes the importance of the ligand introduced into the S1 pocket. While a slight activation by Aoc-CMK was also observed for SaClpP, the more pronounced effects in LmClpP2 suggest a more functionally relevant role in this system. Based on this data, the LmClpP2 active site and substrate pocket seem to control the fate of proteolytic activity. Partial binding of long aliphatic ligands or heterooligomerization with LmClpP1 induce proteolysis via tighter binding to ClpX, which may be necessary under certain stress conditions. For instance, heat stress was previously shown to induce the expression of clpP1 and clpP2, resulting in an increased number of heterocomplexes, which might result in elevated proteolytic rates for removal of misfolded proteins.16 In addition, it is intriguing to speculate that metabolites containing long aliphatic chains may act as native stimulants. While the existence of such metabolites has to be investigated in future studies, it is important to note that activating peptides have previously been reported for heterooligomeric MtClpPs.17,19 In fact, these small molecules bind to active sites and thereby enhance activity.18–20
In total, our in-depth analysis of LmClpP1/2 activity revealed two principles of proteolytic stimulation that both rely on elevated affinity between LmClpPs and LmClpX. This phenomenon appears to be special to the Listeria system, which is already unique through its unusual expression of two ClpP isoforms. Due to the fundamental relevance of ClpP for cell homeostasis under stress conditions, both activation pathways ensure a boost in ClpP activity when needed, while remaining tightly regulated by ClpX interactions in order to prevent uncontrolled damage.
Footnotes |
† Electronic supplementary information (ESI) available: Figures, tables and experimental procedures. See DOI: 10.1039/c6sc03438a |
‡ D. B. and M. D. contributed equally to this study. |
§ Present address: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK. |
This journal is © The Royal Society of Chemistry 2017 |