Structural and functional insights into caseinolytic proteases reveal an unprecedented regulation principle of their catalytic triad

Caseinolytic proteases (ClpPs) are large oligomeric protein complexes that contribute to cell homeostasis as well as virulence regulation in bacteria. Although most organisms possess a single ClpP protein, some organisms encode two or more ClpP isoforms. Here, we elucidated the crystal structures of ClpP1 and ClpP2 from pathogenic Listeria monocytogenes and observe an unprecedented regulation principle by the catalytic triad. Whereas L. monocytogenes (Lm)ClpP2 is both structurally and functionally similar to previously studied tetradecameric ClpP proteins from Escherichia coli and Staphylococcus aureus, heptameric LmClpP1 features an asparagine in its catalytic triad. Mutation of this asparagine to aspartate increased the reactivity of the active site and led to the assembly of a tetradecameric complex. We analyzed the heterooligomeric complex of LmClpP1 and LmClpP2 via coexpression and subsequent labeling studies with natural product-derived probes. Notably, the LmClpP1 peptidase activity is stimulated 75-fold in the complex providing insights into heterooligomerization as a regulatory mechanism. Collectively, our data point toward different preferences for substrates and inhibitors of the two ClpP enzymes and highlight their structural and functional characteristics.The caseinolytic protease P (ClpP) is a highly conserved enzyme present in bacteria and higher organisms (1–3). ClpP is responsible for cell homeostasis and among other duties for the regulation of bacterial virulence in several pathogens including Staphylococcus aureus and Listeria monocytogenes (4, 5). Early structural studies revealed the topology of the Escherichia coli ClpP complex that consists of two heptameric rings building up a 300 kDa cylinder (Fig. 1A) (6). The interior of this proteolytic machinery exhibits 14 active sites flanked by axial pores that allow protein substrates to enter the hydrolytic chamber. ClpP gains its catalytic activity in complex with AAA⁺-chaperones (such as ClpC, ClpE, and ClpX in the case of L. monocytogenes). These ATP-dependent enzymes bind to the axial pores of ClpP, unfold the protein prone to degradation, and direct it into the proteolytic chamber (7–9).Open in a separate windowFig. 1.Main structural elements of ClpP. (A) Top and side view of the tetradecameric ClpP complex from E. coli (10) (EcClpP, PDB ID code 1TYF, surface representation) with one subunit highlighted in dark gray. Each subunit (close-up, ribbon diagram) is made up of seven α-helices (denoted with letters) and 11 β-strands (denoted with numbers) and contains a catalytic triad (highlighted with red circles). Relevant secondary structures (α-helices E and F, β-strand 9) are highlighted in gold. (B) Sequence alignment of EcClpP with LmClpP1 and LmClpP2. The secondary structure elements are depicted for EcClpP. The catalytic triad is framed in red, the residues forming the E-helix are underlined in orange, the conserved proline and the glycins in the Gly-rich loop are colored blue, and the Asp/Arg sensor is shown in green.A close-up view of a single ClpP monomer reveals several characteristic structural features that are conserved among this class of proteases. To harmonize the ClpP nomenclature for all subsequent discussions, we use a general sequence numbering based on the first determined crystal structure of ClpP from E. coli EcClpP, Protein Data Bank (PDB) ID code 1TYF] (10) (Fig. 1B). According to this nomenclature, a catalytic triad (Ser98, His123, Asp172) essential for proteolysis, a central E-helix with a Gly-rich loop region essential for interring contacts between the two heptamers, and a N-terminal region essential for interaction with a AAA⁺-chaperone can be observed in all published X-ray structures to date (Fig. 1A, Fig. S1B) (10–18). Cocrystallization of E. coli ClpP with an irreversible dipeptide chloromethylketone inhibitor confirmed the reactivity of the catalytic triad residues Ser98 and His123 and illustrate a binding site for the dipeptide within the Gly-rich loop region that adopts an antiparallel beta-strand (19) (Fig. 2). Recently, two conformations of ClpP from S. aureus have been reported that are thought to represent physiologically important states with an active and an inactive catalytic triad corresponding to an extended and a bent E-helix, respectively (Fig. S2) (11, 12). In addition, a highly conserved aspartate/arginine sensor (Asp170/Arg171) links oligomerization to the catalytic activity and exhibits characteristic conformations in both states (Fig. S2) (12). In agreement with this model, ClpP heptamers lack the interaction of the sensor residues with their counterparts on the adjacent ring and thus have an inactive triad. In the tetradacameric state, the senor feedbacks the correct assembly to the active sites, thereby ensuring controlled proteolysis.Open in a separate windowFig. 2.Stereo-representation of ClpP monomers. Structural superposition of LmClpP1 (gold), LmClpP2 (green), SaClpP (PDB ID code 3V5E, pale red), and EcClpP (PDB ID code 2FZS, gray) with covalently bound CMK inhibitor.Although most organisms possess a single ClpP protein with a conserved fold (6, 11, 13–16, 18, 20), the genomes of some organisms encode two or more ClpP isoforms (21–24). For a cyanobacterial system, heptameric rings of mixed composition have been reported that interact with different chaperones (22). In contrast, ClpP proteins from L. monocytogenes (LmClpP1 and LmClpP2) as well as from Mycobacterium tuberculosis have been found to assemble into heterooligomeric complexes composed of two homoheptamers (25, 26). Inhibition of LmClpP2 with lactone-based inhibitors led to down-regulation of virulence without affecting viability (27). In contrast, both mycobacterial ClpP subunits are essential for bacterial survival, emphasizing defined functional roles of ClpP proteins among species (26, 28).Interestingly, LmClpP2 shares a high-sequence homology with ClpP enzymes of various organisms that feature one ClpP (Fig. S1 A and C). LmClpP1 exhibits only 41% sequence identity with LmClpP2, raising the question of how these two distinct isoforms interact and how they differ functionally. Furthermore, there is a distinct difference between the two ClpP homologs in the composition of their catalytic triad: Asp172 of LmClpP2 is replaced by an asparagine in LmClpP1, an unusual observation within serine proteases that is, however, conserved in several uncharacterized homologs (Fig. S1 A and C). Although the replacement of an aspartate with an asparagine represents only a moderate structural alteration, it significantly influences the strength of the catalytic triad charge-relay system. The nucleophilicity of the active site Ser98 in LmClpP1 and LmClpP2 was previously monitored and compared by β-lactone activity-based probes (25, 29). Although all monocyclic β-lactones selectively labeled LmClpP2 either as a homooligomer or as part of the heterooligomeric complex, a probe derived from the bicyclic natural product vibralactone (VLP) was able to interact with both LmClpP1 and LmClpP2 catalytic sites. Importantly, binding of the ligand to LmClpP1 was only observed in the presence of LmClpP2 (25).