We thus synthesized two inhibitors that incorporated the best residues from the P1, P2 and P3 library screening. 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 Metoprolol tartrate 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. Introduction ATP-dependent proteolysis represents an important mechanism for removal of misfolded or ribosome-stalled proteins under stress conditions. In prokaryotes AAA+ chaperones (such as ClpX, ClpA and ClpC) recognize and unfold substrate proteins by ATP consumption and direct the linear peptide chain into a proteolytic barrel of caseinolytic protease P (ClpP).1C4 ClpP is a transient tetradecameric serine hydrolase composed of two heptameric rings that are stacked face-to-face. Each subunit carries an active Ser-His-Asp catalytic triad that is essential for activity.5,6 ClpP by itself lacks proteolytic activity but is able to digest small peptides that diffuse into the barrel axial pores.7,8 The first specific inhibitors reported for ClpP include the beta-lactones, which exhibit an irreversible mode of action and, depending on their chemical structure, can cause either retention of the tetradecameric state or deoligomerization of ClpP.9,10 More detailed insights into inhibitor-mediated complex disassembly were provided through a new generation of covalent phenyl esters and relevant modeling studies. These studies suggested that steric clash of the inhibitor within the active site triad triggers a rearrangement at Metoprolol tartrate the heptamer interface, causing dissociation of the ClpP tetradecamer into two heptameric rings.11 Recently, the first reversible ClpP inhibitors were reported, which distort the active site catalytic triad through structural rearrangements.12 However, this inactive state of the ClpP peptidase could be reversed through formation of the ClpXP complex, highlighting the power of conformational control within this dynamic system. ClpXP interaction is mainly mediated by ClpX-loops which bind into hydrophobic clefts located at the ClpP axial surface.13 Interestingly, some bacterial strains such as and encode Metoprolol tartrate two ClpP isoforms (ClpP1 and ClpP2).14C18 While ClpP2 (LmClpP2) resembles related enzymes in other bacteria such as and 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 Metoprolol tartrate 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 and 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 (MtClpP1 and MtClpP2) also assemble into a.LmClpPs assemble into either a LmClpP2 homocomplex or a LmClpP1/2 heterooligomeric complex. 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. Introduction ATP-dependent proteolysis represents an important mechanism for removal of misfolded or ribosome-stalled proteins under stress conditions. In prokaryotes AAA+ chaperones (such as ClpX, ClpA and ClpC) recognize and unfold substrate proteins by ATP consumption and direct the linear peptide chain into a proteolytic barrel of caseinolytic protease P (ClpP).1C4 ClpP is a transient tetradecameric serine hydrolase composed of two heptameric rings that are stacked face-to-face. Each subunit carries an active Ser-His-Asp catalytic triad that is essential for activity.5,6 ClpP by itself lacks proteolytic activity but is able to digest small peptides that diffuse into the barrel axial pores.7,8 The first specific inhibitors reported for ClpP include the beta-lactones, which exhibit an irreversible mode of action and, depending on their chemical structure, can cause either retention of the tetradecameric state or deoligomerization of ClpP.9,10 More detailed insights into inhibitor-mediated complex disassembly were provided through a new generation of covalent phenyl esters and relevant modeling studies. These studies suggested that steric clash of the inhibitor within the active site triad triggers a rearrangement at the heptamer interface, causing dissociation of the ClpP tetradecamer into two heptameric rings.11 Recently, the first reversible ClpP Rabbit Polyclonal to CDC2 inhibitors were reported, which distort the active site catalytic triad through structural rearrangements.12 However, this inactive state of the ClpP peptidase could be reversed through formation of the ClpXP complex, highlighting the power of conformational control within this dynamic system. ClpXP interaction is mainly mediated by ClpX-loops which bind into hydrophobic clefts located at the ClpP axial surface.13 Interestingly, some bacterial strains such as and encode two ClpP isoforms (ClpP1 and ClpP2).14C18 While ClpP2 (LmClpP2) resembles related enzymes in other bacteria such as and Metoprolol tartrate 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-collapse increase in proteolytic activity on the homocomplex was observed when in the presence of LmClpX. From a functional perspective, this implies the cell generates a hyperactive enzyme during stress conditions, when misfolded proteins must be eliminated efficiently. Indeed, quantitative real-time PCR showed increased and manifestation under heat stress.16 So far, no systematic analysis of LmClpP1 and LmClpP2 peptidolytic and proteolytic cleavage specificities has been performed, leaving their part in substrate recognition unresolved..
