Turned on for degradation: ATPase-independent degradation by ClpP

Clp is a barrel-shaped hetero-oligomeric ATP-dependent protease comprising a hexameric ATPase (ClpX or ClpA) that unfolds protein substrates and translocates them into the central chamber of the tetradecameric proteolytic component (ClpP) where they are degraded processively to short peptides. Chamber access is controlled by the N-terminal 20 residues (for Escherichia coli) in ClpP that prevent entry of large polypeptides in the absence of the ATPase subunits and ATP hydrolysis. Remarkably, removal of 10–17 residues from the mature N-terminus allows processive degradation of a large model unfolded substrate to short peptides without the ATPase subunit or ATP hydrolysis; removal of 14 residues is maximal for activation. Furthermore, since the product size distribution of Δ14-ClpP is identical to ClpAP and ClpXP, the ATPases do not play an essential role in determining this distribution. Comparison of the structures of Δ14-ClpP and Δ17-ClpP with other published structures shows R15 and S16 are labile and that residue 17 can adopt a range of rotomers to ensure protection of a hydrophobic pocket formed by I19, R24 and F49 and maintain a hydrophilic character of the pore.     

Functional domains of the ClpA and ClpX molecular chaperones identified by limited proteolysis and deletion analysis

Escherichia coli ClpA and ClpX are ATP-dependent protein unfoldases that each interact with the protease, ClpP, to promote specific protein degradation. We have used limited proteolysis and deletion analysis to probe the conformations of ClpA and ClpX and their interactions with ClpP and substrates. ATP gamma S binding stabilized ClpA and ClpX such that that cleavage by lysylendopeptidase C occurred at only two sites. Both proteins were cleaved within in a loop preceding an alpha-helix-rich C-terminal domain. Although the loop varies in size and composition in Clp ATPases, cleavage occurred within and around a conserved triad, IG(F/L). Binding of ClpP blocked this cleavage, and prior cleavage at this site rendered both ClpA and ClpX defective in binding and activating ClpP, suggesting that this site is involved in interactions with ClpP. ClpA was also cut at a site near the junction of the two ATPase domains, whereas the second cleavage site in ClpX lay between its N-terminal and ATPase domains. ClpP did not block cleavage at these other sites. The N-terminal domain of ClpX dissociated upon cleavage, and the remaining ClpXDeltaN remained as a hexamer, associated with ClpP, and expressed ATPase, chaperone, and proteolytic activity. A truncated mutant of ClpA lacking its N-terminal 153 amino acids also formed a hexamer, associated with ClpP, and expressed these activities. We propose that the N-terminal domains of ClpX and ClpA lie on the outside ring surface of the holoenzyme complexes where they contribute to substrate binding or perform a gating function affecting substrate access to other binding sites and that a loop on the opposite face of the ATPase rings stabilizes interactions with ClpP and is involved in promoting ClpP proteolytic activity.     

Recognition, targeting, and hydrolysis of the lambda O replication protein by the ClpP/ClpX protease.

It has previously been established that sequences at the C termini of polypeptide substrates are critical for efficient hydrolysis by the ClpP/ClpX ATP-dependent protease. We report for the bacteriophage lambda O replication protein, however, that N-terminal sequences play the most critical role in facilitating proteolysis by ClpP/ClpX. The N-terminal portion of lambda O is degraded at a rate comparable with that of wild type O protein, whereas the C-terminal domain of O is hydrolyzed at least 10-fold more slowly. Consistent with these results, deletion of the first 18 amino acids of lambda O blocks degradation of the N-terminal domain, whereas proteolysis of the O C-terminal domain is only slightly diminished as a result of deletion of the C-terminal 15 amino acids. We demonstrate that ClpX retains its capacity to bind to the N-terminal domain following removal of the first 18 amino acids of O. However, ClpX cannot efficiently promote the ATP-dependent binding of this truncated O polypeptide to ClpP, the catalytic subunit of the ClpP/ClpX protease. Based on our results with lambda O protein, we suggest that two distinct structural elements may be required in substrate polypeptides to enable efficient hydrolysis by the ClpP/ClpX protease: (i) a ClpX-binding site, which may be located remotely from substrate termini, and (ii) a proper N- or C-terminal sequence, whose exposure on the substrate surface may be induced by the binding of ClpX.     

The ClpP N-terminus coordinates substrate access with protease active site reactivity

Energy-dependent protein degradation machines, such as the Escherichia coli protease ClpAP, require regulated interactions between the ATPase component (ClpA) and the protease component (ClpP) for function. Recent studies indicate that the ClpP N-terminus is essential in these interactions, yet the dynamics of this region remain unclear. Here, we use synchrotron hydroxyl radical footprinting and kinetic studies to characterize functionally important conformational changes of the ClpP N-terminus. Footprinting experiments show that the ClpP N-terminus becomes more solvent-exposed upon interaction with ClpA. In the absence of ClpA, deletion of the ClpP N-terminus increases the initial degradation rate of large peptide substrates 5-15-fold. Unlike ClpAP, ClpPDeltaN exhibits a distinct slow phase of product formation that is eliminated by the addition of hydroxylamine, suggesting that truncation of the N-terminus leads to stabilization of the acyl-enzyme intermediate. These results indicate that (1) the ClpP N-terminus acts as a "gate" controlling substrate access to the active sites, (2) binding of ClpA opens this "gate", allowing substrate entry and formation of the acyl-enzyme intermediate, and (3) closing of the N-terminal "gate" stimulates acyl-enzyme hydrolysis.     

Both ATPase Domains of ClpA Are Critical for Processing of Stable Protein Structures

ClpA is a ring-shaped hexameric chaperone that binds to both ends of the protease ClpP and catalyzes the ATP-dependent unfolding and translocation of substrate proteins through its central pore into the ClpP cylinder. Here we study the relevance of ATP hydrolysis in the two ATPase domains of ClpA. We designed ClpA Walker B variants lacking ATPase activity in the first (D1) or the second ATPase domain (D2) without impairing ATP binding. We found that the two ATPase domains of ClpA operate independently even in the presence of the protease ClpP or the adaptor protein ClpS. Notably, ATP hydrolysis in the first ATPase module is sufficient to process a small, single domain protein of low stability. Substrate proteins of moderate local stability were efficiently processed when D1 was inactivated. However, ATP hydrolysis in both domains was required for efficiently processing substrates of high local stability. Furthermore, we provide evidence for the ClpS-dependent directional translocation of N-end rule substrates from the N to C terminus and propose a mechanistic model for substrate handover from the adaptor protein to the chaperone.     

Concurrent chaperone and protease activities of ClpAP and the requirement for the N-terminal ClpA ATP binding site for chaperone activity.

ClpA, a member of the Clp/Hsp100 family of ATPases, is both an ATP-dependent molecular chaperone and the regulatory component of ClpAP protease. We demonstrate that chaperone and protease activities occur concurrently in ClpAP complexes during a single round of RepA binding to ClpAP and ATP-dependent release. This result was substantiated with a ClpA mutant, ClpA(K220V), carrying an amino acid substitution in the N-terminal ATP binding site. ClpA(K220V) is unable to activate RepA, but the presence of ClpP or chemically inactivated ClpP restores its ability to activate RepA. The presence of ClpP simultaneously facilitates degradation of RepA. ClpP must remain bound to ClpA(K220V) for these effects, indicating that both chaperone and proteolytic activities of the mutant complex occur concurrently. ClpA(K220V) itself is able to form stable complexes with RepA in the presence of a poorly hydrolyzed ATP analog, adenosine 5'-O-(thiotriphosphate), and to release RepA upon exchange of adenosine 5'-O-(thiotriphosphate) with ATP. However, the released RepA is inactive in DNA binding, indicating that the N-terminal ATP binding site is essential for the chaperone activity of ClpA. Taken together, these results suggest that substrates bound to the complex of the proteolytic and ATPase components can be partitioned between release/reactivation and translocation/degradation.     

Roles of the N-domains of the ClpA unfoldase in binding substrate proteins and in stable complex formation with the ClpP protease

The hexameric cylindrical Hsp100 chaperone ClpA mediates ATP-dependent unfolding and translocation of recognized substrate proteins into the coaxially associated serine protease ClpP. Each subunit of ClpA is composed of an N-terminal domain of approximately 150 amino acids at the top of the cylinder followed by two AAA+ domains. In earlier studies, deletion of the N-domain was shown to have no effect on the rate of unfolding of substrate proteins bearing a C-terminal ssrA tag, but it did reduce the rate of degradation of these proteins (Lo, J. H., Baker, T. A., and Sauer, R. T. (2001) Protein Sci. 10, 551-559; Singh, S. K., Rozycki, J., Ortega, J., Ishikawa, T., Lo, J., Steven, A. C., and Maurizi, M. R. (2001) J. Biol. Chem. 276, 29420-29429). Here we demonstrate, using both fluorescence resonance energy transfer to measure the arrival of substrate at ClpP and competition between wild-type and an inactive mutant form of ClpP, that this effect on degradation is caused by diminished stability of the ClpA-ClpP complex during translocation and proteolysis, effectively disrupting the targeting of unfolded substrates to the protease. We have also examined two larger ssrA-tagged substrates, CFP-GFP-ssrA and luciferase-ssrA, and observed different behaviors. CFP-GFP-ssrA is not efficiently unfolded by the truncated chaperone whereas luciferase-ssrA is, suggesting that the former requires interaction with the N-domains, likely via the body of the protein, to stabilize its binding. Thus, the N-domains play a key allosteric role in complex formation with ClpP and may also have a critical role in recognizing certain tag elements and binding some substrate proteins.     

The heat-shock protein ClpB in Escherichia coli is a protein-activated ATPase.

The clpB gene in Escherichia coli encodes a heat-shock protein that is a close homolog of the clpA gene product. The latter is the ATPase subunit of the multimeric ATP-dependent protease Ti (Clp) in E. coli, which also contains the 21-kDa proteolytic subunit (ClpP). The clpB gene product has been purified to near homogeneity by DEAE-Sepharose and heparin-agarose column chromatographies. The purified ClpB consists of a major 93-kDa protein and a minor 79-kDa polypeptide as analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Upon gel filtration on a Superose-6 column, it behaves as a 350-kDa protein. Thus, ClpB appears to be a tetrameric complex of the 93-kDa subunit. The purified ClpB has ATPase activity which is stimulated 5-10-fold by casein. It is also activated by insulin, but not by other proteins, including globin and denatured bovine serum albumin. ClpB cleaves adenosine 5'-(alpha,beta-methylene)-triphosphate as rapidly as ATP, but not adenosine 5'-(beta,gamma-methylene)-triphosphate. GTP, CTP, and UTP are hydrolyzed 15-25% as well as ATP. ADP strongly inhibits ATP hydrolysis with a Ki of 34 microM. ClpB has a Km for ATP of 1.1 mM, and casein increases its Vmax for ATP without affecting its Km. A Mg2+ concentration of 3 mM is necessary for half-maximal ATP hydrolysis. Mn2+ supports ATPase activity as well as Mg2+, and Ca2+ has about 20% their activity. Anti-ClpB antiserum does not cross-react with ClpA nor does anti-ClpA antiserum react with ClpB. In addition, ClpB cannot replace ClpA in supporting the casein-degrading activity of ClpP. Thus, ClpB is distinct from ClpA in its structural and biochemical properties despite the similarities in their sequences.     

ClpA and ClpX ATPases bind simultaneously to opposite ends of ClpP peptidase to form active hybrid complexes

The Escherichia coli ATP-dependent ClpAP and ClpXP proteases are composed of a single proteolytic component, ClpP, complexed with either of the two related chaperones, ClpA or ClpX. ClpXP and ClpAP complexes interact with different specific substrates and catalyze ATP-dependent protein unfolding and degradation. In vitro in the presence of ATP or ATPgammaS, ClpA and ClpX form homomeric rings of six subunits, which bind to one or both ends of the double heptameric rings of ClpP. We have observed that, when equimolar amounts of ClpA and ClpX hexamers are added to ClpP in vitro in the presence of ATP or ATPgammaS, hybrid complexes in which ClpX and ClpA are bound to opposite ends of the same ClpP are readily formed. The distribution of homomeric and heteromeric complexes was consistent with random binding of ClpA and ClpX to the ends of ClpP. Direct demonstration of the functionality of the heteromeric complexes was obtained by electron microscopy, which allowed us to visualize substrate translocation into proteolytically inactive ClpP chambers. Starting with hybrid complexes to which protein substrates specific to ClpX or ClpA were bound, translocation of both types of substrates was shown to occur without significant redistribution of ClpA or ClpX. The stoichiometric ratios of the ClpA, ClpX, and ClpP oligomeric complexes in vivo are consistent with the predominance of heteromeric complexes in growing cells. Thus, ClpXAP is a bifunctional protease whose two ends can independently target different classes of substrates.     

ClpA and ClpP remain associated during multiple rounds of ATP-dependent protein degradation by ClpAP protease.

The Escherichia coli ClpA and ClpP proteins form a complex, ClpAP, that catalyzes ATP-dependent degradation of proteins. Formation of stable ClpA hexamers and stable ClpAP complexes requires binding of ATP or nonhydrolyzable ATP analogues to ClpA. To understand the order of events during substrate binding, unfolding, and degradation by ClpAP, it is essential to know the oligomeric state of the enzyme during multiple catalytic cycles. Using inactive forms of ClpA or ClpP as traps for dissociated species, we measured the rates of dissociation of ClpA hexamers or ClpAP complexes. When ATP was saturating, the rate constant for dissociation of ClpA hexamers was 0.032 min(-1) (t(1/2) of 22 min) at 37 degrees C, and dissociation of ClpP from the ClpAP complexes occurred with a rate constant of 0. 092 min(-1) (t(1/2) of 7.5 min). Because the k(cat) for casein degradation is approximately 10 min(-1), these results indicate that tens of molecules of casein can be turned over by the ClpAP complex before significant dissociation occurs. Mutations in the N-terminal ATP binding site led to faster rates of ClpA and ClpAP dissociation, whereas mutations in the C-terminal ATP binding site, which cause significant decreases in ATPase activity, led to lower rates of dissociation of ClpA and ClpAP complexes. Dissociation rates for wild-type and first domain mutants of ClpA were faster at low nucleotide concentrations. The t(1/2) for dissociation of ClpAP complexes in the presence of nonhydrolyzable analogues was >/=30 min. Thus, ATP binding stabilizes the oligomeric state of ClpA, and cycles of ATP hydrolysis affect the dynamics of oligomer interaction. However, since the k(cat) for ATP hydrolysis is approximately 140 min(-1), ClpA and the ClpAP complex remain associated during hundreds of rounds of ATP hydrolysis. Our results indicate that the ClpAP complex is the functional form of the protease and as such engages in multiple rounds of interaction with substrate proteins, degradation, and release of peptide products without dissociation.     

The ClpP double ring tetradecameric protease exhibits plastic ring-ring interactions, and the N termini of its subunits form flexible loops that are essential for ClpXP and ClpAP complex formation

ClpP is a conserved serine-protease with two heptameric rings that enclose a large chamber containing the protease active sites. Each ClpP subunit can be divided into a handle region, which mediates ring-ring interactions, and a head domain. ClpP associates with the hexameric ATPases ClpX and ClpA, which can unfold and translocate substrate proteins through the ClpP axial pores into the protease lumen for degradation. We have determined the x-ray structure of Streptococcus pneumoniae ClpP(A153P) at 2.5 A resolution. The structure revealed two novel features of ClpP which are essential for ClpXP and ClpAP functional activities. First, the Ala --> Pro mutation disrupts the handle region, resulting in an altered ring-ring dimerization interface, which, in conjunction with biochemical data, demonstrates the unusual plasticity of this region. Second, the structure shows the existence of a flexible N-terminal loop in each ClpP subunit. The loops line the axial pores in the ClpP tetradecamer and then protrude from the protease apical surface. The sequence of the N-terminal loop is highly conserved in ClpP across all kingdoms of life. These loops are essential determinants for complex formation between ClpP and ClpX/ClpA. Mutation of several amino acid residues in this loop or the truncation of the loop impairs ClpXP and ClpAP complex formation and prevents the coupling between ClpX/ClpA and ClpP activities.     

Role of Clp protease subunits in degradation of carbon starvation proteins in Escherichia coli.

When deprived of a carbon source, Escherichia coli induces the synthesis of a group of carbon starvation proteins. The degradation of proteins labeled during starvation was found to be an energy-dependent process which was inhibited by the addition of KCN and accelerated when cells were resupplied with a carbon source. The degradation of the starvation proteins did not require the ATP-dependent Lon protease or the energy-independent proteases protease I, protease IV, OmpT, and DegP. During starvation, mutants lacking either the ClpA or ClpP subunit of the ATP-dependent Clp protease showed a partial reduction in the degradation of starvation proteins. Strains lacking ClpP failed to increase degradation of starvation proteins when glucose was added to starving cells. The clpP mutants showed a competitive disadvantage compared with wild-type cells when exposed to repeated cycles of carbon starvation and growth. Surprisingly, the glucose-stimulated, ClpP-dependent degradation of starvation proteins did not require either the ClpA or ClpB protein. The patterns of synthesis of starvation proteins were similar in clpP+ and clpP cells. The clpP mutants had reduced rates of degradation of certain starvation proteins in the membrane fraction when a carbon source was resupplied to the starved cells.     

A new component of bacteriophage Mu replicative transposition machinery: the Escherichia coli ClpX protein

We have shown previously that some particular mutations in bacteriophage Mu repressor, the frameshift vir mutations, made the protein very sensitive to the Escherichia coli ATP-dependent Clp protease. This enzyme is formed by the association between a protease subunit (ClpP) and an ATPase subunit. ClpA, the best characterized of these ATPases, is not required for the degradation of the mutant Mu repressors. Recently, a new potential ClpP associated ATPase, ClpX, has been described. We show here that this new subunit is required for Mu vir repressor degradation. Moreover, ClpX (but not ClpP) was found to be required for normal Mu replication. Thus ClpX has activities that do not require its association with ClpP. In the pathway of Mu replicative transposition, the block resides beyond the strand transfer reaction, i.e. after the transposition reaction per se is completed, suggesting that ClpX is required for the transition to the formation of the active replication complex at one Mu end. This is a new clear-cut case of the versatile activity of polypeptides that form multi-component ATP-dependent proteases.     

Binding and degradation of heterodimeric substrates by ClpAP and ClpXP

ClpA and ClpX function both as molecular chaperones and as the regulatory components of ClpAP and ClpXP proteases, respectively. ClpA and ClpX bind substrate proteins through specific recognition signals, catalyze ATP-dependent protein unfolding of the substrate, and when in complexes with ClpP translocate the unfolded polypeptide into the cavity of the ClpP peptidase for degradation. To examine the mechanism of interaction of ClpAP with dimeric substrates, single round binding and degradation experiments were performed, revealing that ClpAP degraded both subunits of a RepA homodimer in one cycle of binding. Furthermore, ClpAP was able to degrade both protomers of a RepA heterodimer in which only one subunit contained the ClpA recognition signal. In contrast, ClpXP degraded both subunits of a dimeric substrate only when both protomers contained a recognition signal. These data suggest that ClpAP and ClpXP may recognize and bind substrates in significantly different ways.     

Functional proteolytic complexes of the human mitochondrial ATP-dependent protease, hClpXP

Human mitochondrial ClpP (hClpP) and ClpX (hClpX) were separately cloned, and the expressed proteins were purified. Electron microscopy confirmed that hClpP forms heptameric rings and that hClpX forms a hexameric ring. Complexes of a double heptameric ring of hClpP with hexameric hClpX rings bound on each side are stable in the presence of ATP or adenosine 5'-(3-thiotriphosphate) (ATPgammaS), indicating that a symmetry mismatch is a universal feature of Clp proteases. hClpXP displays both ATP-dependent proteolytic activity and ATP- or ATPgammaS-dependent peptidase activity. hClpXP cannot degrade lambdaO protein or GFP-SsrA, specific protein substrates recognized by Escherichia coli (e) ClpXP. However, eClpX interacts with hClpP, and, when examined by electron microscopy, the resulting heterologous complexes are indistinguishable from homologous eClpXP complexes. The hybrid eClpX-hClpP complexes degrade eClpX-specific protein substrates. In contrast, eClpA can neither associate with nor activate hClpP. hClpP has an extra C-terminal extension of 28 amino acids. A mutant lacking this C-terminal extension interacts more tightly with both hClpX and eClpX and shows enhanced enzymatic activities but still does not interact with eClpA. Our results establish that human ClpX and ClpP constitute a bone fide ATP-dependent protease and confirm that substrate selection, which differs between human and E. coli ClpX, is dependent solely on the Clp ATPase. Our data also indicate that human ClpP has conserved sites required for interaction with eClpX but not eClpA, implying that the modes of interaction with ClpP may not be identical for ClpA and ClpX.     

Phylogenetic analysis predicts structural divergence for proteobacterial ClpC proteins

Regulated proteolysis is required in all organisms for the removal of misfolded or degradation-tagged protein substrates in cellular quality control pathways. The molecular machines that catalyze this process are known as ATP-dependent proteases with examples that include ClpAP and ClpCP. Clp/Hsp100 subunits form ring-structures that couple the energy of ATP binding and hydrolysis to protein unfolding and subsequent translocation of denatured protein into the compartmentalized ClpP protease for degradation. Copies of the clpA, clpC, clpE, clpK, and clpL genes are present in all characterized bacteria and their gene products are highly conserved in structure and function. However, the evolutionary relationship between these proteins remains unclear. Here we report a comprehensive phylogenetic analysis that suggests divergent evolution yielded ClpA from an ancestral ClpC protein and that ClpE/ClpL represent intermediates between ClpA/ClpC. This analysis also identifies a group of proteobacterial ClpC proteins that are likely not functional in regulated proteolysis. Our results strongly suggest that bacterial ClpC proteins should not be assumed to all function identically due to the structural differences identified here.     

The two-component, ATP-dependent Clp protease of Escherichia coli. Purification, cloning, and mutational analysis of the ATP-binding component

The ATP-binding component (Component II, hereafter referred to as ClpA) of a two-component, ATP-dependent protease from Escherichia coli has been purified to homogeneity. ClpA is a protein with subunit Mr 81,000. It has an intrinsic ATPase activity and activates degradation of protein substrates only in the presence of a second component (Component I, hereafter referred to as ClpP), Mg2+, and ATP. The amount of ClpA varies by less than a factor of 2 in cells grown in different media and at temperatures from 30 to 42 degrees C. ClpA does not appear to be a heat-shock protein since its synthesis is not dependent on htpR. Antibodies against purified ClpA were used to identify lambda transducing phage bearing the clpA gene. The cloned gene contains a DNA sequence expected to code for the first 28 amino acids of ClpA, which were determined by protein sequencing of purified ClpA. The clpA gene in the phage was mutated by insertion of delta kan defective transposons and the mutations were transferred to E. coli by homologous recombination. The clpA gene was mapped to 19 min on the E. coli chromosome. Mutant cells with insertions early in the gene produce no ClpA protein detectable in Western blots, and extracts of such mutant cells have no detectable ClpA activity. clpA- mutants grow well under all conditions tested and are not defective in turnover of proteins during nitrogen starvation nor in the turnover of such highly unstable proteins as the lambda proteins O, N, and cII, or the E. coli proteins SulA, RcsA, and glutamate dehydrogenase. The degradation of abnormal canavanine-containing proteins is defective in clpA mutants especially in cells that also have a lon- mutation. Extracts of clpA- lon- cells have ATP-dependent casein degrading activity.     

Characterization of the N-terminal repeat domain of Escherichia coli ClpA-A class I Clp/HSP100 ATPase

The ClpA, ClpB, and ClpC subfamilies of the Clp/HSP100 ATPases contain a conserved N-terminal region of approximately 150 residues that consists of two approximate sequence repeats. This sequence from the Escherichia coli ClpA enzyme is shown to encode an independent structural domain (the R domain) that is monomeric and approximately 40% alpha-helical. A ClpA fragment lacking the R domain showed ATP-dependent oligomerization, protein-stimulated ATPase activity, and the ability to complex with the ClpP peptidase and mediate degradation of peptide and protein substrates, including casein and ssrA-tagged proteins. Compared with the activities of the wild-type ClpA, however, those of the ClpA fragment missing the R domain were reduced. These results indicate that the R domain is not required for the basic recognition, unfolding, and translocation functions that allow ClpA-ClpP to degrade some protein substrates, but they suggest that it may play a role in modulating these activities.