Clp ATPases Research Articles

Clp ATPases and ClpP proteolytic complexes regulate vital biological processes in low GC, Gram‐positive bacteria

Clp proteolytic complexes consisting of a proteolytic core flanked by Clp ATPases are widely conserved in bacteria, and their biological roles have received considerable interest. In particular, mutants in the clp genes in the low-GC-content Gram-positive phyla Bacillales and Lactobacillales display a diverse range of phenotypic changes including general stress sensitivity, aberrant cell morphology, failure to initiate developmental programs, and for pathogens, severely attenuated virulence. Extensive research dedicated to unravelling the molecular mechanisms underlying these complex phenotypes has led to fascinating new insights that will be covered by this review. First, Clp ATPases and ClpP-containing proteolytic complexes play indispensable roles in cellular protein quality control systems by refolding or degrading damaged proteins in both stressed and non-stressed cells. Secondly, ClpP proteases and the chaperone activity of Clp ATPases are important for controlling stability and activity of central transcriptional regulators, thereby exerting tremendous impact on cell physiology. Targets include major stress regulators like Spx (oxidative stress), the antisigma factor RsiW (alkaline stress) and HdiR (DNA damage) in addition to regulators of developmental programs like ComK (competence development), sigmaH and Sda (sporulation). Thus, Clp proteins are central in co-ordinating developmental decisions and stress response in low GC Gram-positive bacteria.

Specificity in substrate and cofactor recognition by the N-terminal domain of the chaperone ClpX

Clp ATPases are a unique group of ATP-dependent chaperones supporting targeted protein unfolding and degradation in concert with their respective proteases. ClpX is a representative member of these ATPases; it consists of two domains, a zinc-binding domain (ZBD) that forms dimers and a AAA+ ATP-binding domain that arranges into a hexamer. Analysis of the binding preferences of these two domains in ClpX revealed that both domains preferentially bind to hydrophobic residues but have different sequence preferences, with the AAA+ domain preferentially recognizing a wider range of specific sequences than ZBD. As part of this analysis, the binding site of the ClpX dimeric cofactor, SspB2, on ZBD in ClpX was determined by NMR and mutational analysis. The SspB C terminus was found to interact with a hydrophobic patch on the surface of ZBD. The affinity of SspB2 toward ZBD2 and the geometry of the SspB2-ZBD2 complex were investigated by using the newly developed quantitative optical biosensor method of dual polarization interferometry. The data suggest a model for the interaction between SspB2 and the ClpX hexamer.

A camel passes through the eye of a needle: protein unfolding activity of Clp ATPases

Clp ATPases are protein machines involved in protein degradation and disaggregation. The common structural feature of Clp ATPases is the formation of ring-shaped oligomers. Recent work has shown that the function of all Clp ATPases is based on an energy-dependent threading of substrates through the narrow pore at the centre of the ring. This review gives an outline of known mechanistic principles of threading machines that unfold protein substrates either before their degradation (ClpA, ClpX, HslU) or during their reactivation from aggregates (ClpB). The place of Clp ATPases within a broad AAA+ superfamily of ATPases associated with various cellular activities suggests that similar mechanisms can be used by other protein machines to induce conformational rearrangements in a wide variety of substrates.

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.

Clp ATPases are required for stress tolerance, intracellular replication and biofilm formation in Staphylococcus aureus

The Hsp100/Clp ATPases constitute a family of closely related proteins of which some members function solely as chaperones whereas others additionally can associate with the unrelated ClpP peptidase forming a Clp proteolytic complex. We have investigated the role of four Clp ATPases in the versatile pathogen, Staphylococcus aureus. Previously, we showed that ClpX is required for expression of major virulence factors and for virulence of S. aureus, but not for survival during heat shock. In the present study, we have inactivated clpC, clpB and clpL and, while none of these mutations affected toxin production, both ClpC and ClpB and to a minor extent ClpL were required for intracellular multiplication within bovine mammary epithelial cells. These defects were paralleled by an inability of the clpC mutant to grow at high temperature and of the clpB mutant to induce thermotolerance indicating that the protective functions of these proteins are required both at high temperature and during infection. By primer extension analysis and footprint studies, we show that expression of clpC and clpB is controlled by the negative heat-shock regulator, CtsR, and that ClpC is required for its repressor activity. Thus, ClpC is a likely sensor of stress encountered during both environmental stress and infection. In addition to virulence factor production the ability to form biofilms is of importance to S. aureus as a nosocomial pathogen. Interestingly, biofilm formation was reduced in the absence of ClpX or ClpC whereas it was enhanced in the absence of ClpP. Thus, our data show that Clp proteolytic complexes and the Clp ATPases control several key processes of importance to the success of S. aureus as a pathogen.

ClgR, a Novel Regulator of clp and lon Expression in Streptomyces

The clp genes encoding the Clp proteolytic complex are widespread among living organisms. Five clpP genes are present in Streptomyces. Among them, the clpP1 clpP2 operon has been shown to be involved in the Streptomyces growth cycle, as a mutation blocked differentiation at the substrate mycelium step. Four Clp ATPases have been identified in Streptomyces coelicolor (ClpX and three ClpC proteins) which are potential partners of ClpP1 ClpP2. The clpC1 gene appears to be essential, since no mutant has yet been obtained. clpP1 clpP2 and clpC1 are important for Streptomyces growth, and a study of their regulation is reported here. The clpP3 clpP4 operon, which has been studied in Streptomyces lividans, is induced in a clpP1 mutant strain, and regulation of its expression is mediated via PopR, a transcriptional regulator. We report here studies of clgR, a paralogue of popR, in S. lividans. Gel mobility shift assays and DNase I footprinting indicate that ClgR binds not only to the clpP1 and clpC1 promoters, but also to the promoter of the Lon ATP-dependent protease gene and the clgR promoter itself. ClgR recognizes the motif GTTCGC-5N-GCG. In vivo, ClgR acts as an activator of clpC1 gene and clpP1 operon expression. Similarly to PopR, ClgR degradation might be ClpP dependent and could be mediated via recognition of the two carboxy-terminal alanine residues.

Fine-tuning in regulation of Clp protein content in Bacillus subtilis.

Clp-controlled proteolysis in Bacillus subtilis seems to play a substantial role, particularly under stress conditions. Calibrated Western blot analyses were used to estimate the approximate numbers of heat-inducible Clp molecules within a single cell. According to these numbers, the different Clp ATPases do not seem to compete for the proteolytic subunit ClpP. Coimmunoprecipitation experiments revealed the predicted specific ClpX-ClpP, ClpC-ClpP, and ClpE-ClpP interactions. ClpE and ClpX are rapidly degraded in wild-type cells during permanent heat stress but remained almost stable in a clpP mutant, suggesting ClpP-dependent degradation. In particular, ClpCP appeared to be involved in the degradation of the short-lived ClpE ATPase, indicating a negative "autoregulatory" circuit for this particular Clp ATPase at the posttranslational level. Analysis of the half-life of stress-inducible clp mRNAs during exponential growth and heat shock revealed precise regulation of the synthesis of each Clp protein at the posttranscriptional level as well to meet the needs of B. subtilis.

The N-terminal Zinc Binding Domain of ClpX Is a Dimerization Domain That Modulates the Chaperone Function

Clp ATPases are unique chaperones that promote protein unfolding and subsequent degradation by proteases. The mechanism by which this occurs is poorly understood. Here we demonstrate that the N-terminal domain of ClpX is a C4-type zinc binding domain (ZBD) involved in substrate recognition. ZBD forms a very stable dimer that is essential for promoting the degradation of some typical ClpXP substrates such as lambdaO and MuA but not GFP-SsrA. Furthermore, experiments indicate that ZBD contains a primary binding site for the lambdaO substrate and for the cofactor SspB. Removal of ZBD from the ClpX sequence renders the ATPase activity of ClpX largely insensitive to the presence of ClpP, substrates, or the SspB cofactor. All these results indicate that ZBD plays an important role in the ClpX mechanism of function and that ATP binding and/or hydrolysis drives a conformational change in ClpX involving ZBD.

Regulation and Physiological Significance of ClpC and ClpP in Streptococcus mutans.

Tolerance of environmental stress, especially low pH, by Streptococcus mutans is central to the virulence of this organism. The Clp ATPases are implicated in the tolerance of, and regulation of the response to, stresses by virtue of their protein reactivation and remodeling activities and their capacity to target misfolded proteins for degradation by the ClpP peptidase. The purpose of this study was to dissect the role of selected clp genes in the stress responses of S. mutans, with a particular focus on acid tolerance and adaptation. Homologues of the clpB, clpC, clpE, clpL, clpX, and clpP genes were identified in the S. mutans genome. The expression of clpC and clpP, which were chosen as the focus of this study, was induced at low pH and at growth above 40 degrees C. Inactivation of ctsR, the first of two genes in the clpC operon, demonstrated that CtsR acts as a repressor of clp and groES-EL gene expression. Strains lacking ClpP, but not strains lacking ClpC, were impaired in their ability to grow under stress-inducing conditions, formed long chains, aggregated in culture, had reduced genetic transformation efficiencies, and had a reduced capacity to form biofilms. Comparison of two-dimensional protein gels from wild-type cells and the ctsR and clpP mutants revealed many changes in the protein expression patterns. In particular, in the clpP mutant, there was an increased production of GroESL and DnaK, suggesting that cells were stressed, probably due to the accumulation of denatured proteins.

Chaperone-assisted protein folding in the cell cytoplasm.

Folding of polypeptides in the cell typically requires the assistance of a set of proteins termed molecular chaperones. Chaperones are an essential group of proteins necessary for cell viability under both normal and stress conditions. There are several chaperone systems which carry out a multitude of functions all aimed towards insuring the proper folding of target proteins. Chaperones can assist in the efficient folding of newly-translated proteins as these proteins are being synthesized on the ribosome and can maintain pre-existing proteins in a stable conformation. Chaperones can also promote the disaggregation of preformed protein aggregates. Many of the identified chaperones are also heat shock proteins. The general mechanism by which chaperones carry out their function usually involves multiple rounds of regulated binding and release of an unstable conformer of target polypeptides. The four main chaperone systems in the Escherichia coli cytoplasm are as follows. (1) Ribosome-associated trigger factor that assists in the folding of newly-synthesized nascent chains. (2) The Hsp 70 system consisting of DnaK (Hsp 70), its cofactor DnaJ (Hsp 40), and the nucleotide exchange factor GrpE. This system recognizes polypeptide chains in an extended conformation. (3) The Hsp 60 system, consisting of GroEL (Hsp 60) and its cofactor GroES (Hsp 10), which assists in the folding of compact folding intermediates that expose hydrophobic surfaces. (4) The Clp ATPases which are typically members of the Hsp 100 family of heat shock proteins. These ATPases can unfold proteins and disaggregate preformed protein aggregates to target them for degradation. Several advances have recently been made in characterizing the structure and function of all of these chaperone systems. These advances have provided us with a better understanding of the protein folding process in the cell.

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.

Clp ATPases and their role in protein unfolding and degradation

Although much has been learned about the structure and function of Clp chaperones and their role in proteolysis, the mechanism of protein unfolding catalyzed by Clp ATPases and the mechanism of translocation of the unfolded proteins from Clp ATPases to partner proteases remain unsolved puzzles. However, models in which mechanical force is used to destabilize the structure of the substrate in a processive and directional manner are probable. It also seems likely that when ClpA ATPases are associated with proteases, unfolding is coupled to extrusion of the unfolded protein into the proteolytic cavity. In summary, it is anticipated that the large family of Clp ATPases will accomplish their many important cellular functions by similar mechanisms and what has been learned by studying the prokaryotic members reviewed here will shed a great deal of light on all members of the family.

CtsR controls class III heat shock gene expression in the human pathogen Listeria monocytogenes.

Stress proteins play an important role in virulence, yet little is known about the regulation of stress response in pathogens. In the facultative intracellular pathogen Listeria monocytogenes, the Clp ATPases, including ClpC, ClpP and ClpE, are required for stress survival and intracellular growth. The first gene of the clpC operon of L. monocytogenes encodes a homologue of the Bacillus subtilis CtsR repressor of stress response genes. An L. monocytogenes ctsR-deleted mutant displayed enhanced survival under stress conditions (growth in the presence of 2% NaCl or at 42 degrees C), but its level of virulence in the mouse was not affected. The virulence of a wild-type strain constitutively expressing CtsR is significantly attenuated, presumably because of repression of the stress response. Regulation of the L. monocytogenes clpC, clpP and clpE genes was investigated using transcriptional fusions in B. subtilis as a host. The L. monocytogenes ctsR gene was placed under the control of an inducible promoter, and regulation by CtsR and heat shock was demonstrated in vivo in B. subtilis. The purified CtsR protein of L. monocytogenes binds specifically to the clpC, clpP and clpE regulatory regions, and the extent of the CtsR binding sites was defined by DNase I footprinting. Our results demonstrate that this human pathogen possesses a CtsR regulon controlling class III heat shock genes, strikingly similar to that of the saprophyte B. subtilis. This is the first description of a stress response regulatory gene in a pathogen.

Redundant in vivo proteolytic activities of Escherichia coli Lon and the ClpYQ (HslUV) protease.

The ClpYQ (HslUV) ATP-dependent protease of Escherichia coli consists of an ATPase subunit closely related to the Clp ATPases and a protease component related to those found in the eukaryotic proteasome. We found that this protease has a substrate specificity overlapping that of the Lon protease, another ATP-dependent protease in which a single subunit contains both the proteolytic active site and the ATPase. Lon is responsible for the degradation of the cell division inhibitor SulA; lon mutants are UV sensitive, due to the stabilization of SulA. lon mutants are also mucoid, due to the stabilization of another Lon substrate, the positive regulator of capsule transcription, RcsA. The overproduction of ClpYQ suppresses both of these phenotypes, and the suppression of UV sensitivity is accompanied by a restoration of the rapid degradation of SulA. Inactivation of the chromosomal copy of clpY or clpQ leads to further stabilization of SulA in a lon mutant but not in lon+ cells. While either lon, lon clpY, or lon clpQ mutants are UV sensitive at low temperatures, at elevated temperatures the lon mutant loses its UV sensitivity, while the double mutants do not. Therefore, the degradation of SulA by ClpYQ at elevated temperatures is sufficient to lead to UV resistance. Thus, a protease with a structure and an active site different from those of Lon is capable of recognizing and degrading two different Lon substrates and appears to act as a backup for Lon under certain conditions.

ClpE, a novel type of HSP100 ATPase, is part of the CtsR heat shock regulon of Bacillus subtilis.

Clp ATPases, which include the ubiquitous HSP100 family, are classified according to their structural features and sequence similarities. During the course of the Bacillus subtilis genome sequencing project, we identified a gene encoding a new member of the HSP100 family. We designated this protein ClpE, as it is the prototype of a novel subfamily among the Clp ATPases, and have identified homologues in several bacteria, including Listeria monocytogenes, Enterococcus faecalis, Streptococcus pyogenes, Streptococcus pneumoniae, Lactobacillus sakei and Clostridium acetobutylicum. A unique feature of these Hsp100-type Clp ATPases is their amino-terminal zinc finger motif. Unlike the other class III genes of B. subtilis (clpC and clpP ), clpE does not appear to be required for stress tolerance. Transcriptional analysis revealed two sigmaA-type promoters, expression from which was shown to be inducible by heat shock and puromycin treatment. Investigation of the regulatory mechanism controlling clpE expression indicates that this gene is controlled by CtsR and is thus a member of the class III heat shock genes of B. subtilis. CtsR negatively regulates clpE expression by binding to the promoter region, in which five CtsR binding sites were identified through DNase I footprinting and sequence analysis.

ClpE, a novel member of the HSP100 family, is involved in cell division and virulence of Listeria monocytogenes.

We identified, in the facultative intracellular pathogen Listeria monocytogenes, a previously unknown Clp ATPase, unique among the HSP100 proteins because of the presence of a short N-terminal region with a potential zinc finger motif. This protein of 726 amino acids is highly homologous to ClpE of Bacillus subtilis, and is a member of a new subfamily of HSP100/Clp ATPases. The clpE gene is transcribed as a monocistronic mRNA from a typical consensus sigma A promoter. clpE is not stimulated by various stresses, but is upregulated in a clpC mutant. This is the first example of cross-regulation between Clp ATPases. By constructing a clpE mutant of L. monocytogenes, we found that ClpE is required for prolonged survival at 42 degrees C and is involved in the virulence of this pathogen. A double mutant deficient in both ClpE and ClpC was avirulent in a mouse model and completely eliminated in the liver. Electron microscopy studies did not show any morphological alterations in clpE or clpC mutants. In the clpE-clpC double mutant, however, cell division was affected, indicating that ClpE acts synergistically with ClpC in cell septation. These results show that the Clp chaperones play a crucial role in both cell division and virulence of L. monocytogenes.

A regulatory switch involving a Clp ATPase.

Clp ATPase chaperone proteins are found in procaryotes and eucaryotes. Recently, ClpC of Bacillus subtilis was found to be part of a regulatory switch(1). ClpC, in combination with the MecA and ComS proteins, regulates the activity of a transcription factor, ComK, which is necessary for the development of genetic competence (the ability to bind and take up exogenous DNA). The complex of ClpC:MecA:ComK renders ComK inactive. Interaction between ComS and the ternary complex releases active ComK. This regulatory switch controls ComK activity in response to cell density signals that affect production of ComS. Regulated interaction between Clp ATPase and target proteins might prove to be widespread.

The Clp ATPases define a novel class of molecular chaperones.

The Clp ATPases were originally identified as a regulatory component of the bacterial ATP-dependent Clp serine proteases. Proteins homologous to the Escherichia coli Clp ATPases (ClpA, B, X or Y) have been identified in every organism examined so far. Recent data suggest that the Clp ATPases are not only specificity factors which help to 'present' various protein substrates to the ClpP or other catalytic proteases, but are also molecular chaperones which can function independently of ClpP. This review discusses the recent evidence that the Clp ATPases are indeed molecular chaperones capable of either repairing proteins damaged during stress conditions or activating the initiation proteins for Mu, lambda or P1 DNA replication. A mechanism is suggested to explain how the Clp ATPases 'decide' whether to repair or destroy their protein substrates.

Stress induction of clpC in Bacillus subtilis and its involvement in stress tolerance.

A member of the clpC subfamily of stress response-related Clp ATPases was cloned from Bacillus subtilis. The B. subtilis clpC gene was induced in response to various stresses, including heat shock. Its product was identified as a general stress protein (Gsp12) described previously. A dramatic increase in the amount of clpC mRNA immediately after exposure to multiple stresses suggested regulation on a transcriptional level. Induction by heat shock was independent of the alternative sigma factor SigB, indicating a new mechanism of heat shock induction in B. subtilis. A clpC insertional mutant had an impaired tolerance for heat shock and salt stress. Furthermore, the mutation triggered the formation of elongated cells, a phenomenon particularly pronounced during stress.