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.     

Visualization of substrate binding and translocation by the ATP-dependent protease, ClpXP

Binding and internalization of a protein substrate by E. coli ClpXP was investigated by electron microscopy. In sideviews of ATP gamma S-stabilized ClpXP complexes, a narrow axial channel was visible in ClpX, surrounded by protrusions on its distal surface. When substrate lambda O protein was added, extra density attached to this surface. Upon addition of ATP, this density disappeared as lambda O was degraded. When ATP was added to proteolytically inactive ClpXP-lambda O complexes, the extra density transferred to the center of ClpP and remained inside ClpP after separation from ClpX. We propose that substrates of ATP-dependent proteases bind to specific sites on the distal surface of the ATPase, and are subsequently unfolded and translocated into the internal chamber of the protease.     

Crystallography and mutagenesis point to an essential role for the N-terminus of human mitochondrial ClpP

We have determined a 2.1 A crystal structure for human mitochondrial ClpP (hClpP), the proteolytic component of the ATP-dependent ClpXP protease. HClpP has a structure similar to that of the bacterial enzyme, with the proteolytic active sites sequestered within an aqueous chamber formed by face-to-face assembly of the two heptameric rings. The hydrophobic N-terminal peptides of the subunits are bound within the narrow (12 A) axial channel, positioned to interact with unfolded substrates translocated there by the associated ClpX chaperone. Mutation or deletion of these residues causes a drastic decrease in ClpX-mediated protein and peptide degradation. Residues 8-16 form a mobile loop that extends above the ring surface and is also required for activity. The 28 amino acid C-terminal domain, a unique feature of mammalian ClpP proteins, lies on the periphery of the ring, with its proximal portion forming a loop that extends out from the ring surface. Residues at the start of the C-terminal domain impinge on subunit interfaces within the ring and affect heptamer assembly and stability. We propose that the N-terminal peptide of ClpP is a structural component of the substrate translocation channel and may play an important functional role as well.     

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.     

Structural determinants stabilizing the axial channel of ClpP for substrate translocation

Acyldepsipeptides (ADEPs) antibiotics bind to Escherichia coli ClpP mimicking the interactions that the IGL/F loops in ClpA or ClpX ATPases establish with the hydrophobic pockets surrounding the axial pore of the tetradecamer that the protease forms. ADEP binding induces opening of the gates blocking the axial channel of ClpP and allowing protein substrates to be translocated and hydrolysed in the degradation chamber. To identify the structural determinants stabilizing the open conformation of the axial channel for efficient substrate translocation, we constructed ClpP variants with amino acid substitutions in the N‐terminal region that forms the axial gates. We found that adoption of a β‐hairpin loop by this region and the integrity of the hydrophobic cluster at the base of this loop are necessary elements for the axial gate to efficiently translocate protein substrates. Analysis of ClpP variants from Bacillus subtilis suggested that the identified structural requirements of the axial channel for efficient translocation are conserved between Gram‐positive and Gram‐negative bacteria. These findings provide mechanistic insights into the activation of ClpP by ADEPs as well as the gating mechanism of the protease in the context of the ClpAP and ClpXP complexes.     

Roles of the ClpX IGF loops in ClpP association,dissociation, and protein degradation

IGF‐motif loops project from the hexameric ring of ClpX and are required for docking with the self‐compartmentalized ClpP peptidase, which consists of heptameric rings stacked back‐to‐back. Here, we show that ATP or ATPγS support assembly by changing the conformation of the ClpX ring, bringing the IGF loops closer to each other and allowing efficient multivalent contacts with docking clefts on ClpP. In single‐chain ClpX pseudohexamers, deletion of one or two IGF loops modestly slows association with ClpP but strongly accelerates dissociation of ClpXP complexes. We probe how changes in the sequence and length of the IGF loops affect ClpX–ClpP interactions and show that deletion of one or two IGF loops slows ATP‐dependent proteolysis by ClpXP. We also find that ClpXP degradation is less processive when two IGF loops are deleted.     

ClpP: a structurally dynamic protease regulated by AAA+ proteins

Proteolysis is an important process for many aspects of bacterial physiology. Clp proteases carry out a large proportion of protein degradation in bacteria. These enzymes assemble in complexes that combine the protease ClpP and the unfoldase, ClpA or ClpX. ClpP oligomerizes as two stacked heptameric rings enclosing a central chamber containing the proteolytic sites. ClpX and ClpA assemble into hexameric rings that bind both axial surfaces of the ClpP tetradecamer forming a barrel-like complex. ClpP requires association with ClpA or ClpX to unfold and thread protein substrates through the axial pore into the inner chamber where degradation occurs. A gating mechanism regulated by the ATPase exists at the entry of the ClpP axial pore and involves the N-terminal regions of the ClpP protomers. These gating motifs are located at the axial regions of the tetradecamer but in most crystal structures they are not visible. We also lack structural information about the ClpAP or ClpXP complexes. Therefore, the structural details of how the axial gate in ClpP is regulated by the ATPases are unknown. Here, we review our current understanding of the conformational changes that ClpA or ClpX induce in ClpP to open the axial gate and increase substrate accessibility into the degradation chamber. Most of this knowledge comes from the recent crystal structures of ClpP in complex with acyldepsipeptides (ADEP) antibiotics. These small molecules are providing new insights into the gating mechanism of this protease because they imitate the interaction of ClpA/ClpX with ClpP and activate its protease activity.     

Control of Substrate Gating and Translocation into ClpP by Channel Residues and ClpX Binding

ClpP is a self-compartmentalized protease, which has very limited degradation activity unless it associates with ClpX to form ClpXP or with ClpA to form ClpAP. Here, we show that ClpX binding stimulates ClpP cleavage of peptides larger than a few amino acids and enhances ClpP active-site modification. Stimulation requires ATP binding but not hydrolysis by ClpX. The magnitude of this enhancement correlates with increasing molecular weight of the molecule entering ClpP. Amino-acid substitutions in the channel loop or helix A of ClpP enhance entry of larger substrates into the free enzyme, eliminate ClpX binding in some cases, and are not further stimulated by ClpX binding in other instances. These results support a model in which the channel residues of free ClpP exclude efficient entry of all but the smallest peptides into the degradation chamber, with ClpX binding serving to relieve these inhibitory interactions. Specific ClpP channel variants also prevent ClpXP translocation of certain amino-acid sequences, suggesting that the wild-type channel plays an important role in facilitating broad translocation specificity. In combination with previous studies, our results indicate that collaboration between ClpP and its partner ATPases opens a gate that functions to exclude larger substrates from isolated ClpP.     

Communication between ClpX and ClpP during substrate processing and degradation

In the ClpXP compartmental protease, ring hexamers of the AAA(+) ClpX ATPase bind, denature and then translocate protein substrates into the degradation chamber of the double-ring ClpP(14) peptidase. A key question is the extent to which functional communication between ClpX and ClpP occurs and is regulated during substrate processing. Here, we show that ClpX-ClpP affinity varies with the protein-processing task of ClpX and with the catalytic engagement of the active sites of ClpP. Functional communication between symmetry-mismatched ClpXP rings depends on the ATPase activity of ClpX and seems to be transmitted through structural changes in its IGF loops, which contact ClpP. A conserved arginine in the sensor II helix of ClpX links the nucleotide state of ClpX to the binding of ClpP and protein substrates. A simple model explains the observed relationships between ATP binding, ATP hydrolysis and functional interactions between ClpX, protein substrates and ClpP.     

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.     

Dynamics of substrate denaturation and translocation by the ClpXP degradation machine

ClpXP is a protein machine composed of the ClpX ATPase, a member of the Clp/Hsp100 family of remodeling enzymes, and the ClpP peptidase. Here, ClpX and ClpXP are shown to catalyze denaturation of GFP modified with an ssrA degradation tag. ClpX translocates this denatured protein into the proteolytic chamber of ClpP and, when proteolysis is blocked, also catalyzes release of denatured GFP-ssrA from ClpP in a reaction that requires ATP and additional substrate. Kinetic experiments reveal that multiple reaction steps require collaboration between ClpX and ClpP and that denaturation is the rate-determining step in degradation. These insights into the mechanism of ClpXP explain how it executes efficient degradation in a manner that is highly specific for tagged proteins, irrespective of their intrinsic stabilities.     

Large nucleotide-dependent movement of the N-terminal domain of the ClpX chaperone

The ClpXP ATPase-protease complex is a major component of the protein quality control machinery in the cell. A ClpX subunit consists of an N-terminal zinc binding domain (ZBD) and a C-terminal AAA+ domain. ClpX oligomerizes into a hexamer with the AAA+ domains forming the base of the hexamer and the ZBDs extending out of the base. Here, we report that ClpX switches between a capture and a feeding conformation. ZBDs in ClpX undergo large nucleotide-dependent block movement towards ClpP and into the AAA+ ring. This motion is modulated by the ClpX cofactor, SspB. Evidence for this movement was initially obtained by the surprising observation that an N-terminal extension on ClpX is clipped by bound ClpP in functional ClpXP complexes. Protease-protection, crosslinking, and light scattering experiments further support these findings.     

ClpXP, an ATP-powered unfolding and protein-degradation machine

ClpXP is a AAA+ protease that uses the energy of ATP binding and hydrolysis to perform mechanical work during targeted protein degradation within cells. ClpXP consists of hexamers of a AAA+ ATPase (ClpX) and a tetradecameric peptidase (ClpP). Asymmetric ClpX hexamers bind unstructured peptide tags in protein substrates, unfold stable tertiary structure in the substrate, and then translocate the unfolded polypeptide chain into an internal proteolytic compartment in ClpP. Here, we review our present understanding of ClpXP structure and function, as revealed by two decades of biochemical and biophysical studies.     

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.     

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.     

Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine

All cells employ ATP-powered proteases for protein-quality control and regulation. In the ClpXP protease, ClpX is a AAA+ machine that recognizes specific protein substrates, unfolds these molecules, and then translocates the denatured polypeptide through a central pore and into ClpP for degradation. Here, we use optical-trapping nanometry to probe the mechanics of enzymatic unfolding and translocation of single molecules of a multidomain substrate. Our experiments demonstrate the capacity of ClpXP and ClpX to perform mechanical work under load, reveal very fast and highly cooperative unfolding of individual substrate domains, suggest a translocation step size of 5-8 amino acids, and support a power-stroke model of denaturation in which successful enzyme-mediated unfolding of stable domains requires coincidence between mechanical pulling by the enzyme and a transient stochastic reduction in protein stability. We anticipate that single-molecule studies of the mechanical properties of other AAA+ proteolytic machines will reveal many shared features with ClpXP.     

An essential protease involved in bacterial cell-cycle control.

Proteolytic inactivation of key regulatory proteins is essential in eukaryotic cell-cycle control. We have identified a protease in the eubacterium Caulobacter crescentus that is indispensable for viability and cell-cycle progression, indicating that proteolysis is also involved in controlling the bacterial cell cycle. Mutants of Caulobacter that lack the ATP-dependent serine protease ClpXP are arrested in the cell cycle before the initiation of chromosome replication and are blocked in the cell division process. ClpXP is composed of two types of polypeptides, the ClpX ATPase and the ClpP peptidase. Site-directed mutagenesis of the catalytically active serine residue of ClpP confirmed that the proteolytic activity of ClpXP is essential. Analysis of mutants lacking ClpX or ClpP revealed that both proteins are required in vivo for the cell-cycle-dependent degradation of the regulatory protein CtrA. CtrA is a member of the response regulator family of two-component signal transduction systems and controls multiple cell-cycle processes in Caulobacter. In particular, CtrA negatively controls DNA replication and our findings suggest that specific degradation of the CtrA protein by the ClpXP protease contributes to G1-to-S transition in this organism.     

Distinct static and dynamic interactions control ATPase-peptidase communication in a AAA+ protease

In the ClpXP proteolytic machine, ClpX uses the energy of ATP hydrolysis to unfold protein substrates and translocate them through a central pore and into the degradation chamber of ClpP. Here, we demonstrate a bipartite system of ClpX-ClpP interactions that serves multiple functional roles. High-affinity contacts between six loops near the periphery of the hexameric ClpX ring and a ClpP ring establish correct positioning and increase degradation activity but are insensitive to nucleotide state. These static peripheral interactions maintain a stable ClpXP complex, while other parts of this machine change conformation hundreds of times per minute. By contrast, relatively weak axial contacts between loops at the bottom of the ClpX central channel and N-terminal loops of ClpP vary dynamically with the nucleotide state of individual ClpX subunits, control ATP-hydrolysis rates, and facilitate efficient protein unfolding. Thus, discrete static and dynamic interactions mediate binding and communication between ClpX and ClpP.     

The structural basis for the activation and peptide recognition of bacterial ClpP

ClpP and its ATPase compartment, ClpX or ClpA, remove misfolded proteins in cells and are of utmost importance in protein quality control. The ring hexamers of ClpA or ClpX recognize, unfold, and translocate target substrates into the degradation chamber of the double-ring tetradecamer of ClpP. The overall reaction scheme catalyzed by ClpXP or ClpAP has been proposed; however, the molecular mechanisms associated with substrate recognition and degradation have not yet been clarified in detail. To investigate these mechanisms, we determined the crystal structures of ClpP from Helicobacter pylori in complex with product peptides bound to the active site as well as in the apo state. In the complex structure, the peptides are zipped with two antiparallel strands of ClpP and point to the adjacent active site, thus providing structural explanations for the broad substrate specificity, the product inhibition and the processive degradation of substrates in the chamber. The structures also suggest that substrate binding causes local conformational changes around the active site that ultimately induce the active conformation of ClpP.     

The Mycobacterium tuberculosis ClpP1P2 Protease Interacts Asymmetrically with Its ATPase Partners ClpX and ClpC1

Clp chaperone-proteases are cylindrical complexes built from ATP-dependent chaperone rings that stack onto a proteolytic ClpP double-ring core to carry out substrate protein degradation. Interaction of the ClpP particle with the chaperone is mediated by an N-terminal loop and a hydrophobic surface patch on the ClpP ring surface. In contrast to E. coli, Mycobacterium tuberculosis harbors not only one but two ClpP protease subunits, ClpP1 and ClpP2, and a homo-heptameric ring of each assembles to form the ClpP1P2 double-ring core. Consequently, this hetero double-ring presents two different potential binding surfaces for the interaction with the chaperones ClpX and ClpC1. To investigate whether ClpX or ClpC1 might preferentially interact with one or the other double-ring face, we mutated the hydrophobic chaperone-interaction patch on either ClpP1 or ClpP2, generating ClpP1P2 particles that are defective in one of the two binding patches and thereby in their ability to interact with their chaperone partners. Using chaperone-mediated degradation of ssrA-tagged model substrates, we show that both Mycobacterium tuberculosis Clp chaperones require the intact interaction face of ClpP2 to support degradation, resulting in an asymmetric complex where chaperones only bind to the ClpP2 side of the proteolytic core. This sets the Clp proteases of Mycobacterium tuberculosis, and probably other Actinobacteria, apart from the well-studied E. coli system, where chaperones bind to both sides of the protease core, and it frees the ClpP1 interaction interface for putative new binding partners.