Human mitochondrial ClpP is a stable heptamer that assembles into a tetradecamer in the presence of ClpX

The functional form of ClpP, the proteolytic component of ATP-dependent Clp proteases, is a hollow-cored particle composed of two heptameric rings joined face-to-face forming an aqueous chamber containing the proteolytic active sites. We have found that isolated human mitochondrial ClpP (hClpP) is stable as a heptamer and remains a monodisperse species (s(20,w) 7.0 S; M(app) 169, 200) at concentrations > or = 3 mg/ml. Heptameric hClpP has no proteolytic activity and very low peptidase activity. In the presence of ATP, hClpX interacts with hClpP forming a complex, which by equilibrium sedimentation measurements has a M(app) of 1 x 10(6). Electron microscopy confirmed that the complex consisted of a double ring of hClpP with an hClpX ring axially aligned on each end. The hClpXP complex has protease activity and greatly increased peptidase activity, indicating that interaction with hClpX affects the conformation of the hClpP catalytic active site. A mutant of hClpP, in which a cysteine residue was introduced into the handle region at the interface between the two rings formed stable tetradecamers under oxidizing conditions but spontaneously dissociated into two heptamers upon reduction. Thus, hClpP rings interact transiently but very weakly in solution, and hClpX must exert an allosteric effect on hClpP to promote a conformation that stabilizes the tetradecamer. These data suggest that hClpX can regulate the appearance of hClpP peptidase activity in mitochondria and might affect the nature of the degradation products released during ATP-dependent proteolytic cycles.     

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.     

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.     

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.     

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.     

Molecular determinants of complex formation between Clp/Hsp100 ATPases and the ClpP peptidase

The Clp/Hsp100 ATPases are hexameric protein machines that catalyze the unfolding, disassembly and disaggregation of specific protein substrates in bacteria, plants and animals. Many family members also interact with peptidases to form ATP-dependent proteases. In Escherichia coli, for instance, the ClpXP protease is assembled from the ClpX ATPase and the ClpP peptidase. Here, we have used multiple sequence alignments to identify a tripeptide 'IGF' in E. coli ClpX that is essential for ClpP recognition. Mutations in this IGF sequence, which appears to be part of a surface loop, disrupt ClpXP complex formation and prevent protease function but have no effect on other ClpX activities. Homologous tripeptides are found only in a subset of Clp/Hsp100 ATPases and are a good predictor of family members that have a ClpP partner. Mapping of the IGF loop onto a homolog of known structure suggests a model for ClpX-ClpP docking.     

Recent structural insights into the mechanism of ClpP protease regulation by AAA+ chaperones and small molecules

ClpP is a highly conserved serine protease that is a critical enzyme in maintaining protein homeostasis and is an important drug target in pathogenic bacteria and various cancers. In its functional form, ClpP is a self-compartmentalizing protease composed of two stacked heptameric rings that allow protein degradation to occur within the catalytic chamber. ATPase chaperones such as ClpX and ClpA are hexameric ATPases that form larger complexes with ClpP and are responsible for the selection and unfolding of protein substrates prior to their degradation by ClpP. Although individual structures of ClpP and ATPase chaperones have offered mechanistic insights into their function and regulation, their structures together as a complex have only been recently determined to high resolution. Here, we discuss the cryoelectron microscopy structures of ClpP-ATPase complexes and describe findings previously inaccessible from individual Clp structures, including how a hexameric ATPase and a tetradecameric ClpP protease work together in a functional complex. We then discuss the consensus mechanism for substrate unfolding and translocation derived from these structures, consider alternative mechanisms, and present their strengths and limitations. Finally, new insights into the allosteric control of ClpP gained from studies using small molecules and gain or loss-of-function mutations are explored. Overall, this review aims to underscore the multilayered regulation of ClpP that may present novel ideas for structure-based drug design.     

Alternating translocation of protein substrates from both ends of ClpXP protease

In ClpXP protease complexes, hexameric rings of the ATP-dependent ClpX chaperone stack on one or both faces of the double-heptameric rings of ClpP. We used electron microscopy to record the initial binding of protein substrates to ClpXP and their accumulation inside proteolytically inactive ClpP. Proteins with N- or C-terminal recognition motifs bound to complexes at the distal surface of ClpX and, upon addition of ATP, were translocated to ClpP. With a partially translocated substrate, the non-translocated portion remained on the surface of ClpX, aligned with the central axis of the complex, confirming that translocation proceeds through the axial channel of ClpXP. Starting with substrate bound on both ends, most complexes translocated substrate from only one end, and rarely (<5%) from both ends. We propose that translocation from one side is favored for two reasons: initiation of translocation is infrequent, making the probability of simultaneous initiation low; and, further, the presence of protein within the cis side translocation channel or within ClpP generates an inhibitory signal blocking translocation from the trans side.     

Structural and functional insights into the chloroplast ATP-dependent Clp protease in Arabidopsis

In contrast with the model Escherichia coli Clp protease, the ATP-dependent Clp protease in higher plants has a remarkably diverse proteolytic core consisting of multiple ClpP and ClpR paralogs, presumably arranged within a dual heptameric ring structure. Using antisense lines for the nucleus-encoded ClpP subunit, ClpP6, we show that the Arabidopsis thaliana Clp protease is vital for chloroplast development and function. Repression of ClpP6 produced a proportional decrease in the Clp proteolytic core, causing a chlorotic phenotype in young leaves that lessened upon maturity. Structural analysis of the proteolytic core revealed two distinct subcomplexes that likely correspond to single heptameric rings, one containing the ClpP1 and ClpR1-4 proteins, the other containing ClpP3-6. Proteomic analysis revealed several stromal proteins more abundant in clpP6 antisense lines, suggesting that some are substrates for the Clp protease. A proteolytic assay developed for intact chloroplasts identified potential substrates for the stromal Clp protease in higher plants, most of which were more abundant in young Arabidopsis leaves, consistent with the severity of the chlorotic phenotype observed in the clpP6 antisense lines. The identified substrates all function in more general housekeeping roles such as plastid protein synthesis, folding, and quality control, rather than in metabolic activities such as photosynthesis.     

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.     

Crystal structure of ClpX molecular chaperone from Helicobacter pylori

ClpX, a heat shock protein 100 chaperone, which acts as the regulatory subunit of the ATP-dependent ClpXP protease, is responsible for intracellular protein remodeling and degradation. To provide a structural basis for a better understanding of the function of the Clp ATPase family, the crystal structures of Helicobacter pylori ClpX, lacking an N-terminal Cys cluster region complexed with ADP, was determined. The overall structure of ClpX is similar to that of heat shock locus U (HslU), consisting of two subdomains, with ADP bound at the subdomain interface. The crystal structure of ClpX reveals that a conserved tripeptide (LGF) is located on the tip of ClpP binding loop extending from the N-terminal subdomain. A hexameric model of ClpX suggests that six tripeptides make hydrophobic contacts with the hydrophobic clefts of the ClpP heptmer asymmetrically. In addition, the nucleotide binding environment provides the structural explanation for the hexameric assembly and the modulation of ATPase activity.     

Subunit stoichiometry, evolution, and functional implications of an asymmetric plant plastid ClpP/R protease complex in Arabidopsis

The caseinolytic protease (Clp) protease system has been expanded in plant plastids compared with its prokaryotic progenitors. The plastid Clp core protease consists of five different proteolytic ClpP proteins and four different noncatalytic ClpR proteins, with each present in one or more copies and organized in two heptameric rings. We determined the exact subunit composition and stoichiometry for the intact core and each ring. The chloroplast ClpP/R protease was affinity purified from clpr4 and clpp3 Arabidopsis thaliana null mutants complemented with C-terminal StrepII-tagged versions of CLPR4 and CLPP3, respectively. The subunit stoichiometry was determined by mass spectrometry-based absolute quantification using stable isotope-labeled proteotypic peptides generated from a synthetic gene. One heptameric ring contained ClpP3,4,5,6 in a 1:2:3:1 ratio. The other ring contained ClpP1 and ClpR1,2,3,4 in a 3:1:1:1:1 ratio, resulting in only three catalytic sites. These ClpP1/R1-4 proteins are most closely related to the two subunits of the cyanobacterial P3/R complex and the identical P:R ratio suggests conserved adaptation. Furthermore, the plant-specific C-terminal extensions of the ClpP/R subunits were not proteolytically removed upon assembly, suggesting a regulatory role in Clp chaperone interaction. These results will now allow testing ClpP/R structure-function relationships using rationale design. The quantification workflow we have designed is applicable to other protein complexes.     

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.     

New insights into the ATP-dependent Clp protease: Escherichia coli and beyond

Proteolysis functions as a precise regulatory mechanism for a broad spectrum of cellular processes. Such control impacts not only on the stability of key metabolic enzymes but also on the effective removal of terminally damaged polypeptides. Much of this directed protein turnover is performed by proteases that require ATP and, of those in bacteria, the Clp protease from Escherichia coli is one of the best characterized to date. The Clp holoenzyme consists of two adjacent heptameric rings of the proteolytic subunit known as ClpP, which are flanked by a hexameric ring of a regulatory subunit from the Clp/Hsp100 chaperone family at one or both ends. The recently resolved three-dimensional structure of the E. coli ClpP protein has provided new insights into its interaction with the regulatory/chaperone subunits. In addition, an increasing number of studies over the last few years have recognized the added complexity and functional importance of ClpP proteins in other eubacteria and, in particular, in photosynthetic organisms ranging from cyanobacteria to higher plants. The goal of this review is to summarize these recent findings and to highlight those areas that remain unresolved.     

Distinctive types of ATP-dependent Clp proteases in cyanobacteria

Cyanobacteria are the only prokaryotes that perform oxygenic photosynthesis and are thought to be ancestors to plant chloroplasts. Like chloroplasts, cyanobacteria possess a diverse array of proteolytic enzymes, with one of the most prominent being the ATP-dependent Ser-type Clp protease. The model Clp protease in Escherichia coli consists of a single ClpP proteolytic core flanked on one or both ends by a HSP100 chaperone partner. In comparison, cyanobacteria have multiple ClpP paralogs plus a ClpP variant (ClpR), which lacks the catalytic triad typical of Ser-type proteases. In this study, we reveal that two distinct soluble Clp proteases exist in the unicellular cyanobacterium Synechococcus elongatus. Each protease consists of a unique proteolytic core comprised of two separate Clp subunits, one with ClpP1 and ClpP2, the other with ClpP3 and ClpR. Each core also associates with a particular HSP100 chaperone partner, ClpC in the case of the ClpP3/R core, and ClpX for the ClpP1/P2 core. The two adaptor proteins, ClpS1 and ClpS2 also interact with the ClpC chaperone protein, likely increasing the range of protein substrates targeted by the Clp protease in cyanobacteria. We also reveal the possible existence of a third Clp protease in Synechococcus, one which associates with the internal membrane network. Altogether, we show that presence of several distinctive Clp proteases in cyanobacteria, a feature which contrasts from that in most other organisms.     

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.     

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.     

The asymmetry in the mature amino-terminus of ClpP facilitates a local symmetry match in ClpAP and ClpXP complexes

ClpP is a self-compartmentalized proteolytic assembly comprised of two, stacked, heptameric rings that, when associated with its cognate hexameric ATPase (ClpA or ClpX), form the ClpAP and ClpXP ATP-dependent protease, respectively. The symmetry mismatch is an absolute feature of this large energy-dependent protease and also of the proteasome, which shares a similar barrel-shaped architecture, but how it is accommodated within the complex has yet to be understood, despite recent structural investigations, due in part to the conformational lability of the N-termini. We present the structures of Escherichia coli ClpP to 1.9A and an inactive variant that provide some clues for how this might be achieved. In the wild type protein, the highly conserved N-terminal 20 residues can be grouped into two major structural classes. In the first, a loop formed by residues 10-15 protrudes out of the central access channel extending approximately 12-15A from the surface of the oligomer resulting in the closing of the access channel observed in one ring. Similar loops are implied to be exclusively observed in human ClpP and a variant of ClpP from Streptococcus pneumoniae. In the other ring, a second class of loop is visible in the structure of wt ClpP from E. coli that forms closer to residue 16 and faces toward the interior of the molecule creating an open conformation of the access channel. In both classes, residues 18-20 provide a conserved interaction surface. In the inactive variant, a third class of N-terminal conformation is observed, which arises from a conformational change in the position of F17. We have performed a detailed functional analysis on each of the first 20 amino acid residues of ClpP. Residues that extend beyond the plane of the molecule (10-15) have a lesser effect on ATPase interaction than those lining the pore (1-7 and 16-20). Based upon our structure-function analysis, we present a model to explain the widely disparate effects of individual residues on ClpP-ATPase complex formation and also a possible functional reason for this mismatch.     

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.