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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Diverse pore loops of the AAA+ ClpX machine mediate unassisted and adaptor-dependent recognition of ssrA-tagged substrates

ClpX, an archetypal proteolytic AAA+ unfoldase, must engage the ssrA tags of appropriate substrates prior to ATP-dependent unfolding and translocation of the denatured polypeptide into ClpP for degradation. Here, specificity-transplant and disulfide-crosslinking experiments reveal that the ssrA tag interacts with different loops that form the top, middle, and lower portions of the central channel of the ClpX hexamer. Our results support a two-step binding mechanism, in which the top loop serves as a specificity filter and the remaining loops form a binding site for the peptide tag relatively deep within the pore. Crosslinking experiments suggest a staggered arrangement of pore loops in the hexamer and nucleotide-dependent changes in pore-loop conformations. This mechanism of initial tag binding would allow ATP-dependent conformational changes in one or more pore loops to drive peptide translocation, force unfolding, and mediate threading of the denatured protein through the ClpX pore.     

Cytoplasmic degradation of ssrA-tagged proteins

Degradation of ssrA-tagged proteins is a central feature of protein-quality control in all bacteria. In Escherichia coli, the ATP-dependent ClpXP and ClpAP proteases are thought to participate in this process, but their relative contributions to degradation of ssrA-tagged proteins in vivo have been uncertain because two adaptor proteins, ClpS and SspB, can modulate proteolysis of these substrates. Here, intracellular levels of these protease components and adaptors were determined during exponential growth and as cells entered early stationary phase. Levels of ClpA and ClpP increased about threefold during this transition, whereas ClpX, ClpS and SspB levels remained nearly constant. Using GFP-ssrA expressed from the chromosome as a degradation reporter, the effects of altered concentrations of different protease components or adaptor proteins were explored. Both ClpXP and ClpAP degraded GFP-ssrA in the cell, demonstrating that wild-type levels of SspB and ClpS do not inhibit ClpAP completely. Upon entry into stationary phase, increased levels of ClpAP resulted in increased degradation of ssrA-tagged substrates. As measured by maximum turnover rates, ClpXP degradation of GFP-ssrA in vivo was significantly more efficient than in vitro. Surprisingly, ClpX-dependent ClpP-independent degradation of GFP-ssrA was also observed. Thus, unfolding of this substrate by ClpX appears to enhance intracellular degradation by other proteases.     

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.     

Energy-dependent degradation: Linkage between ClpX-catalyzed nucleotide hydrolysis and protein-substrate processing

ClpX requires ATP to unfold protein substrates and translocate them into the proteolytic chamber of ClpP for degradation. The steady-state parameters for hydrolysis of ATP and ATPgammaS by ClpX were measured with different protein partners and the kinetics of degradation of ssrA-tagged substrates were determined with both nucleotides. ClpX hydrolyzed ATPgammaS to ADP and thiophosphate at a rate (6/min) significantly slower than ATP hydrolysis (140/min), but the hydrolysis of both nucleotides was increased by ssrA-tagged substrates and decreased by ClpP. K(M) and k(cat) for hydrolysis of ATP and ATPgammaS were linearly correlated over a 200-fold range, suggesting that protein partners largely affect k(cat) rather than nucleotide binding, indicating that most bound ATP leaves the enzyme by hydrolysis rather than dissociation, and placing an upper limit of approximately 15 micro M on K(D) for both nucleotides. Competition studies with ClpX and fluorescently labeled ADP gave inhibition constants for ATPgammaS ( approximately 2 micro M) and ADP ( approximately 3 micro M) under the reaction conditions used for steady-state kinetics. In the absence of Mg(2+), where hydrolysis does not occur, the inhibition constant for ATP ( approximately 55 micro M) was weaker but very similar to the value for ATPgammaS ( approximately 45 micro M). Compared with ATP, ATPgammaS supported slow but roughly comparable rates of ClpXP degradation for two Arc-ssrA substrates and denatured GFP-ssrA, but not of native GFP-ssrA. These results show that the processing of protein substrates by ClpX is closely coupled to the maximum rate of nucleotide hydrolysis.     

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.     

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.     

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.     

Asymmetric interactions of ATP with the AAA+ ClpX6 unfoldase: allosteric control of a protein machine

ATP hydrolysis by AAA+ ClpX hexamers powers protein unfolding and translocation during ClpXP degradation. Although ClpX is a homohexamer, positive and negative allosteric interactions partition six potential nucleotide binding sites into three classes with asymmetric properties. Some sites release ATP rapidly, others release ATP slowly, and at least two sites remain nucleotide free. Recognition of the degradation tag of protein substrates requires ATP binding to one set of sites and ATP or ADP binding to a second set of sites, suggesting a mechanism that allows repeated unfolding attempts without substrate release over multiple ATPase cycles. Our results rule out concerted hydrolysis models involving ClpX(6)*ATP(6) or ClpX(6)*ADP(6) and highlight structures of hexameric AAA+ machines with three or four nucleotides as likely functional states. These studies further emphasize commonalities between distant AAA+ family members, including protein and DNA translocases, helicases, motor proteins, clamp loaders, and other ATP-dependent enzymes.