Michael R. Maurizi

Enzymatic and Structural Similarities between the Escherichia coli ATP-dependent Proteases, ClpXP and ClpAP*

Escherichia coli ClpX, a member of the Clp family of ATPases, has ATP-dependent chaperone activity and is required for specific ATP-dependent proteolytic activities expressed by ClpP. Gel filtration and electron microscopy showed that ClpX subunits (M r46,000) associate to form a six-membered ring (M r ∼ 280,000) that is stabilized by binding of ATP or nonhydrolyzable analogs of ATP. ClpP, which is composed of two seven-membered rings stacked face-to-face, interacts with the nucleotide-stabilized hexamer of ClpX to form a complex that could be isolated by gel filtration. Electron micrographs of negatively stained ClpXP preparations showed side views of 1:1 and 2:1 ClpXP complexes in which ClpP was flanked on either one or both sides by a ring of ClpX. Thus, as was seen for ClpAP, a symmetry mismatch exists in the bonding interactions between the seven-membered rings of ClpP and the six-membered rings of ClpX. Competition studies showed that ClpA may have a slightly higher affinity (∼2-fold) for binding to ClpP. Mixed complexes of ClpA, ClpX, and ClpP with the two ATPases bound simultaneously to opposite faces of a single ClpP molecule were seen by electron microscopy. In the presence of ATP or nonhydrolyzable analogs of ATP, ClpXP had nearly the same activity as ClpAP against oligopeptide substrates (>10,000 min−1/tetradecamer of ClpP). Thus, ClpX and ClpA interactions with ClpP result in structurally analogous complexes and induce similar conformational changes that affect the accessibility and the catalytic efficiency of ClpP active sites.

ATP-dependent degradation of CcdA by Lon protease. Effects of secondary structure and heterologous subunit interactions.

CcdA, the antidote protein of the ccd post-segregational killing system carried by the F plasmid, was degraded in vitro by purified Lon protease from Escherichia coli. CcdA had a low affinity for Lon (Km ≥200 µM), and the peptide bond turnover number was ∼10 min−1. CcdA formed tight complexes with purified CcdB, the killer protein encoded in the ccd operon, and fluorescence and hydrodynamic measurements suggested that interaction with CcdB converted CcdA to a more compact conformation. CcdB prevented CcdA degradation by Lon and blocked the ability of CcdA to activate the ATPase activity of Lon, suggesting that Lon may recognize bonding domains of proteins exposed when their partners are absent. Degradation of CcdA required ATP hydrolysis; however, CcdA41, consisting of the carboxyl-terminal 41 amino acids of CcdA and lacking the α-helical secondary structure present in CcdA, was degraded without ATP hydrolysis. Lon cleaved CcdA primarily between aliphatic and hydrophilic residues, and CcdA41 was cleaved at the same peptide bonds, indicating that ATP hydrolysis does not affect cleavage specificity. CcdA lost α-helical structure at elevated temperatures (Tm ∼50°C), and its degradation became independent of ATP hydrolysis at this temperature. ATP hydrolysis may be needed to disrupt interactions that stabilize the secondary structure of proteins allowing the disordered protein greater access to the proteolytic active sites.

Regulatory Subunits of Energy-Dependent Proteases

The regulatory components of the energy-dependent proteases provide controlled access to the proteolytic components, which innately possess broad specificity of peptide bond cleavage. The existence of multiple regulatory complexes capable of interacting with the same proteolytic component (e.g., ClpAP and ClpXP) provides a means of further regulation by increasing the range of substrate specificity of the protease without losing selectivity. Such a combinatorial approach to assembling different types of regulatory complexes may be used to direct degradative activity toward specific proteins or classes of proteins in different cell types or in response to regulatory signals. Once a substrate is recognized, ATP-dependent unfolding and translocation moves it into the proteolytic cavity. Interactions with additional factors can further modify both substrate selection and the specificity of peptide bond cleavage to control not only the proteins targeted but also the peptide output from proteolysis. By controlling these instruments of protein destruction, the cell has added enormously to its ability to regulate the levels and activities of important regulatory proteins.‡To whom correspondence should be addressed.

[25] ATP-dependent protease La (Lon) from Escherichia coli

Publisher Summary This chapter describes the isolation and assay of protease La from Escherichia coli and summarizes some of its enzymatic properties. Adenosine triphosphate (ATP)-dependent cytosolic protease La is product of the lon gene and plays an important role in intracellular protein degradation. Protease La serves as a paradigm for other ATP-dependent proteases from prokaryotic and eukaryotic cells, all of which use the energy of ATP hydrolysis to accelerate the rate-limiting steps in the kinetically driven degradation of proteins. This enzyme catalyzes the rate-limiting steps in the degradation of highly abnormal proteins in E. coli and certain short-lived regulatory proteins. This enzyme has ATPase and proteolytic activity and is a multimeric structure of high molecular mass. Protease La also has multiple modes of interaction with proteins such that the proteolytic active site remains in an inactive state until an appropriate substrate binds to an allosteric site on the enzyme. This binding step temporarily activates the enzyme and leads to rapid degradation of the bound protein. Enzymes closely homologous to protease La (Lon protease) appear to be widespread in nature.

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.

[23] Endopeptidase Clp: ATP-dependent Clp protease from Escherichia coli

Publisher Summary Escherichia coli Clp protease is a multicomponent protease that has an adenosine triphosphate (ATP)-activated proteolytic activity and an ATPase activity that is activated by proteins and peptides. This chapter describes purification and properties of two components of Clp protease—namely, ClpP and ClpA. These two components by themselves form an active complex, referred as “ClpAP protease,” responsible for degradation of specific classes of proteins. The regulatory subunit of Clp protease, ClpA, can be overexpressed in mostly soluble form in E. coli cells, both under its own promoter and under strong promoters such as p L and p tac on multicopy plasmids. Repeated freezing and thawing of purified ClpA and relatively short exposures to temperatures above 10° lead to losses of activity. ATP and nonhydrolyzable analogs of ATP stabilize ClpA. ClpA and ClpP form a tight complex in the presence of MgCl 2 and ATP or the nonhydrolyzable analog, ATP γ S. The ClpAP complex is composed of a dodecamer of ClpP and a hexamer of ClpA.

Translocation pathway of protein substrates in ClpAP protease.

Intracellular protein degradation, which must be tightly controlled to protect normal proteins, is carried out by ATP-dependent proteases. These multicomponent enzymes have chaperone-like ATPases that recognize and unfold protein substrates and deliver them to the proteinase components for digestion. In ClpAP, hexameric rings of the ClpA ATPase stack axially on either face of the ClpP proteinase, which consists of two apposed heptameric rings. We have used cryoelectron microscopy to characterize interactions of ClpAP with the model substrate, bacteriophage P1 protein, RepA. In complexes stabilized by ATPγS, which bind but do not process substrate, RepA dimers are seen at near-axial sites on the distal surface of ClpA. On ATP addition, RepA is translocated through ≈150 Å into the digestion chamber inside ClpP. Little change is observed in ClpAP, implying that translocation proceeds without major reorganization of the ClpA hexamer. When translocation is observed in complexes containing a ClpP mutant whose digestion chamber is already occupied by unprocessed propeptides, a small increase in density is observed within ClpP, and RepA-associated density is also seen at other axial sites. These sites appear to represent intermediate points on the translocation pathway, at which segments of unfolded RepA subunits transiently accumulate en route to the digestion chamber.

Subunit‐specific degradation of the UmuD/D′ heterodimer by the ClpXP protease: the role of trans recognition in UmuD′ stability

The Escherichia coli UmuD′ protein is a subunit of the recently described error‐prone DNA polymerase, pol V. UmuD′ is initially synthesized as an unstable and mutagenically inactive pro‐protein, UmuD. Upon processing, UmuD′ assumes a relatively stable conformation and becomes mutagenically active. While UmuD and UmuD′ by themselves exist in vivo as homodimers, when together they preferentially interact to form heterodimers. Quite strikingly, it is in this context that UmuD′ becomes susceptible to ClpXP‐mediated proteolysis. Here we report a novel targeting mechanism designed for degrading the mutagenically active UmuD′ subunit of the UmuD/D′ heterodimer complex, while leaving the UmuD protein intact. Surprisingly, a signal that is essential and sufficient for targeting UmuD′ for degradation was found to reside on UmuD not UmuD′. UmuD was also shown to be capable of channeling an excess of UmuD′ to ClpXP for degradation, thereby providing a mechanism whereby cells can limit error‐prone DNA replication.

Six‐fold rotational symmetry of ClpQ, the E. coli homolog of the 20S proteasome, and its ATP‐dependent activator, ClpY

ClpQ (HsIV) is a homolog of the β‐subunits of the 20S proteasome. In E. coli, it is expressed from an operon that also encodes ClpY (HsIU), an ATPase homologous to the protease chaperone, ClpX. ClpQ (subunit M r 19 000) and ClpY (subunit M r 49 000) were purified separately as oligomeric proteins with molecular weights of ∼220 000 and ∼350 000, respectively, estimated by gel filtration. Mixtures of ClpY and ClpQ displayed ATP‐dependent proteolytic activity against casein, and a complex of the two proteins was isolated by gel filtration in the presence of ATP. Image processing of negatively stained electron micrographs revealed strong six‐fold rotational symmetry for both ClpY and ClpQ, suggesting that the subunits of both proteins are arranged in hexagonal rings. The molecular weight of ClpQ combined with its symmetry is consistent with a double hexameric ring, whereas the data on ClpY suggest only one such ring. The symmetry mismatch previously observed between hexameric ClpA and heptameric ClpP in the related ClpAP protease is apparently not reproduced in the symmetry‐matched ClpYQ system.

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) (ATPγS), indicating that a symmetry mismatch is a universal feature of Clp proteases. hClpXP displays both ATP-dependent proteolytic activity and ATP- or ATPγS-dependent peptidase activity. hClpXP cannot degrade λO 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 abone 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.