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M R Maurizi

Publications and source records attributed to M R Maurizi.

At least 19 recordsLinked to original sources
PubMed2001

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.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2001

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 ATPgammaS, 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 approximately 150 A 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.

Adenosine Triphosphatases↗
PubMed2001

The RssB response regulator directly targets sigma(S) for degradation by ClpXP.

The sigma(S) subunit of Escherichia coli RNA polymerase regulates the expression of stationary phase and stress response genes. Control over sigma(S) activity is exercised in part by regulated degradation of sigma(S). In vivo, degradation requires the ClpXP protease together with RssB, a protein homologous to response regulator proteins. Using purified components, we reconstructed the degradation of sigma(S) in vitro and demonstrate a direct role for RssB in delivering sigma(S) to ClpXP. RssB greatly stimulates sigma(S) degradation by ClpXP. Acetyl phosphate, which phosphorylates RssB, is required. RssB participates in multiple rounds of sigma(S) degradation, demonstrating its catalytic role. RssB promotes sigma(S) degradation specifically; it does not affect degradation of other ClpXP substrates or other proteins not normally degraded by ClpXP. sigma(S) and RssB form a stable complex in the presence of acetyl phosphate, and together they form a ternary complex with ClpX that is stabilized by ATP[gamma-S]. Alone, neither sigma(S) nor RssB binds ClpX with high affinity. When ClpP is present, a larger sigma(S)--RssB--ClpXP complex forms. The complex degrades sigma(S) and releases RssB from ClpXP in an ATP-dependent reaction. Our results illuminate an important mechanism for regulated protein turnover in which a unique targeting protein, whose own activity is regulated through specific signaling pathways, catalyzes the delivery of a specific substrate to a specific protease.

Adenosine Triphosphatases↗
PubMed2000

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.

Adenosine Triphosphatases↗
PubMed2000

Protein binding and unfolding by the chaperone ClpA and degradation by the protease ClpAP.

ClpA, a bacterial member of the Clp/Hsp100 chaperone family, is an ATP-dependent molecular chaperone and the regulatory component of the ATP-dependent ClpAP protease. To study the mechanism of binding and unfolding of proteins by ClpA and translocation to ClpP, we used as a model substrate a fusion protein that joined the ClpA recognition signal from RepA to green fluorescent protein (GFP). ClpAP degrades the fusion protein in vivo and in vitro. The substrate binds specifically to ClpA in a reaction requiring ATP binding but not hydrolysis. Binding alone is not sufficient to destabilize the native structure of the GFP portion of the fusion protein. Upon ATP hydrolysis the GFP fusion protein is unfolded, and the unfolded intermediate can be sequestered by ClpA if a nonhydrolyzable analog is added to displace ATP. ATP is required for release. We found that although ClpA is unable to recognize native proteins lacking recognition signals, including GFP and rhodanese, it interacts with those same proteins when they are unfolded. Unfolded GFP is held in a nonnative conformation while associated with ClpA and its release requires ATP hydrolysis. Degradation of unfolded untagged proteins by ClpAP requires ATP even though the initial ATP-dependent unfolding reaction is bypassed. These results suggest that there are two ATP-requiring steps: an initial protein unfolding step followed by translocation of the unfolded protein to ClpP or in some cases release from the complex.

Adenosine Triphosphatases↗
PubMed2000

Unfolding and internalization of proteins by the ATP-dependent proteases ClpXP and ClpAP.

ClpX and ClpA are molecular chaperones that interact with specific proteins and, together with ClpP, activate their ATP-dependent degradation. The chaperone activity is thought to convert proteins into an extended conformation that can access the sequestered active sites of ClpP. We now show that ClpX can catalyze unfolding of a green fluorescent protein fused to a ClpX recognition motif (GFP-SsrA). Unfolding of GFP-SsrA depends on ATP hydrolysis. GFP-SsrA unfolded either by ClpX or by treatment with denaturants binds to ClpX in the presence of adenosine 5'-O-(3-thiotriphosphate) and is released slowly (t(1/2) approximately 15 min). Unlike ClpA, ClpX cannot trap unfolded proteins in stable complexes unless they also have a high-affinity binding motif. Addition of ATP or ADP accelerates release (t(1/2) approximately 1 min), consistent with a model in which ATP hydrolysis induces a conformation of ClpX with low affinity for unfolded substrates. Proteolytically inactive complexes of ClpXP and ClpAP unfold GFP-SsrA and translocate the protein to ClpP, where it remains unfolded. Complexes of ClpXP with translocated substrate within the ClpP chamber retain the ability to unfold GFP-SsrA. Our results suggest a bipartite mode of interaction between ClpX and substrates. ClpX preferentially targets motifs exposed in specific proteins. As the protein is unfolded by ClpX, additional motifs are exposed that facilitate its retention and favor its translocation to ClpP for degradation.

Adenosine Triphosphatases↗
PubMed2000

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.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1999

Posttranslational quality control: folding, refolding, and degrading proteins.

Polypeptides emerging from the ribosome must fold into stable three-dimensional structures and maintain that structure throughout their functional lifetimes. Maintaining quality control over protein structure and function depends on molecular chaperones and proteases, both of which can recognize hydrophobic regions exposed on unfolded polypeptides. Molecular chaperones promote proper protein folding and prevent aggregation, and energy-dependent proteases eliminate irreversibly damaged proteins. The kinetics of partitioning between chaperones and proteases determines whether a protein will be destroyed before it folds properly. When both quality control options fail, damaged proteins accumulate as aggregates, a process associated with amyloid diseases.

Adenosine Triphosphatases↗
PubMed1999

ClpA and ClpP remain associated during multiple rounds of ATP-dependent protein degradation by ClpAP protease.

The Escherichia coli ClpA and ClpP proteins form a complex, ClpAP, that catalyzes ATP-dependent degradation of proteins. Formation of stable ClpA hexamers and stable ClpAP complexes requires binding of ATP or nonhydrolyzable ATP analogues to ClpA. To understand the order of events during substrate binding, unfolding, and degradation by ClpAP, it is essential to know the oligomeric state of the enzyme during multiple catalytic cycles. Using inactive forms of ClpA or ClpP as traps for dissociated species, we measured the rates of dissociation of ClpA hexamers or ClpAP complexes. When ATP was saturating, the rate constant for dissociation of ClpA hexamers was 0.032 min(-1) (t(1/2) of 22 min) at 37 degrees C, and dissociation of ClpP from the ClpAP complexes occurred with a rate constant of 0. 092 min(-1) (t(1/2) of 7.5 min). Because the k(cat) for casein degradation is approximately 10 min(-1), these results indicate that tens of molecules of casein can be turned over by the ClpAP complex before significant dissociation occurs. Mutations in the N-terminal ATP binding site led to faster rates of ClpA and ClpAP dissociation, whereas mutations in the C-terminal ATP binding site, which cause significant decreases in ATPase activity, led to lower rates of dissociation of ClpA and ClpAP complexes. Dissociation rates for wild-type and first domain mutants of ClpA were faster at low nucleotide concentrations. The t(1/2) for dissociation of ClpAP complexes in the presence of nonhydrolyzable analogues was >/=30 min. Thus, ATP binding stabilizes the oligomeric state of ClpA, and cycles of ATP hydrolysis affect the dynamics of oligomer interaction. However, since the k(cat) for ATP hydrolysis is approximately 140 min(-1), ClpA and the ClpAP complex remain associated during hundreds of rounds of ATP hydrolysis. Our results indicate that the ClpAP complex is the functional form of the protease and as such engages in multiple rounds of interaction with substrate proteins, degradation, and release of peptide products without dissociation.

Adenosine Triphosphatases↗
PubMed1999

Concurrent chaperone and protease activities of ClpAP and the requirement for the N-terminal ClpA ATP binding site for chaperone activity.

ClpA, a member of the Clp/Hsp100 family of ATPases, is both an ATP-dependent molecular chaperone and the regulatory component of ClpAP protease. We demonstrate that chaperone and protease activities occur concurrently in ClpAP complexes during a single round of RepA binding to ClpAP and ATP-dependent release. This result was substantiated with a ClpA mutant, ClpA(K220V), carrying an amino acid substitution in the N-terminal ATP binding site. ClpA(K220V) is unable to activate RepA, but the presence of ClpP or chemically inactivated ClpP restores its ability to activate RepA. The presence of ClpP simultaneously facilitates degradation of RepA. ClpP must remain bound to ClpA(K220V) for these effects, indicating that both chaperone and proteolytic activities of the mutant complex occur concurrently. ClpA(K220V) itself is able to form stable complexes with RepA in the presence of a poorly hydrolyzed ATP analog, adenosine 5'-O-(thiotriphosphate), and to release RepA upon exchange of adenosine 5'-O-(thiotriphosphate) with ATP. However, the released RepA is inactive in DNA binding, indicating that the N-terminal ATP binding site is essential for the chaperone activity of ClpA. Taken together, these results suggest that substrates bound to the complex of the proteolytic and ATPase components can be partitioned between release/reactivation and translocation/degradation.

Adenosine Triphosphatases↗
PubMed1999

Nucleotide-dependent oligomerization of ClpB from Escherichia coli.

Self-association of ClpB (a mixture of 95- and 80-kDa subunits) has been studied with gel filtration chromatography, analytical ultracentrifugation, and electron microscopy. Monomeric ClpB predominates at low protein concentration (0.07 mg/mL), while an oligomeric form is highly populated at >4 mg/mL. The oligomer formation is enhanced in the presence of 2 mM ATP or adenosine 5'-O-thiotriphosphate (ATPgammaS). In contrast, 2 mM ADP inhibits full oligomerization of ClpB. The apparent size of the ATP- or ATPgammaS-induced oligomer, as determined by gel filtration, sedimentation velocity and electron microscopy image averaging, and the molecular weight, as determined by sedimentation equilibrium, are consistent with those of a ClpB hexamer. These results indicate that the oligomerization reactions of ClpB are similar to those of other Hsp100 proteins.

Adenosine Triphosphate↗
PubMed1998

Importance of heptameric ring integrity for activity of Escherichia coli ClpP.

Radiation target analysis has been used to identify the minimal functional unit for expression of activity of ClpP, the proteolytic component of the ATP-dependent ClpAP protease. Radiation target sizes determined for small peptide hydrolysis, for ClpA activated and nucleotide-activated oligopeptide cleavage, and for ClpA-activated ATP-dependent protein degradation were 154, 118, and 160 kDa, respectively. Thus, the hydrolytic activity of ClpP, subunit M, 21,500, is dependent on the native oligomeric structure. The quaternary structure of ClpP determined by electron microscopy and hydrodynamic studies consists of two face-to-face seven-membered rings. The radiation target sizes are consistent with a requirement for conformational integrity of an entire ring for expression of hydrolytic activity. Radiation damage led to disruption of inter-ring contacts, giving rise to isolated rings of ClpP. Thus, contacts between rings of ClpP are less stable and more easily disrupted than contacts between subunits within the rings. Our data suggest that cooperative interactions between subunits within the ClpP rings are important for maintaining the active conformation of the proteolytic active site.

Adenosine Triphosphatases↗
PubMed1998

The role of the ClpA chaperone in proteolysis by ClpAP.

ClpA, a member of the Clp/Hsp100 family of ATPases, is a molecular chaperone and, in combination with a proteolytic component ClpP, participates in ATP-dependent proteolysis. We investigated the role of ClpA in protein degradation by ClpAP by dissociating the reaction into several discrete steps. In the assembly step, ClpA-ClpP-substrate complexes assemble either by ClpA-substrate complexes interacting with ClpP or by ClpA-ClpP complexes interacting with substrate; ClpP in the absence of ClpA is unable to bind substrates. Assembly requires ATP binding but not hydrolysis. We discovered that ClpA translocates substrates from their binding sites on ClpA to ClpP. The translocation step specifically requires ATP; nonhydrolyzable ATP analogs are ineffective. Only proteins that are degraded by ClpAP are translocated. Characterization of the degradation step showed that substrates can be degraded in a single round of ClpA-ClpP-substrate binding followed by ATP hydrolysis. The products generated are indistinguishable from steady-state products. Taken together, our results suggest that ClpA, through its interaction with both the substrate and ClpP, acts as a gatekeeper, actively translocating specific substrates into the proteolytic chamber of ClpP where degradation occurs. As multicomponent ATP-dependent proteases are widespread in nature and share structural similarities, these findings may provide a general mechanism for regulation of substrate import into the proteolytic chamber.

Adenosine Triphosphatases↗
PubMed1998

Proteasome assembly: biting the hand.

Proteasome assembly is regulated to ensure the enzyme is inactive until its active sites are compartmentalized within an interior aqueous chamber. In yeast, this depends on a dedicated chaperone that is trapped within the nascent proteasome, and degraded on maturation of the proteolytic subunits.

Animals↗