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M R Maurizi

Publications and source records attributed to M R Maurizi.

At least 37 records · Page 2Linked to original sources
PubMed1998

Molecular properties of ClpAP protease of Escherichia coli: ATP-dependent association of ClpA and clpP.

The ClpAP protease from Escherichia coli consists of the ATP-binding regulatory component, ClpA (subunit Mr 84 165), and the proteolytic component, ClpP (subunit Mr 21 563). Our hydrodynamic studies demonstrate that the predominant forms of these proteins in solution correspond to those observed by electron microscopy. ClpP and proClpP(SA), which in electron micrographs appear to have subunits arranged in rings of seven subunits, were found by ultracentrifugation to have s20,w values of 12.2 and 13.2 S and molecular weights of 300 000 and 324 000 +/- 3000, respectively, indicating that the native form of each consists of two such rings. The two intact rings of ClpP were separated in the presence of >/= 0.1 M sulfate at low temperatures, suggesting that ring-ring contacts are polar in nature and more easily disrupted than subunit contacts within individual rings. Sedimentation equilibrium analysis indicated that ClpA purified without nucleotide exists as an equilibrium mixture of monomers and dimers with Ka = (1.0 +/- 0.2) x 10(5) M-1 and that, upon addition of MgATP or adenosine 5'-O-(3-thiotriphosphate), ClpA subunits associated to a form with Mr 505 000 +/- 5000, consistent with the hexameric structure seen by electron microscopy. Sedimentation velocity and gel-filtration analysis showed that the nucleotide-promoted hexamer of ClpA (s20,w = 17.2 S) binds tightly to ClpP producing species with s20,w values of 21 and 27 S (f/f0 = 1.5 and 1.8, respectively), consistent with electron micrographs of ClpAP that show a single tetradecamer of ClpP associated with either one or two ClpA hexamers [Kessel et al. (1995) J. Mol. Biol. 250, 587-594]. Under assay conditions in the presence of ATP and Mg2+, the apparent dissociation constant of hexameric ClpA and tetradecameric ClpP was approximately 4 +/- 2 nM. By the method of continuous variation, the optimal ratio of ClpA to ClpP in the active complex was 2:1. The specific activities of limiting ClpA and ClpP determined in the presence of an excess of the other component indicated that the second molecule of ClpA provides very little additional activation of ClpP.

Adenosine Triphosphatases↗
PubMed1998

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 (Mr 46, 000) associate to form a six-membered ring (Mr approximately 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 (approximately 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.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1998

Isolation and characterization of the phage T4 PinA protein, an inhibitor of the ATP-dependent lon protease of Escherichia coli.

The bacteriophage T4 PinA protein, expression of which leads to inhibition of protein degradation in Escherichia coli cells, has been purified from cells carrying multiple copies of the pinA gene. PinA is a heat-stable protein with a subunit Mr of 18,800 and an isoelectric point of 4.6. Under nondenaturing conditions on a gel filtration column, PinA migrated in two peaks corresponding to a dimer and a tetramer. Purified PinA inhibited ATP-dependent protein degradation by Lon protease in vitro; it did not inhibit the activity of other E. coli ATP-dependent proteases, ClpAP or ClpYQ. Furthermore, PinA did not inhibit ATP-independent proteolysis in E. coli cell extracts. PinA binds with high affinity to Lon protease (Kd approximately 10 nM for dimer binding), and a complex with approximately 1 dimer of PinA per tetramer of Lon protease could be isolated by gel filtration. Lon activity was partially restored upon dilution of the PinA-Lon complex to subnanomolar concentrations, indicating that inhibition was reversible and that PinA did not covalently modify Lon protease. PinA was not cleaved by Lon protease, and heating the Lon-PinA complex at 65 degrees C denatured Lon protease and released active PinA. The properties of PinA in vitro suggest that PinA inhibits protein degradation in vivo by forming a tight, reversible complex with Lon protease.

ATP-Dependent Proteases↗
PubMed1998

PinA inhibits ATP hydrolysis and energy-dependent protein degradation by Lon protease.

The bacteriophage T4 PinA protein inhibited degradation of [3H]alpha-methyl casein by purified Lon protease from Escherichia coli, but inhibition was noncompetitive with respect to casein. PinA did not inhibit cleavage of the fluorogenic peptide, N-glutaryl-alanylalanylphenylalanyl-3-methoxynaphthylamide and, moreover, did not block the ability of protein substrates, such as casein, to activate cleavage of fluorogenic peptides by Lon. Thus, PinA does not block the proteolytic active site or the allosteric protein-binding site on Lon. Inhibition of basal ATPase activity was variable (50-90%), whereas inhibition of protein-activated ATPase activity was usually 80-95%. Inhibition was noncompetitive with respect to ATP. PinA did not block activation of peptide cleavage by nonhydrolyzable analogs of ATP. These data suggest that PinA does not bind at the ATPase active site of Lon and does not interfere with nucleotide binding to the enzyme. PinA inhibited cleavage of the 72-amino acid protein, CcdA, degradation of which requires ATP hydrolysis, but did not inhibit cleavage of the carboxyl-terminal 41-amino acid fragment of CcdA, degradation of which does not require ATP hydrolysis. PinA thus appears to interact at a novel regulatory or enzymatic site involved in the coupling between ATP hydrolysis and proteolysis, possibly blocking the protein unfolding or remodeling step essential for degradation of high molecular weight protein substrates by Lon.

ATP-Dependent Proteases↗
PubMed1998

At sixes and sevens: characterization of the symmetry mismatch of the ClpAP chaperone-assisted protease.

ClpAP, a typical energy-dependent protease, consists of a proteolytic component (ClpP) and a chaperone-like ATPase (ClpA). ClpP is composed of two apposed heptameric rings, whereas in the presence of ATP or ATPgammaS, ClpA is a single hexameric ring. Formation of ClpAP complexes involves a symmetry mismatch as sixfold ClpA stacks axially on one or both faces of sevenfold ClpP. We have analyzed these structures by cryo-electron microscopy. Our three-dimensional reconstruction of ClpA at 29-A resolution shows the monomer to be composed of two domains of similar size that, in the hexamer, form two tiers enclosing a large cavity. Cylindrical reconstruction of ClpAP reveals three compartments: the digestion chamber inside ClpP; a compartment between ClpP and ClpA; and the cavity inside ClpA. They are connected axially via narrow apertures, implying that substrate proteins should be unfolded to allow translocation into the digestion chamber. The cavity inside ClpA is structurally comparable to the "Anfinsen cage" of other chaperones and may play a role in the unfolding of substrates. A geometrical description of the symmetry mismatch was obtained by using our model of ClpA and the crystal structure of ClpP (Wang et al., 1997, Cell 91, 447-456) to identify the particular side views presented by both molecules in individual complexes. The interaction is characterized by a key pair of subunits, one of each protein. A small turn (8.6(o) = 2pi/42; equivalent to a 4-A shift) would transfer the key interaction to another pair of subunits. We propose that nucleotide hydrolysis results in rotation, facilitating the processive digestion of substrate proteins.

Adenosine Triphosphatases↗
PubMed1996

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

ClpQ (HslV) is a homolog of the beta-subunits of the 20S proteasome. In E. coli, it is expressed from an operon that also encodes ClpY (HslU), an ATPase homologous to the protease chaperone, ClpX. ClpQ (subunit Mr 19,000) and ClpY (subunit Mr 49,000) were purified separately as oligomeric proteins with molecular weights of approximately 220,000 and approximately 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.

ATP-Dependent Proteases↗
PubMed1996

Role of the heat shock protein DnaJ in the lon-dependent degradation of naturally unstable proteins.

We have investigated the role of DnaJ in protein degradation by examining the degradation of intrinsically unstable proteins by Lon protease in vivo. In Escherichia coli, Lon protease is responsible for the rate-limiting step in degradation of highly unstable proteins such as SulA, RcsA, and lambdaN protein, as well as for about 50% of the rapid degradation of abnormal proteins such as canavanine-containing proteins. We found that Lon-dependent degradation of both SulA and lambdaN protein was unaffected in cells lacking functional DnaJ, whereas Lon-dependent turnover of canavanine-containing proteins was slower in dnaJ mutant cells. DnaJ also affected the slow SulA degradation seen in the absence of Lon. The rate of degradation of RcsA was reduced in dnaJ mutants, but both Lon-dependent and Lon-independent degradation was affected; abnormal, canavanine-containing proteins were similarly affected. Both the RcsA that accumulated in dnaJ mutant cells and the SulA that accumulated in lon dnaJ mutant cells was aggregated. The abnormal proteins that partitioned to the insoluble pellet became solubilized over time in dnaJ+ cells but not in dnaJ- cells. Therefore, the co-chaperone DnaJ is not essential for Lon-dependent degradation and may act in protein turnover only as an accessory factor helping to maintain substrates in a soluble form. Such an accessory factor is apparently necessary for abnormal proteins and for RcsA. The relative rates of degradation and aggregation of specific protein targets may determine the importance of the chaperone systems in turnover of a given protein.

ATP-Dependent Proteases↗
PubMed1996

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 microM), and the peptide bond turnover number was approximately 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 alpha-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 alpha-helical structure at elevated temperatures (Tm approximately 50 degrees 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.

ATP-Dependent Proteases↗
PubMed1995

Homology in structural organization between E. coli ClpAP protease and the eukaryotic 26 S proteasome.

Energy-dependent protein degradation is carried out by large multimeric protein complexes such as the proteasomes of eukaryotic and archaeal cells and the ATP-dependent proteases of eubacterial cells. Clp protease, a major multicomponent protease of Escherichia coli, consists of a proteolytic component, ClpP, in association with an ATP-hydrolyzing, chaperonin-like component, ClpA. To provide a structural basis for understanding the regulation and mechanism of action of Clp protease, we have used negative staining electron microscopy and image analysis to examine ClpA and ClpP separately, as well as active ClpAP complexes. Digitized images of ClpP and ClpA were analyzed using a novel algorithm designed to detect rotational symmetries. ClpP is composed of two rings of seven subunits superimposed in bipolar fashion along the axis of rotational symmetry. This structure is similar to that formed by the beta subunits of the eukaryotic and archaeal proteasomes. In the presence of MgATP, ClpA forms an oligomer with 6-fold symmetry when viewed en face. Side views of ClpA indicate that the subunits are bilobed with the respective domains forming two stacked rings. ClpAP complexes contain a tetradecamer of ClpP flanked at one or both ends with a hexamer of ClpA, resulting in a symmetry mismatch between the axially aligned molecules. Our findings demonstrate that, despite the lack of sequence similarity between ClpAP and proteasomes, these multimeric proteases nevertheless have a profound similarity in their underlying architecture that may reflect a common mechanism of action.

Adenosine Triphosphatases↗
PubMed1994

A molecular chaperone, ClpA, functions like DnaK and DnaJ.

The two major molecular chaperone families that mediate ATP-dependent protein folding and refolding are the heat shock proteins Hsp60s (GroEL) and Hsp70s (DnaK). Clp proteins, like chaperones, are highly conserved, present in all organisms, and contain ATP and polypeptide binding sites. We discovered that ClpA, the ATPase component of the ATP-dependent ClpAP protease, is a molecular chaperone. ClpA performs the ATP-dependent chaperone function of DnaK and DnaJ in the in vitro activation of the plasmid P1 RepA replication initiator protein. RepA is activated by the conversion of dimers to monomers. We show that ClpA targets RepA for degradation by ClpP, demonstrating a direct link between the protein unfolding function of chaperones and proteolysis. In another chaperone assay, ClpA protects luciferase from irreversible heat inactivation but is unable to reactivate luciferase.

Adenosine Triphosphatases↗
PubMed1994

Mutational analysis demonstrates different functional roles for the two ATP-binding sites in ClpAP protease from Escherichia coli.

ClpA, the regulatory subunit of Clp protease from Escherichia coli, has two ATP-binding sites in non-homologous regions of the protein, referred to as domain I and domain II. We have mutated the invariant lysine in the ATP-binding sites of domain I and domain II and studied the enzymatic properties of the purified mutant ClpA proteins. The domain I mutant, ClpA-K220Q, was unable to form a hexamer in the presence of nucleotide, but the comparable domain II mutant, ClpA-K501Q, associated into a hexamer in the presence of ATP, indicating that nucleotide binding to domain I favors a conformation required to stabilize the quaternary structure of ClpA. ClpA-K220Q was defective in ATPase activity and in the ability to activate protein and peptide degradation by ClpP, but the defects could be partially overcome by formation of hybrid hexamers with wild-type ClpA. Another domain I mutant, ClpA-K220R, readily formed hexamers in the presence of ATP and retained > or = 60% of the wild-type ATPase activity and ability to activate ClpP. These results indicate that hexamer formation is a prerequisite for expression of enzymatic activity. Domain II mutants ClpA-K501Q and ClpA-K501R had very low ATPase activity (< 10% of wild-type) and a severe defect in activation of protein degradation, which requires ATP hydrolysis. Domain II mutants were able to activate ClpP to degrade a peptide whose degradation required nucleotide binding but not hydrolysis. Nucleotide binding to domain II of ClpA is important to form a productive complex with ClpP, and domain II appears to be primarily responsible for an energy-requiring step in the catalytic cycle unique to the degradation of large proteins.

Adenosine Triphosphatases↗
PubMed1994

Synthesis, processing, and localization of human Lon protease.

The synthesis, maturation, and localization of the human homolog of the bacterial ATP-dependent Lon protease have been studied in cultured cells and in a cell-free system. Immunofluorescence microscopy of cells transfected with a recombinant human Lon protease-FLAG epitope chimeric protein confirmed the mitochondrial location of human Lon protease. The primary product of transcription and translation directed by a human LON cDNA in vitro had an apparent mass of approximately 107 kDa, and incubation of the translation product with isolated rat liver mitochondria converted the precursor into the approximately 100-kDa mature form. The latter pelleted with mitochondria and was resistant to trypsin digestion. Maturation of human Lon protease in vitro was dependent on mitochondrial inner membrane potential, as the uncoupling agent 2,4-dinitrophenol (DNP) completely blocked this process. In intact cultured cells treated with DNP, newly synthesized Lon protease also accumulated as the precursor form, and subsequent removal of DNP allowed the precursor to be translocated into mitochondria and cleaved to its mature form. A pulse-chase experiment in the absence of DNP showed that the Lon protease precursor is converted to the mature form with a half-time of < 2 min and that the mature human Lon protease is indefinitely stable in intact cells. Submitochondrial fractionation and immunoblot analysis of rat liver mitochondrial proteins using the polyclonal anti-human Lon protease antiserum indicated that the protease is located exclusively in the matrix. These data demonstrate that the maturation of human Lon protease is characterized by steps similar to those reported for other mitochondrial matrix proteins.

ATP-Dependent Proteases↗
PubMed1994

Activity and specificity of Escherichia coli ClpAP protease in cleaving model peptide substrates.

Escherichia coli ClpAP protease is an ATP-dependent protease composed of the proteolytic component ClpP and a regulatory ATPase, ClpA. ClpAP protease degraded a variety of peptide bonds in protein and peptide substrates at a slow rate (kcat < or = 30 min-1/subunit of ClpP), but showed very high activity (kcat > or = 800 min-1) for a synthetic peptide composed of the first 19 amino acids of ClpP, MSYSGERDNFAPHMALVPV, referred to as the propeptide. The propeptide was not degraded by ClpP alone, but was degraded in the presence of ClpA and ClpP. Degradation was activated by nonhydrolyzable analogs of ATP, indicating that nucleotide-promoted interaction between ClpA and ClpP is sufficient to activate ClpP for propeptide cleavage. The propeptide, as well as truncated forms lacking either the first 9 or the last 3 amino acids, was cleaved at the same Met-Ala bond at which autoprocessing occurs in vivo. No hydrolysis of FAPHMALVPV derivatives was observed when Met was replaced by Glu, Lys, Ser, Tyr, Ile, and D-Met, but cleavage at the same position did occur with Leu or Trp substitutions. A peptide composed of a tandem repeat of FAPHMALVPV was cleaved between both Met-Ala bonds (Kcat values > or = 39 min-1). Propeptides inhibited degradation of alpha-casein by competition for a binding site on ClpA, and they stimulated the basal ATPase activity of ClpA in the absence of ClpP. Peptides and protein substrates interact at an allosteric site on ClpA, which appears to be the site at which specific substrates are recognized by the Clp protease.

ATP-Dependent Proteases↗
PubMed1994

Processive degradation of proteins by the ATP-dependent Clp protease from Escherichia coli. Requirement for the multiple array of active sites in ClpP but not ATP hydrolysis.

ClpP, the proteolytic component of the ATP-dependent ClpAP protease, is composed of 12 identical subunits and has intrinsic degradative activity against short peptides. Degradation of proteins and some peptides by ClpP requires the regulatory component ClpA and ATP. Peptide and protein substrates have been used to distinguish the roles of nucleotide binding and nucleotide hydrolysis in the activation of ClpAP protease. ATP binding alone promoted interaction between ClpA and ClpP, affected the substrate response curves for very short peptides, and activated degradation of larger peptides that were not degraded by ClpP alone. ATP hydrolysis did not increase in proportion to the increase in peptide bond hydrolysis of short peptides. However, ATP hydrolysis was strictly required for degradation of proteins such as alpha-casein; there was no indication of even limited cleavage of protein substrates when nonhydrolyzable analogs of ATP were used. Most large peptides and proteins were degraded in multiple sites without release of high molecular weight intermediates. Partial inactivation of ClpP with diisopropyl fluorophosphate produced ClpP with one to three active subunits/dodecamer. When only a few active sites were available in the active complex of ClpAP, degradation of large peptides and proteins released significant amounts of high molecular weight intermediates. Thus, processive degradation of protein substrates is a function of the multiple array of proteolytic active sites within the ClpP dodecamer.

ATP-Dependent Proteases↗