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Information on EC 3.4.21.92 - Endopeptidase Clp and Organism(s) Escherichia coli and UniProt Accession P0A6G7
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3.4.21.92 Endopeptidase ClpSpecify your search results
Word Map
- 3.4.21.92
- chloroplast
- adaptor
- tuberculosis
- mycobacterium
- atpases
- plastid
- aureus
- sigma
-
misfolded
-
hexamer
-
adeps
-
ssra-tagged
-
heptameric
- caulobacter
- unfoldase
-
proteostasis
- crescentus
-
tetradecameric
-
energy-dependent
- apicoplasts
- dnak
-
self-compartmentalized
-
n-degrons
- medicine
- ctra
- spx
-
atp-fueled
- pyrazinamide
-
toxin-antitoxin
-
double-ring
- tmrna
-
plastid-encoded
-
barrel-shaped
- drug development
- analysis
The taxonomic range for the selected organisms is: Escherichia coli
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
Hydrolysis of proteins to small peptides in the presence of ATP and Mg2+. alpha-Casein is the usual test substrate. In the absence of ATP, only oligopeptides shorter than five residues are hydrolysed (such as succinyl-Leu-Tyr-/-NHMec, and Leu-Tyr-Leu-/-Tyr-Trp, in which cleavage of the -Tyr-/-Leu- and -Tyr-/-Trp bonds also occurs)
Synonyms
clp protease, clpap, clpxp protease, clpc1, clpp1, caseinolytic protease, clpp protease, clpp2, clpp1p2, atp-dependent clp protease, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
ATP-dependent Clp protease
-
-
-
-
Caseinolytic protease
-
-
-
-
Clp protease
ClpP
ClpX
ClpXP
endopeptidase Clp
-
-
-
-
endopeptidase Ti
-
-
-
-
Heat shock protein F21.5
-
-
-
-
Protease Ti
-
-
-
-
stress protein G7
-
-
-
-
Clp protease
-
-
-
-
ClpP
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Hydrolysis of proteins to small peptides in the presence of ATP and Mg2+. alpha-Casein is the usual test substrate. In the absence of ATP, only oligopeptides shorter than five residues are hydrolysed (such as succinyl-Leu-Tyr-/-NHMec, and Leu-Tyr-Leu-/-Tyr-Trp, in which cleavage of the -Tyr-/-Leu- and -Tyr-/-Trp bonds also occurs)
dual mechanism in which translocation alternates with proteolysis, allowing peptides of more or less unifirm length to be cleaved processively from a translocating substrate
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis of peptide bond
hydrolysis of peptide bond
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
110910-59-3
-
131017-00-0
-
131017-01-1
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
fEGFP-ssrA + H2O
?
i.e. N-terminal His-tagged superfolder enhanced green fluorescent protein with the ssrA tag sequence at the C-terminus
-
-
?
31-knotted methyltransferase YbeA-ssrA + H2O
?
-
substrate contains a deep trefoil knot, with 70 and 34 residues lying to the N- and C-terminus of the knotted core, and is fused to the 11-amino acid ssrA degron
-
-
?
52-knotted ubiquitin C-terminal hydrolase L1-ssrA
?
-
substrate is fused to the 11-amino acid ssrA degron
-
-
?
Bacteriophage lambdaO-DNA replication protein + H2O
Hydrolyzed bacteriophage lambdaO-DNA replication protein
-
degraded by ClpXP
-
-
?
beta-Galactosidase fusion proteins + H2O
Hydrolyzed beta-galactosidase fusion protein
-
-
-
-
?
Lambda O Arc + H2O
?
-
Arc repressor with a N-terminal lambda O degradation tag
-
-
?
Leu-Tyr-Leu-Tyr-Trp + H2O
Leu-Tyr-Leu + Tyr-Trp
-
cleavage occurs primarily at Leu3-Tyr4, but significant cleavage also at Tyr2-Leu3 and Leu4-Trp5 bond
-
?
Mutated repressor of Mu prophage + H2O
Hydrolyzed mutated repressor of Mu prophage
-
high susceptibility to the Clp-dependent degradation
-
-
?
N-succinyl-Leu-Tyr 4-methylcoumarin 7-amide + H2O
N-succinyl-Leu-Tyr + 7-amino-4-methylcoumarin
-
-
-
-
?
N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin + H2O
?
-
initial degradation rate is the same within error for wild-type ClpP, ClpAP, and ClpPDELTAN
-
-
?
N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin + H2O
N-succinyl-Leu-Tyr + 7-amino-4-methylcoumarin
-
-
-
-
?
Oxidized insulin B-chain + H2O
Hydrolyzed insulin B-chain
-
cleavage at multiple sites
-
-
?
Phe-Ala-Pro-His-Met-Ala-Leu-Val-Pro-Val + H2O
?
-
synthetic polypeptide that corresponds to the 10 amino acids surrounding the in vivo processing site in ClpP subunit
-
-
?
ssrA-dabsyl + H2O
?
-
initial rate of degradation of this intermediate-sized substrate is 3fold faster with ClpAP as compared to wild-type Clp and 5fold faster with ClpPDELTAN as compared to wild-type ClpP
-
-
?
Starvation proteins + H2O
?
-
the ClpP proteolytic subunit plays a subtle but important role when cells are recovering from starvation. This enzyme is important in the selective degradation of starvation proteins when growth resumes
-
-
?
Succinyl-Ala-Ala-Phe 4-methylcoumarin 7-amide + H2O
Succinyl-Ala-Ala + Phe 4-methylcoumarin 7-amide
Succinyl-Leu-Leu-Val-Tyr 4-methylcoumarin 7-amide + H2O
Succinyl-Leu + Leu + Val-Tyr 4-methylcoumarin 7-amide
Succinyl-Leu-Tyr 4-methylcoumarin 7-amide + H2O
Succinyl-Leu-Tyr + 7-amino-4-methylcoumarin
-
ClpP subunit alone
-
?
casein + H2O
small peptides derived from casein
-
-
-
-
?
Succinyl-Ala-Ala-Phe 4-methylcoumarin 7-amide + H2O
Succinyl-Ala-Ala + Phe 4-methylcoumarin 7-amide
-
ClpP subunit alone
-
?
Succinyl-Ala-Ala-Phe 4-methylcoumarin 7-amide + H2O
Succinyl-Ala-Ala + Phe 4-methylcoumarin 7-amide
-
ClpP subunit alone
-
?
Succinyl-Leu-Leu-Val-Tyr 4-methylcoumarin 7-amide + H2O
Succinyl-Leu + Leu + Val-Tyr 4-methylcoumarin 7-amide
-
ClpP subunit alone
-
?
Succinyl-Leu-Leu-Val-Tyr 4-methylcoumarin 7-amide + H2O
Succinyl-Leu + Leu + Val-Tyr 4-methylcoumarin 7-amide
-
ClpP subunit alone
-
?
additional information
?
-
ClpP requires association with ClpA or ClpX to unfold and thread protein substrates through the axial pore into the inner chamber where degradation occurs
-
-
?
additional information
?
-
-
role for the Clp protease in activating Mu-mediated DNA rearrangements
-
-
?
additional information
?
-
-
ClpP subunit has peptidase activity against very short peptides, with fewer than five amino acid residues in the absence of ClpA and nucleotide
-
-
?
additional information
?
-
-
when activated by ClpA subunit, ClpP can degrade longer polypeptides and proteins
-
-
?
additional information
?
-
-
physiological activation of Mu-dependent DNA rearrangements requires Clp functions. Clp plays a role in monitoring the physiological status of the cell
-
-
?
additional information
?
-
-
ClpXP appears to be involved in plasmid maintenance and in phage Mu virulence
-
-
?
additional information
?
-
-
the high degree of similarity among the ClpA-like proteins suggests that Clp-like proteases are likely to be important participants in energy-dependent proteolysis in prokaryotic and eukaryotic cells
-
-
?
additional information
?
-
-
selectivity of degradation by ClpP in vivo is determined by interaction of ClpP with different regulatory ATPase subunits
-
-
?
additional information
?
-
-
ClpP is present in a wide range of prokaryotic and eukaryotic cells and is highly conserved in plant chloroplasts
-
-
?
additional information
?
-
-
removing of irreversibly damaged polypeptides
-
?
additional information
?
-
-
the ClpP N-terminus acts as a gate controlling substrate access to the active sites, binding of ClpA opens this gate, allowing substrate entry and formation of the acyl-enzyme intermediate, and closing of the N-terminal gate stimulates acyl-enzyme hydrolysis
-
-
?
additional information
?
-
-
ClpP associates with ClpX or ClpA to form the AAA+ ClpXP or ClpAP proteases
-
-
?
additional information
?
-
-
ClpP binds to AAA+ ATPase/unfoldase, ClpA or ClpX
-
-
?
additional information
?
-
-
phosphate release is the force-generating step of the ATPase cycle. Protease ClpXP translocates substrate polypeptides by highly coordinated conformational changes in up to four ATPase subunits. To unfold stable substrates like GFP, ClpXP must use this maximum successive firing capacity. The dwell duration between individual bursts of translocation is constant and governed by an internal clock, regardless of the number of translocating subunits
-
-
?
additional information
?
-
protease ClpXP unfolds most domains by a single pathway, with kinetics that depend on the native fold and structural stability. Subsequent translocation or pausing occurs at rates that vary with the sequence of the unfolded substrate. During translocation, ClpXP does not exhibit a sequential pattern of step sizes, supporting a fundamentally stochastic reaction, but a mechanism of enzymatic memory results in short physical steps being more probable after short steps and longer physical steps being more likely after longer steps, allowing the enzyme to run at different speeds. Two ATP-hydrolysis events can drive more than two power strokes. Solution proteolysis is many times slower than predicted from single-molecule results
-
-
?
additional information
?
-
-
ClpXP can easily degrade a deeply 31-knotted protein and is able to degrade 52-knotted proteins. The degradation depends critically on the location of the degradation tag and the local stability near the tag
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
Starvation proteins + H2O
?
-
the ClpP proteolytic subunit plays a subtle but important role when cells are recovering from starvation. This enzyme is important in the selective degradation of starvation proteins when growth resumes
-
-
?
additional information
?
-
ClpP requires association with ClpA or ClpX to unfold and thread protein substrates through the axial pore into the inner chamber where degradation occurs
-
-
?
additional information
?
-
-
physiological activation of Mu-dependent DNA rearrangements requires Clp functions. Clp plays a role in monitoring the physiological status of the cell
-
-
?
additional information
?
-
-
ClpXP appears to be involved in plasmid maintenance and in phage Mu virulence
-
-
?
additional information
?
-
-
the high degree of similarity among the ClpA-like proteins suggests that Clp-like proteases are likely to be important participants in energy-dependent proteolysis in prokaryotic and eukaryotic cells
-
-
?
additional information
?
-
-
selectivity of degradation by ClpP in vivo is determined by interaction of ClpP with different regulatory ATPase subunits
-
-
?
additional information
?
-
-
ClpP is present in a wide range of prokaryotic and eukaryotic cells and is highly conserved in plant chloroplasts
-
-
?
additional information
?
-
-
removing of irreversibly damaged polypeptides
-
?
additional information
?
-
-
ClpP associates with ClpX or ClpA to form the AAA+ ClpXP or ClpAP proteases
-
-
?
additional information
?
-
-
ClpP binds to AAA+ ATPase/unfoldase, ClpA or ClpX
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ATP
-
cleavage of peptides of more than five amino acids residues by ClpP requires activation by ClpA and either ATP or a nonhydrolyzable analog of ATP such as ATPgammaS or 5'-adenylyl imidodiphosphate
ATP
-
ATP hydrolysis is required for protein breakdown but not for hydrolysis of small peptides
ATP
-
ATP has two functions in activating Clp protease: an allosteric role in promoting association between the subunits and a mechanistically undefined role in promoting continuous rounds of peptide bond cleavage. The functions may be separately fulfilled by the two ATP-binding sites identified by sequence analysis on each ClpA subunit
ATP
-
ATP dependent, for full proteolytic activity ClpP must associate with one of two related ATPase subunits, ClpA or ClpX, other nucleoside triphosphates cannot substitute for ATP
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
Mg2+
-
proteolytic activity of ClpAP is dependent on, concentrations higher than about 30 mM are inhibitory
Mg2+
-
ClpA degrades large proteins down to short peptides of 7 to 10 amino acids, the process requires both Mg2+ and ATP hydrolysis
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
High salt concentrations
-
chloride is much more inhibitory than acetate, divalent anions are also very inhibitory
-
Mg2+
-
proteolytic activity of ClpAP is dependent on, but concentrations higher than about 30 mM are inhibitory
Succinyl-Leu-Tyr 4-methylcoumarin 7-amide
-
at high concentrations complete inhibition of casein breakdown
Xaa-Tyr-Leu-Tyr-Trp
-
competitive to succinyl-Leu-Tyr 4-methylcoumarin 7-amide degradation
additional information
-
ClpP of E. coli has a serine and a histidine that are necessary for activity and probably represent two elements of the active site triad found in most serine proteases
-
diisopropyl fluorophosphate
-
blocks similarly the hydrolysis of both protein and peptide substrates
diisopropyl fluorophosphate
-
inhibits both oligopeptidase activity of ClpP and proteinase activity of ClpAP
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
acyldepsipeptide
-
allow the protease to degrade folded native proteins in the absence of its cognate chaperones
ClpX
-
ClpX binding stimulates ClpP cleavage of peptides larger than a few amino acids and enhances ClpP active-site modification. Stimulation requires ATP binding but not hydrolysis by ClpX
-
additional information
-
carbonyl cyanide m-chloro phenylhydrazone and 2,4-dinitrophenol induce the heat-shock-like response, cellular level of the heat shock regulator protein sigma-32 also increases
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.8
N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin
-
wild-type, non-reducing conditions, 4°C, pH not specified in the publication
1
N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin
-
wild-type, reducing conditions, 4°C, pH not specified in the publication
1.1
N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin
-
mutant A153C, reducing conditions, 4°C, pH not specified in the publication
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.5
N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin
-
mutant A153C, reducing conditions, 4°C, pH not specified in the publication
9.6
N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin
-
wild-type, non-reducing conditions, 4°C, pH not specified in the publication
15.8
N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin
-
wild-type, reducing conditions, 4°C, pH not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
42
-
active in degrading alpha-casein up to 42°C, no proteinase activity at 55°C
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
malfunction
metabolism
-
presence of ATP favours assembly and ADP dissociation of the hexameric assembly. Subunit exchange kinetics is at least one order of magnitude slower than the ATP hydrolysis rate, and ClpB dynamics and activity are related processe. DnaK and substrate proteins regulate the ATPase activity and dynamics of ClpB
physiological function
physiological function
ClpXP protease consists of the ClpX hexamer and the ClpP peptidase. Small-molecule acyldepsipeptides such as ADEP-2B compete with the IGF loops of ClpX for ClpP-cleft binding and cause exceptionally rapid dissociation of otherwise stable ClpXP complexes, suggesting that the IGF-loop interactions with ClpP must be highly dynamic
physiological function
purified ClpXP added to a cell-free transcription-translation system that uses Escherichia coli S30 cell extract, has very low proteolytic activity. Addition of exogenous ATP and an energy regeneration system improves activity
malfunction
-
both FlhD and FlhC regulator proteins accumulate markedly following ClpXP depletion, and their half-lives are significantly longer in the mutant cells
malfunction
-
deletion of clpX and clpP suppresses temperature-sensitive filamentation of cells carrying the ftsZ84 allele and reduces FtsZ84 degradation, consistent with ClpXP (two-component protease composed of ClpX and ClpP) playing a role in modulating the level of FtsZ (a tubulin-like protein)
malfunction
-
in a division-defective strain, DELTAminC, the additional deletion of clpX or clpP delays cell division and exacerbates filamentation
physiological function
-
ClpXP protease of Escherichia coli consists of the AAA+ ClpX unfoldase and the associated ClpP compartmental peptidase. Once the substrate is unfolded, ClpX translocates the unfolded polypeptide into the degradation chamber of ClpP in steps of five to eight amino acids per power stroke
physiological function
-
ClpXP protease tightly regulates the flagellar expression by degrading the FlhD/FlhC master regulator in EHEC. The flagellar regulon in EHEC might be controlled by ClpXP protease through two pathways, namely post-translational control of the FlhD/FlhC master regulator by degrading them and transcriptional control of the flhDC operon through the locus of enterocyte effacement (LEE)-encoded GrlR-GrlA regulatory system
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
21000
-
1 * 230000, subunit ClpP (12 * 21000, amino acid sequence, subunit of ClpP)
23000
230000
-
1 * 230000, subunit ClpP (12 * 21000, amino acid sequence, subunit of ClpP)
240000
-
240000 (ClpP with the subunit structure 12 * 23000, SDS-PAGE), gel filtration in presence of more than or at 0.1 M KCl, in absence of KCl, native ClpP appears to dimerize giving a structure with a MW of 500000
300000
346000
-
sedimentation velocity analytical ultracentrifugation, tetradecamer
80000
-
x * 80000 (ClpA, SDS-PAGE, behaves as a dimer of MW 140000 Da on gel filtration) + x * 23000 (ClpP, SDS-PAGE, behaves as a complex of 10-12 subunits, MW 260000 Da)
83000
-
x * 120000-140000, subunit ClpA, gel filtration, x * 83000, subunit ClpA, amino acid sequence
23000
-
x * 80000 (ClpA, SDS-PAGE, behaves as a dimer of MW 140000 Da on gel filtration) + x * 23000 (ClpP, SDS-PAGE, behaves as a complex of 10-12 subunits, MW 260000 Da)
23000
-
240000 (ClpP with the subunit structure 12 * 23000, SDS-PAGE), gel filtration in presence of more than or at 0.1 M KCl, in absence of KCl, native ClpP appears to dimerize giving a structure with a MW of 500000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
?
-
1 * 230000, subunit ClpP (12 * 21000, amino acid sequence, subunit of ClpP)
?
-
x * 120000-140000, subunit ClpA, gel filtration, x * 83000, subunit ClpA, amino acid sequence
?
-
ClpP is composed of two superimposed rings of six subunits each, ClpA and ClpP form a tight complex in the presence of MgCl2 and ATP, the ClpAP complex is composed of a dodecamer of ClpP and a hexamer of ClpA
?
-
x * 80000 (ClpA, SDS-PAGE, behaves as a dimer of MW 140000 Da on gel filtration) + x * 23000 (ClpP, SDS-PAGE, behaves as a complex of 10-12 subunits, MW 260000 Da)
?
-
240000 (ClpP with the subunit structure 12 * 23000, SDS-PAGE), gel filtration in presence of more than or at 0.1 M KCl, in absence of KCl, native ClpP appears to dimerize giving a structure with a MW of 500000
additional information
-
the enzyme is a complex consisting of two components I and II, that can be separated from each other
additional information
-
bacteria, tomatoes and Trypanosomes all contain genes for a large protein with extensive homology to the regulartory subunit, ClpA
additional information
-
ClpP subunits (MW 21500 Da) are arranged in two hexagonal rings directly superimposed on each other, ClpA (subunit 83000) and ClpP do not associate in absence of ATP
additional information
-
ClpA subunit has ATPase activity, ClpP subunit has the proteolytic active site and is activated by ClpA in the presence of ATP
additional information
-
enzyme consists of two components: ClpP and ClpA or ClpX
additional information
-
enzyme consists of two components: ClpP and ClpA or ClpX
additional information
-
enzyme consists of two components: ClpP and ClpA or ClpX
additional information
-
enzyme consists of two components: ClpP and ClpA or ClpX
additional information
-
enzyme consists of two components: ClpP and ClpA or ClpX
additional information
-
enzyme consists of two components: ClpP and ClpA or ClpX
additional information
-
enzyme consists of two components: ClpP and ClpA or ClpX
additional information
-
ClpX participates with ClpP in the rapid and specific degradation of the lambda O protein
additional information
-
ClpX participates with ClpP in the rapid and specific degradation of the lambda O protein
additional information
-
ClpX participates with ClpP in the rapid and specific degradation of the lambda O protein
additional information
-
ClpA bound with ATP associates with ClpP to form an active proteolytic complex with MW 700000
additional information
-
ClpP and ClpA interact to form the active protease, this complex degrades a number of proteins, such as alpha-casein into small peptides and can hydrolyze ATP
additional information
-
but the combination of ClpX and ClpP has very little activity against alpha-casein
additional information
-
ClpP is synthesized with a 14-amino acid leader which is rapidly cleaved in vivo yielding the in vitro active protein of 193 amino acids
additional information
-
effective degradation by enzyme requires an intact complex with N-terminal domains of ClpA chaperone protein to ensure the unfolding of substrates and targeting of unfolded substrates to the protease
additional information
-
tetradecamer, sedimentation velocity analytical ultracentrifugation
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
cryoelectron microscopy at 11 A resolution is used to visualize ClpAP (AAA+ ATPase/unfoldase), aiming to gain insight into ClpP as complexed with ClpA and the structural changes that their interaction may entail. The availability of crystal structures for ClpP that could be fitted into the cryoelectron microscopy density maps shows its changes in conformation that accompany binding of ClpA
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A153C
-
the structure of a crosslinked Escherichia coli ClpP are determined in which the two heptameric rings of ClpP are held together by disulfide bonds. While all Escherichia coli ClpP structures solved to date are in the extended state, the crosslinked ClpP structure is found to be in the compact state. Under reducing condition Km (N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin) is similar to wild-type but kcat is 32fold lower. Under non-reducing conditions mutant is inactive and does not bind its cognate chaperone
Delta1-10
-
deletion of the N-terminal 10, 14, or 17 residues of mature ClpP allows these mutants to degrade alpha-casein, a natively unfolded protein
DELTA1-14
-
deletion of the N-terminal 10, 14, or 17 residues of mature ClpP allows these mutants to degrade alpha-casein, a natively unfolded protein
DELTA1-17
-
deletion of the N-terminal 10, 14, or 17 residues of mature ClpP allows these mutants to degrade alpha-casein, a natively unfolded protein
E8A/R12A/E14A/R15A
-
residues 8-15 form the channel loop of the pore: when charged residues in the channel (amino acids 8-15) are changed with alanine this mutant cleaves the decapeptide at a rate 8fold faster than observed with wild-type ClpP but cleaves the dipeptide at a comparable rate. Mutant shows a substantial GFP-ssrA degradation similar to wild-type
E8G/Q9G/T10G/S11G/R12G/G13G/E14G/R15G
-
residues 8-15 form the channel loop of the pore: when replaced with eight glycines this mutant cleaves the decapeptide at a rate 8fold faster than observed with wild-type ClpP but cleaves the dipeptide at a comparable rate. Mutant shows a much slower degradation of GFP-ssrA
E8G/R12G/E14G/R15G
-
residues 8-15 form the channel loop of the pore: when charged residues in the channel (amino acids 8-15) are changed with glycine this mutant cleaves the decapeptide at a rate 8fold faster than observed with wild-type ClpP but cleaves the dipeptide at a comparable rate. Mutant shows a much slower degradation of GFP-ssrA
E8R/R12E/E14R/R15E
-
residues 8-15 form the channel loop of the pore: when charged residues in the channel (amino acids 8-15) are reversed this mutant cleaves the decapeptide at a rate 8fold faster than observed with wild-type ClpP but cleaves the dipeptide at a comparable rate. Mutant shows a much slower degradation of GFP-ssrA
I19A
-
mutant shows wild-type level of dipeptide cleavage, 20fold increase in decapeptide cleavage compared to wild-type, in contrast to wild-type mutant degrades 113-residue unfolded I27 domain of human titin. Mutant shows high decrease in ClpX affinity
I19D
-
mutant shows 16fold increase in decapeptide cleavage compared to wild-type
I19L
-
mutant shows 6fold increase in decapeptide cleavage compared to wild-type
K25A
-
mutant shows wild-type level of dipeptide cleavage, 1.5fold increase in decapeptide cleavage compared to wild-type. Mutant shows only moderate decrease in ClpX affinity
L23A
-
mutant shows wild-type level of dipeptide cleavage, 5fold increase in decapeptide cleavage compared to wild-type. Mutant shows only moderate decrease in ClpX affinity
L24A
-
mutant shows wild-type level of dipeptide cleavage, 20fold increase in decapeptide cleavage compared to wild-type. Mutant shows high decrease in ClpX affinity
R22A
-
mutant shows wild-type level of dipeptide cleavage, 1.5fold increase in decapeptide cleavage compared to wild-type. Mutant shows high decrease in ClpX affinity
S21A
-
mutant shows wild-type level of dipeptide cleavage, 5fold increase in decapeptide cleavage compared to wild-type. Mutant shows high decrease in ClpX affinity
S499C
-
mutation introduced for FRET measurements. Labelling of S499C with fluorescent probes induces a 10-20fold increase in both chaperone and ATPase activities
Y20A
-
mutant shows wild-type level of dipeptide cleavage, 5fold increase in decapeptide cleavage compared to wild-type. Mutant shows high decrease in ClpX affinity
additional information
additional information
-
RKH loops are important for in substrate recognition
additional information
-
in the absence of ClpA, deletion of the ClpP N-terminus increases the initial degradation rate of large peptide substrates 5-15fold. ClpPDELTAN accelerates degradation of insulin chain B and induces distinct rapid and slow phases of product formation. The distinct slow phase of product formation is eliminated by the addition of hydroxylamine, truncation of the N-terminus leads to stabilization of the acyl-enzyme intermediate
additional information
-
in the clpP mutant (strain SG22159), no degradation of the alkaline phosphatase-aggregate takes place
additional information
-
labeling with 13Cmethyl-methanethiosulfonate (13C-MMTS) at the N-termini of the subunits comprising the Escherichia coli ClpP protease that reveals multiple conformations of gating residues in this complex. These N-terminal residues adopt a single conformation upon gate opening
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
65
-
10 min, 20 mM Tris-HCl, pH 7.8, 5 mM MgCl2, 0.5 mM EDTA, 10% glycerol, little or no loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
Dilution, particularly in plastic tubes inactivates, stabilization by including 0.05% v/v Triton X-100 and 0.1 M KCl stabilizes subunit ClpA
-
In buffers containing Triton X-100, subunit ClpA is stable for several days
-
Repeated freezing and thawing leads to loss of activity of subunit ClpA
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-30°C, subunit ClpA, 20 mM Tris-HCl, pH 7.8, 5 mM MgCl2, 2 mM DTT, 0.5 mM EDTA, 20% v/v glycerol, 1 month, 50% loss of activity,
-
-70°C, 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM DTT, 10% v/v glycerol, stable for extended periods
-
4°C, 20 mM Tris-HCl, pH 7.8, 5 mM MgCl2, 0.5 mM EDTA, 10% glycerol, subunit ClpA stable for several months
-
4°C, 20 mM Tris-HCl, pH 7.8, 5 mM MgCl2, 2 mM DTT, 0.5 mM EDTA, 20% v/v glycerol, 80% loss of activity after 1 week, subunit ClpA
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
of the recombinant proteins by Ni2+-NTA chromatography and gel filtration, untagged protein by phenyl sepharose and Sephacryl 16/60 S-300 column chromatography
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
plasmid encoding ClpPDELTAN transformed into Escherichia coli SG1146GaBL21(DE3) cells
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
analysis
development of a ClpXP protein degradation systemusing purified ClpXP in a cell-free transcription-translation system
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Gottesman, S.; Clark, W.P.; Maurizi, M.R.
The ATP-dependent Clp protease of Escherichia coli. Sequence of clpA and identification of a Clp-specific substrate
J. Biol. Chem.
265
7886-7893
1990
Escherichia coli
Maurizi, M.R.; Clark, W.P.; Katayama, Y.; Rudikoff, S.; Pumphrey, J.; Bowers, B.; Gottesman, S.
Sequence and structure of Clp P, the proteolytic component of the ATP-dependent Clp protease of Escherichia coli
J. Biol. Chem.
265
12536-12545
1990
Escherichia coli
Maurizi, M.R.; Thompson, M.W.; Singh, S.K.; Kim, S.H.
Endopeptidase Clp: ATP-dependent Clp protease from Escherichia coli
Methods Enzymol.
244
314-331
1994
Escherichia coli, Escherichia coli CSH100 (ClpA)
Gottesman, S.; Squires, C.; Pichersky, E.; Carrington, M.; Hobbs, M.; Mattick, J.S.; Dalrymple, B.; Kuramitsu, H.; Shiroza, T.; Foster, T.; Clark, W.P.; Ross, B.; Squires, C.L.; Maurizi, M.R.
Conservation of the regulatory subunit for the Clp ATP-dependent protease in prokaryotes and eukaryotes
Proc. Natl. Acad. Sci. USA
87
3513-3517
1990
Escherichia coli
Hwang, B.J.; Woo, K.M.; Goldberg, A.L.; Chung, C.H.
Protease Ti, a new ATP-dependent protease in Escherichia coli, contains protein-activated ATPase and proteolytic functions in distinct subunits
J. Biol. Chem.
263
8727-8734
1988
Escherichia coli, Escherichia coli RGC125 (lon-)
Katayama-Fujimura, Y.; Gottesman, S.; Maurizi, M.R.
A multiple-component, ATP-dependent protease from Escherichia coli
J. Biol. Chem.
262
4477-4485
1987
Escherichia coli
Katayama, Y.; Gottesman, S.; Pumphrey, J.; Rudikoff, S.; Clark, W.P.; Mauritzi, M.R.
The two-component, ATP-dependent Clp protease of Escherichia coli. Purification, cloning, and mutational analysis of the ATP-binding component
J. Biol. Chem.
263
15226-15236
1988
Escherichia coli
Damerau, K.; St.John, A.C.
Role of Clp protease subunits in degradation of carbon starvation proteins in Escherichia coli
J. Bacteriol.
175
53-63
1993
Escherichia coli
Woo, K.M.; Chung, W.J.; Ha, D.B.; Goldberg, A.L.; Chung, C.H.
Protease Ti from Escherichia coli requires ATP hydrolysis for protein breakdown but not for hydrolysis of small peptides
J. Biol. Chem.
264
2088-2091
1989
Escherichia coli, Escherichia coli RGC125 (lon-)
Wojtkowiak, D.; Geogopoulos, C.; Zylicz, M.
Isolation and characterization of ClpX, a new ATP-dependent specificity component of the Clp protease of Escherichia coli
J. Biol. Chem.
268
22609-22617
1993
Escherichia coli, Escherichia coli W3110 B178
Gottesman, S.; Clark, W.P.; de Crecy-Lagard, V.; Maurizi, M.R.
ClpX, an alternative subunit for the ATP-dependent Clp protease of Escherichia coli. Sequence and in vivo activities
J. Biol. Chem.
268
22618-22626
1993
Escherichia coli
Maurizi, M.R.
ATP-promoted interaction between Clp A and Clp P in activation of Clp protease from Escherichia coli
Biochem. Soc. Trans.
19
719-723
1991
Escherichia coli
Maurizi, M.R.; Clark, W.P.; Kim, S.H.; Gottesman, S.
Clp P represents a unique family of serine proteases
J. Biol. Chem.
265
12546-12552
1990
Escherichia coli
Geuskens, V.; Mhammedi-Alaoui, A.; Desmet, L.; Toussaint, A.
Virulence in bacteriophage Mu: a case of trans-dominant proteolysis by the Escherichia coli Clp serine protease
EMBO J.
11
5121-5127
1992
Escherichia coli
Shapito, J.A.
A role for the Clp protease in activating Mu-mediated DNA rearrangements
J. Bacteriol.
175
2625-2631
1993
Escherichia coli
Porankiewicz, J.; Wang, J.; Clarke, A.K.
New insights into the ATP-dependent Clp protease: Escherichia coli and beyond
Mol. Microbiol.
32
449-458
1999
Aquifex aeolicus (O67357), Arabidopsis thaliana (P56772), Arabidopsis thaliana (Q787X4), Bacillus subtilis (P80244), Bordetella pertussis, Borreliella burgdorferi (O51698), Caenorhabditis elegans (Q27539), Caulobacter vibrioides (B8GX16), Chlamydia trachomatis (P38002), Chlamydomonas moewusii (P42379), Chlamydomonas reinhardtii (P42380), Chlorella vulgaris (P56317), Chlorobaculum tepidum, Clostridium acetobutylicum, Cyanophora paradoxa (Q36863), Deinococcus radiodurans, Enterococcus faecalis, Epifagus virginiana (P30063), Escherichia coli, Fritillaria agrestis (O49081), Haemophilus influenzae (P43867), Helicobacter pylori, Homo sapiens (Q16740), Lactococcus lactis (Q9ZAB0), Listeria monocytogenes, Marchantia polymorpha (P12208), Mus musculus (O88696), Mycobacterium tuberculosis (P9WPC5 and P9WPC3), Mycobacterium tuberculosis H37Rv (P9WPC5 and P9WPC3), Myxococcus xanthus, Nicotiana tabacum (P12210), Oryza sativa (P0C312), Paracoccus denitrificans (P54414), Pinus contorta (P36387), Pinus thunbergii (P41609), Populus tremula, Porphyromonas gingivalis, Pseudomonas aeruginosa, Rhodobacter capsulatus, Salmonella enterica subsp. enterica serovar Typhimurium, Shewanella putrefaciens, Solanum lycopersicum (Q42886), Streptococcus pyogenes, Streptococcus salivarius (P36398), Streptomyces coelicolor, Synechococcus sp., Synechococcus sp. (O34125), Synechococcus sp. (P54415), Synechocystis sp. (P54416), Synechocystis sp. (P74467), Synechocystis sp. (Q59993), Treponema pallidum (O84003), Triticum aestivum (P24064), Yersinia enterocolitica (O30612), Yersinia enterocolitica (Q60107), Zea mays (P12340)
Smith, C.K.; Baker, T.A.; Sauer, R.T.
Lon and Clp family proteases and chaperones share homologous substrate-recognition domains
Proc. Natl. Acad. Sci. USA
96
6678-6682
1999
Escherichia coli
Ishikawa, T.; Beuron, F.; Kessel, M.; Wickner, S.; Maurizi, M.R.; Steven, A.C.
Translocation pathway of protein substrates in ClpAP protease
Proc. Natl. Acad. Sci. USA
98
4328-4333
2001
Escherichia coli
Choi, K.H.; Licht, S.
Control of peptide product sizes by the energy-dependent protease ClpAP
Biochemistry
44
13921-13931
2005
Escherichia coli
Hinnerwisch, J.; Reid, B.G.; Fenton, W.A.; Horwich, A.L.
Roles of the N-domains of the ClpA unfoldase in binding substrate proteins and in stable complex formation with the ClpP protease
J. Biol. Chem.
280
40838-40844
2005
Escherichia coli
Yu, A.Y.; Houry, W.A.
ClpP: a distinctive family of cylindrical energy-dependent serine proteases
FEBS Lett.
581
3749-3757
2007
Streptococcus pneumoniae, Escherichia coli, Homo sapiens, Mycobacterium tuberculosis, Plasmodium falciparum
Farrell, C.M.; Baker, T.A.; Sauer, R.T.
Altered specificity of a AAA+ protease
Mol. Cell
25
161-166
2007
Escherichia coli
Jennings, L.D.; Bohon, J.; Chance, M.R.; Licht, S.
The ClpP N-terminus coordinates substrate access with protease active site reactivity
Biochemistry
47
11031-11040
2008
Escherichia coli
Jana, B.; Panja, S.; Saha, S.; Basu, T.
Mechanism of protonophores-mediated induction of heat-shock response in Escherichia coli
BMC Microbiol.
9
20
2009
Escherichia coli
Maillard, R.A.; Chistol, G.; Sen, M.; Righini, M.; Tan, J.; Kaiser, C.M.; Hodges, C.; Martin, A.; Bustamante, C.
ClpX(P) generates mechanical force to unfold and translocate its protein substrates
Cell
145
459-469
2011
Escherichia coli
Religa, T.L.; Ruschak, A.M.; Rosenzweig, R.; Kay, L.E.
Site-directed methyl group labeling as an NMR probe of structure and dynamics in supramolecular protein systems: applications to the proteasome and to the ClpP protease
J. Am. Chem. Soc.
133
9063-9068
2011
Escherichia coli
Camberg, J.L.; Hoskins, J.R.; Wickner, S.
The interplay of ClpXP with the cell division machinery in Escherichia coli
J. Bacteriol.
193
1911-1918
2011
Escherichia coli
Effantin, G.; Maurizi, M.R.; Steven, A.C.
Binding of the ClpA unfoldase opens the axial gate of ClpP peptidase
J. Biol. Chem.
285
14834-14840
2010
Escherichia coli
Lee, M.E.; Baker, T.A.; Sauer, R.T.
Control of substrate gating and translocation into ClpP by channel residues and ClpX binding
J. Mol. Biol.
399
707-718
2010
Escherichia coli
Nager, A.R.; Baker, T.A.; Sauer, R.T.
Stepwise unfolding of a beta-barrel protein by the AAA+ ClpXP protease
J. Mol. Biol.
413
4-16
2011
Escherichia coli
Kitagawa, R.; Takaya, A.; Yamamoto, T.
Dual regulatory pathways of flagellar gene expression by ClpXP protease in enterohaemorrhagic Escherichia coli
Microbiology
157
3094-3103
2011
Escherichia coli
Kimber, M.S.; Yu, A.Y.; Borg, M.; Leung, E.; Chan, H.S.; Houry, W.A.
Structural and theoretical studies indicate that the cylindrical protease ClpP samples extended and compact conformations
Structure
18
798-808
2010
Escherichia coli
Sen, M.; Maillard, R.; Nyquist, K.; Rodriguez-Aliaga, P.; Presse, S.; Martin, A.; Bustamante, C.
The ClpXP protease unfolds substrates using a constant rate of pulling but different gears
Cell
155
X636-X646
2013
Escherichia coli
-
Cordova, J.C.; Olivares, A.O.; Shin, Y.; Stinson, B.M.; Calmat, S.; Schmitz, K.R.; Aubin-Tam, M.E.; Baker, T.A.; Lang, M.J.; Sauer, R.T.
Stochastic but highly coordinated protein unfolding and translocation by the ClpXP proteolytic machine
Cell
158
647-658
2014
Escherichia coli (P0A6H1)
Alexopoulos, J.A.; Guarne, A.; Ortega, J.
ClpP: a structurally dynamic protease regulated by AAA+ proteins
J. Struct. Biol.
179
202-210
2012
Escherichia coli (P0A6G7), Bacillus subtilis (P80244)
Amor, A.J.; Schmitz, K.R.; Sello, J.K.; Baker, T.A.; Sauer, R.T.
Highly dynamic interactions maintain kinetic stability of the ClpXP protease during the ATP-fueled mechanical cycle
ACS Chem. Biol.
11
1552-1560
2016
Escherichia coli (P0A6G7)
Aguado, A.; Fernandez-Higuero, J.A.; Cabrera, Y.; Moro, F.; Muga, A.
ClpB dynamics is driven by its ATPase cycle and regulated by the DnaK system and substrate proteins
Biochem. J.
466
561-570
2015
Escherichia coli, Escherichia coli BB4561
Shi, X.; Wu, T.; M Cole, C.; K Devaraj, N.; Joseph, S.
Optimization of ClpXP activity and protein synthesis in an E. coli extract-based cell-free expression system
Sci. Rep.
8
3488
2018
Escherichia coli (P0A6G7), Escherichia coli
EXTERNAL LINKS (specific for EC-Number 3.4.21.92)
Transporter Classification Database (TCDB): 3.A.26.1.1
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