Information on EC 3.4.21.92 - Endopeptidase Clp

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
3.4.21.92
-
RECOMMENDED NAME
GeneOntology No.
Endopeptidase Clp
REACTION
REACTION DIAGRAM
COMMENTARY hide
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)
show the reaction diagram
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)
show the reaction diagram
in presence of ATP and Mg2+. In the absence of ATP only oligopeptides shorter than five residues are hydrolyzed
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
CAS REGISTRY NUMBER
COMMENTARY hide
110910-59-3
-
131017-00-0
-
131017-01-1
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SwissProt
Manually annotated by BRENDA team
proteolytic subunit
UniProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
strain NCTC11168, mutants are constructed by replacing the central parts of the C. jejuni clpP genes with a cat gene
-
-
Manually annotated by BRENDA team
strain NCTC11168, mutants are constructed by replacing the central parts of the C. jejuni clpP genes with a cat gene
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
CSH100 (ClpA)
-
-
Manually annotated by BRENDA team
RGC125 (lon-)
-
-
Manually annotated by BRENDA team
W3110 B178
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
ATP-dependent subunit 2
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
tobacco
SwissProt
Manually annotated by BRENDA team
rice
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
pea, L. cv. Alaska
-
-
Manually annotated by BRENDA team
x tremuloides
-
-
Manually annotated by BRENDA team
SS101
UniProt
Manually annotated by BRENDA team
serovar typhimurium
UniProt
Manually annotated by BRENDA team
serovar typhimurium
UniProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
corn, maize
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
Abz-KASPVSLGY(NO2)D + H2O
?
show the reaction diagram
-
-
-
-
?
alkaline phosphatase + H2O
?
show the reaction diagram
-
-
-
-
?
alpha-casein + H2O
?
show the reaction diagram
-
is completely degraded by ClpC and ClpP3/R within 20 min
-
-
?
antitoxin epsilon + H2O
?
show the reaction diagram
-
Epsilon is an antitoxin of the Epsilon/Zeta toxin-antitoxin system family, purified Zeta toxin protects the Epsilon protein from rapid ClpXP-catalyzed degradation
-
-
?
Arc-ssrA + H2O
peptides
show the reaction diagram
-
Arc repressor with a C-terminal ssrA tag
-
-
-
Bacteriophage lambdaO-DNA replication protein + H2O
Hydrolyzed bacteriophage lambdaO-DNA replication protein
show the reaction diagram
beta-casein + H2O
?
show the reaction diagram
-
-
-
?
beta-Galactosidase fusion proteins + H2O
Hydrolyzed beta-galactosidase fusion protein
show the reaction diagram
-
-
-
-
-
casein + H2O
?
show the reaction diagram
casein + H2O
small peptides derived from casein
show the reaction diagram
casein-fluorescein isothiocyanate + H2O
?
show the reaction diagram
-
-
-
?
central competence regulator sigmax + H2O
?
show the reaction diagram
chlorophyll + H2O
?
show the reaction diagram
-
-
-
-
?
chlorophyllide a oxygenase + H2O
?
show the reaction diagram
-
ClpC1 regulates the level of chlorophyllide a oxygenase, chloroplast ClpC1 regulates chlorophyll b biosynthesis
-
-
-
CM-titin-ssrA + H2O
?
show the reaction diagram
-
-
-
-
-
COMK + H2O
?
show the reaction diagram
-
ClpCP, MecA required
-
-
-
CtrA + H2O
?
show the reaction diagram
-
-
-
-
?
elongation factor Ts + H2O
?
show the reaction diagram
-
clpP6 mutant have impaired photosynthesis and chloroplast development
-
-
?
FITC-casein + H2O
?
show the reaction diagram
-
neither ClpC nor ClpP3/R alone degrade FITC-casein but they do when added together. No proteolytic activity when ClpP3 alone is combined with ClpC
-
-
?
FixK2 + H2O
?
show the reaction diagram
substrate is a CRP-like transcription factor that controls the endosymbiotic lifestyle of Bradyrhizobium japonicum. Degradation occurs by the ClpAP1 chaperone-protease complex, but not by the ClpXP1 chaperone-protease complex, and is inhibited by the ClpS1 adaptor protein. The last 12 amino acids of FixK2 are recognized by ClpA
-
-
?
FlhC subunit + H2O + ATP
?
show the reaction diagram
FR-GFP + H2O
?
show the reaction diagram
-
ClpCP3/R with ClpS1 take over 20 min to completely degrade FR-GFP, whereas the ClpAP protease degrades all FR-GFP within 2 min
-
-
?
GFP-K17 fusion protein + H2O
?
show the reaction diagram
-
-
-
-
-
GFP-ssrA + H2O
?
show the reaction diagram
-
-
-
-
?
Glucagon + H2O
Hydrolyzed glucagon
show the reaction diagram
Gly-L-Arg-7-amido-4-methylcoumarin + H2O
Gly-L-Arg + 7-amino-4-methylcoumarin
show the reaction diagram
substrate for the recombinant ClpP
-
-
?
green-fluorescent-protein-ssrA + H2O
?
show the reaction diagram
-
-
-
-
?
Hsp90 + H2O
?
show the reaction diagram
-
-
-
-
?
insulin chain B + H2O
?
show the reaction diagram
-
-
-
-
?
LacZ + H2O
?
show the reaction diagram
Lambda O Arc + H2O
?
show the reaction diagram
-
Arc repressor with a N-terminal lambda O degradation tag
-
-
-
Lambda O CM-titin + H2O
?
show the reaction diagram
-
-
-
-
-
Leu-Tyr-Leu-Tyr-Trp + H2O
Leu-Tyr-Leu + Tyr-Trp
show the reaction diagram
-
cleavage occurs primarily at Leu3-Tyr4, but significant cleavage also at Tyr2-Leu3 and Leu4-Trp5 bond
-
-
LexA N-terminal domain + H2O
?
show the reaction diagram
-
-
-
-
?
luciferase-ssrA + H2O
?
show the reaction diagram
-
-
-
-
?
MurAA + H2O
?
show the reaction diagram
-
MecA not required for degradation
-
-
-
Mutated repressor of Mu prophage + H2O
Hydrolyzed mutated repressor of Mu prophage
show the reaction diagram
-
high susceptibility to the Clp-dependent degradation
-
-
-
N-succinyl-Ile-Ile-Trp-7-amido-4-methylcoumarin + H2O
N-succinyl-Ile-Ile-Trp + 7-amino-4-methylcoumarin
show the reaction diagram
-
throughout the 5 min time course, ClpP readily degrades the dipeptide, whereas ClpP3/R does not. Prolonging the incubation time with ClpP3/R to 20 min does not result in any visible degradation. Addition of ClpC to the assays also fails to produce any degradation
-
-
?
N-succinyl-L-isoleucine-L-isoleucine-L-tryptophan-7-amido-4-methylcoumarin + H2O
?
show the reaction diagram
-
-
-
?
N-succinyl-Leu-Tyr 4-methylcoumarin 7-amide + H2O
N-succinyl-Leu-Tyr + 7-amino-4-methylcoumarin
show the reaction diagram
N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin + H2O
?
show the reaction diagram
N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin + H2O
N-succinyl-Leu-Tyr + 7-amino-4-methylcoumarin
show the reaction diagram
N-succinyl-LLVY-7-amido-4-methylcoumarin + H2O
?
show the reaction diagram
-
-
-
?
N-succinyl-Val-Lys-Met-7-amido-4-methylcoumarin + H2O
N-succinyl-Val-Lys-Met + 7-amino-4-methylcoumarin
show the reaction diagram
-
throughout the 5 min time course, ClpP readily degrades the dipeptide, whereas ClpP3/R does not. Prolonging the incubation time with ClpP3/R to 20 min does not result in any visible degradation. Addition of ClpC to the assays also fails to produce any degradation
-
-
?
ornithine decarboxylase CC030 + H2O
?
show the reaction diagram
-
-
CC0360 is rapidly degraded by ClpP protease in vitro. CC0360 is exclusively degraded by the full-length ClpXP complex and not by a version of ClpX lacking the Nterminal domain
-
?
Oxidized insulin B-chain + H2O
Hydrolyzed insulin B-chain
show the reaction diagram
-
cleavage at multiple sites
-
-
-
Phe-Ala-Pro-His-Met-Ala-Leu-Val-Pro-Val + H2O
?
show the reaction diagram
-
synthetic polypeptide that corresponds to the 10 amino acids surrounding the in vivo processing site in ClpP subunit
-
-
-
protein RepA + H2O
?
show the reaction diagram
-
model substrate from bacteriophage P1
-
?
RNA Helicase + H2O
?
show the reaction diagram
-
-
-
-
?
RpoS sigma factor + H2O
?
show the reaction diagram
-
with the assistance of recognition factor RssB, ClpXP degrades the RpoS sigma factor
-
-
?
RsiW + H2O
?
show the reaction diagram
-
ClXCP, AA at C-terminal as degradation tag
-
-
-
Sda + H2O
?
show the reaction diagram
-
ClpXP, VSS at C-terminal as degradation tag
-
-
-
SpollAB + H2O
?
show the reaction diagram
-
ClpCP, LCN at C-terminal as degradation tag, MecA not required, production of ClpP is strongly increased in response to heat shock or other stress signals, ClpP removes heat damaged proteins
-
-
-
Spx + H2O
?
show the reaction diagram
SsrA tagged proteins + H2O
?
show the reaction diagram
-
ClpXP, AA at C-terminal as degradation tag
-
-
-
ssrA-dabsyl + H2O
?
show the reaction diagram
-
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
-
-
?
SsrA-tagged LacZ + H2O
?
show the reaction diagram
stalk synthesis transcription factor TacA + H2O
?
show the reaction diagram
-
TacA degradation is controlled during the cell cycle dependent on the ClpXP regulator CpdR and stabilization of TacA increases degradation of another ClpXP substrate, CtrA, while restoring deficiencies associated with prolific CpdR activity
-
-
?
Starvation proteins + H2O
?
show the reaction diagram
-
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
-
-
-
Suc-AAPF-4-methylcoumarin-7-amide + H2O
?
show the reaction diagram
-
-
-
?
Suc-AFK-4-methylcoumarin-7-amide + H2O
?
show the reaction diagram
-
-
-
?
Suc-IA-4-methylcoumarin-7-amide + H2O
?
show the reaction diagram
-
-
-
?
Suc-IIW-4-methylcoumarin-7-amide + H2O
?
show the reaction diagram
-
-
-
?
Suc-LY-4-methylcoumarin-7-amide + H2O
?
show the reaction diagram
-
-
-
?
Succinyl-Ala-Ala-Phe 4-methylcoumarin 7-amide + H2O
Succinyl-Ala-Ala + Phe 4-methylcoumarin 7-amide
show the reaction diagram
succinyl-L-Leu-L-Lys-7-amido-4-methylcoumarin + H2O
?
show the reaction diagram
recombinant mature ClpP is most active against succinyl-L-Leu-L-Lys-7-amido-4-methylcoumarin
-
-
?
succinyl-L-Leu-L-Tyr-7-amido-4-methylcoumarin + H2O
succinyl-L-Leu-L-Tyr + 7-amino-4-methylcoumarin
show the reaction diagram
recombinant ClpP does not cleave the known ClpP substrate succinyl-L-Leu-L-Tyr-7-amido-4-methylcoumarin
-
-
?
Succinyl-Leu-Leu-Val-Tyr 4-methylcoumarin 7-amide + H2O
Succinyl-Leu + Leu + Val-Tyr 4-methylcoumarin 7-amide
show the reaction diagram
Succinyl-Leu-Tyr 4-methylcoumarin 7-amide + H2O
Succinyl-Leu-Tyr + 7-amino-4-methylcoumarin
show the reaction diagram
-
ClpP subunit alone
-
-
succinyl-LY-4-methylcoumarin-7-amide + H2O
?
show the reaction diagram
-
-
-
-
?
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
antitoxin epsilon + H2O
?
show the reaction diagram
-
Epsilon is an antitoxin of the Epsilon/Zeta toxin-antitoxin system family, purified Zeta toxin protects the Epsilon protein from rapid ClpXP-catalyzed degradation
-
-
?
central competence regulator sigmax + H2O
?
show the reaction diagram
FixK2 + H2O
?
show the reaction diagram
Q89KG1
substrate is a CRP-like transcription factor that controls the endosymbiotic lifestyle of Bradyrhizobium japonicum. Degradation occurs by the ClpAP1 chaperone-protease complex, but not by the ClpXP1 chaperone-protease complex, and is inhibited by the ClpS1 adaptor protein. The last 12 amino acids of FixK2 are recognized by ClpA
-
-
?
FlhC subunit + H2O + ATP
?
show the reaction diagram
Starvation proteins + H2O
?
show the reaction diagram
-
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
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
poor substitute for Mg2+
Mn2+
-
can partially substitute for Mg2+
Zn2+
-
poor substitute for Mg2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(4R)-3-(4-methoxyphenyl)-4-(pent-4-yn-1-yl)oxetan-2-one
-
inhibitor efficiently alters the oligomerization of the enzyme to smaller species, almost quantitative shift from the tetradecamer to the heptamer with modification of 35% of the active sites
1-(1,1-dioxido-1,2-thiazetidin-2-yl)hexan-1-one
-
alkyne-free beta-sultam analogue. Treatment leads to dehydroalanine formation of the active site serine. The reaction proceeds through sulfonylation and subsequent elimination, thereby obliterating the catalytic charge relay system
1-(4-benzoyl-1,1-dioxido-1,2-thiazetidin-2-yl)ethanone
-
alkyne-free beta-sultam analogue. Treatment leads to dehydroalanine formation of the active site serine. The reaction proceeds through sulfonylation and subsequent elimination, thereby obliterating the catalytic charge relay system
1-[4-(4-ethynylbenzoyl)-1,1-dioxido-1,2-thiazetidin-2-yl]ethanone
-
treatment results in almost instant covalent modification of all 14 active sites and complete inhibition of peptidase activity
1-[4-(4-ethynylbenzoyl)-1,1-dioxido-1,2-thiazetidin-2-yl]undec-10-en-1-one
-
inhibitor efficiently alters the oligomerization of the enzyme to smaller species, almost quantitative shift from the tetradecamer to the heptamer with modification of 63% of the active sites
1-[4-benzoyl-1,1-dioxido-1,2-thiazetidin-2-yl]undec-10-en-1-one
-
alkyne-free beta-sultam analogue. Treatment leads to dehydroalanine formation of the active site serine. The reaction proceeds through sulfonylation and subsequent elimination, thereby obliterating the catalytic charge relay system
3-(4-methoxyphenyl)-4-(pent-4-ynyl)oxetan-2-one
-
shows stronger inhibitory effect
3-(non-8-ynyl)-4-(pent-4-ynyl)oxetan-2-one
-
exerts the weakest effect on peptidase activity
3-butyl-4-(pent-4-ynyl)oxetan-2-one
-
shows stronger inhibitory effect
CAANDENYALAA
-
-
CAANDENYALAA-NH2
-
-
diisopropyl fluorophosphate
diisopropylfluorophosphate
-
-
fluorosulfonylbenzoyladenosine
-
-
High salt concentrations
-
chloride is much more inhibitory than acetate, divalent anions are also very inhibitory
-
kappa-casein
-
strong, competitive
-
Mg2+
-
proteolytic activity of ClpAP is dependent on, but concentrations higher than about 30 mM are inhibitory
Neohydrin
-
-
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
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
acyldepsipeptide
acyldepsipeptides
-
ADEP series 2, 3, 4 as well as two other synthesized derivatives, IDR-10001 and IDR-10011, which incorporate N-methylalanine instead of the more rigid homoproline in the depsipeptide core structure. All five compound are active against Mycobacterium tuberculosis, ADEP2 is the most active
-
ATP
stimulates proteolytic activity of ClpP in heart mitochondria of muscle creatine kinase mutants
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
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0005 - 0.14
Arc-ssrA
-
0.21
ATP
-
for hydrolysis of both casein and ATP
0.0024 - 0.08
CM-titin-ssrA
-
0.04 - 0.3167
insulin chain B
-
0.0044 - 0.013
Lambda O Arc
-
0.011 - 0.041
Lambda O CM-titiin
-
1.3
N-Succinyl-Leu-Tyr 4-methylcoumarin 7-amide
-
-
0.52 - 1.1
N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin
0.065
Suc-LLVY-4-methylcoumarin-7-amide
30°C, pH 7.0
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.17 - 2.17
ATP
0.5
glucagon
Escherichia coli
-
value below
0.33 - 0.35
insulin chain B
-
0.003 - 15.8
N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin
0.5
oxidized insulin B-chain
Escherichia coli
-
value below
-
13.3
Phe-Ala-Pro-His-Met-Ala-Leu-Val-Pro-Val
Escherichia coli
-
-
0.000036
Suc-LLVY-4-methylcoumarin-7-amide
Plasmodium falciparum
O97252
30°C, pH 7.0, PfClpP is a very weak peptidase on its own
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.004
3-(4-methoxyphenyl)-4-(pent-4-ynyl)oxetan-2-one
-
-
0.031
3-(non-8-ynyl)-4-(pent-4-ynyl)oxetan-2-one
-
-
0.006
3-butyl-4-(pent-4-ynyl)oxetan-2-one
-
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
98
1-[4-(4-ethynylbenzoyl)-1,1-dioxido-1,2-thiazetidin-2-yl]ethanone
Staphylococcus aureus
-
pH not specified in the publication, temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
-
succinyl-Leu-Tyr 4-methylcoumarin 7-amide
7.5 - 9.5
-
alpha-casein
7.6
-
assay at
additional information
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
42
-
active in degrading alpha-casein up to 42°C, no proteinase activity at 55°C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
reduced level of the Lon and ClpP proteins in SPG13 patient cells as compared with controls
Manually annotated by BRENDA team
-
reduced level of the Lon and ClpP proteins in SPG13 patient cells as compared with controls
Manually annotated by BRENDA team
additional information
-
decreased expression of the mitochondrial protein quality control proteases Lon and ClpP, both at the RNA and protein level, in patients with an autosomal dominant form of hereditary spastic paraplegia (SPG13)
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Coxiella burnetii (strain RSA 493 / Nine Mile phase I)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Francisella tularensis subsp. tularensis (strain SCHU S4 / Schu 4)
Francisella tularensis subsp. tularensis (strain SCHU S4 / Schu 4)
Francisella tularensis subsp. tularensis (strain SCHU S4 / Schu 4)
Francisella tularensis subsp. tularensis (strain SCHU S4 / Schu 4)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Listeria monocytogenes serovar 1/2a (strain ATCC BAA-679 / EGD-e)
Listeria monocytogenes serovar 1/2a (strain ATCC BAA-679 / EGD-e)
Listeria monocytogenes serovar 1/2a (strain ATCC BAA-679 / EGD-e)
Listeria monocytogenes serovar 1/2a (strain ATCC BAA-679 / EGD-e)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain CDC 1551 / Oshkosh)
Mycobacterium tuberculosis (strain CDC 1551 / Oshkosh)
Neisseria meningitidis serogroup B (strain MC58)
Staphylococcus aureus (strain NCTC 8325)
Staphylococcus aureus (strain NCTC 8325)
Staphylococcus aureus (strain NCTC 8325)
Staphylococcus aureus (strain NCTC 8325)
Staphylococcus aureus (strain NCTC 8325)
Staphylococcus aureus (strain NCTC 8325)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
12620
-
ClpA, analytical ultracentrifugation
12640
-
ClpB, analytical ultracentrifugation
13940
-
ClpX, analytical ultracentrifugation
14230
-
ClpY, analytical ultracentrifugation
21000
-
1 * 230000, subunit ClpP (12 * 21000, amino acid sequence, subunit of ClpP)
22000
-
ClpP1(pClpP), MALDI-TOF, ClpS1(nClpC like), MALDI-TOF, ClpP5 (nClpP1), MALDI-TOF, ClpP6 (nClpP6), MALDI-TOF
23000 - 25000
-
ClpP2 (nClpP7), MALDI-TOF
25000
Western blot
26000 - 29000
-
2 isoenzymes, immunoblot analysis, antibody against plastid-encoded rice ClpP
26000
-
ClpR2 (nClpP2), MALDI-TOF, 6,13,21 ClpP4 (nClpP4), MALDI-TOF
27000
-
ClpR3 (nClpP8), MALDI-TOF
28000
-
ClpR1 (nClpP5), MALDI-TOF
29000
-
ClpP3 (nClpP3), MALDI-TOF
43000
calculated from cDNA, unprocessed protein
46000
-
x * 46000, ClpX, SDS-PAGE
46300
-
x * 46300, ClpX, calculation from amino acid sequence
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)
81000
-
x * 81000, ClpA, SDS-PAGE
83000
-
x * 120000-140000, subunit ClpA, gel filtration, x * 83000, subunit ClpA, amino acid sequence
140000
-
native Page, ClpP1; native Page, ClpP2
150000
-
gel filtration, enzyme treated with inhibitor diisopropyl fluorophosphate, 1-[4-(4-ethynylbenzoyl)-1,1-dioxido-1,2-thiazetidin-2-yl]undec-10-en-1-one, or beta-lactone (4R)-3-(4-methoxyphenyl)-4-(pent-4-yn-1-yl)oxetan-2-one
180000
-
n * 230000, n * 180000, ClpP1, ClpP3, clpP4, ClpP5, clpP6, clpR1, clpR2, ClpR3, cClR4, ClpS1
230000
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
270000
300000
304000
-
gel filtration, native enzyme
335000
-
wild type, native Page, decreased by 80% in clpP6 antisense mutants, ClpP6 is necessary for the formation of the Clp proteolytic core complex
340000
-
E. coli
346000
-
sedimentation velocity analytical ultracentrifugation, tetradecamer
350000
-
ClpP protease complex, gel filtration
700000
-
E. coli, complex of subunits ClpA with ClpP in presence of ATP
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heptamer
multimer
-
n * 230000, n * 180000, ClpP1, ClpP3, clpP4, ClpP5, clpP6, clpR1, clpR2, ClpR3, cClR4, ClpS1
oligomer
tetradecamer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of ClpP from Bacillus subtilis shows a significantly compressed shape along the axial direction. A portion of the handle regions comprising the heptameric ring-ring contacts shows structural transition from an ordered to a disordered state, which triggers the large conformational change from an extended to an overall compressed structure. Along with this structural change, 14 side pores are generated for product release and the catalytic triad adopts an inactive orientation
-
ClpP structures have been solved from five different organisms
-
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
-
by the microbatch method at 14°C. Crystal structure of ClpP, at a resolution of 2.6 A, in complex with product peptides (heptapeptide) bound to the active site as well as in the apo state and crystal structure of the ClpP mutant, at 2.5 A resolution, and in complex with a tetrapeptide. In the complex structure, the peptides are zipped with two antiparallel strands of ClpP and point to the adjacent active site, explaining the broad substrate specificity, the product inhibition and the processive degradation of substrates in the chamber. Substrate binding causes local conformational changes around the active site that ultimately induce the active conformation of ClpP. The peptide binds to the active site of ClpP via hydrogen-bond networks
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ClpP structures have been solved from five different organisms, human ClpP can form a complex with Escherichia coli ClpX in vitro
-
ClpP structures have been solved from five different organisms
the X-ray structure of PfClpP shows the protein as a compacted tetradecamer
SaClpP consists of two heptameric rings that form a tetradecameric barrel. Compared to other ClpP structures, the barrel is compressed by roughly 10 A along the axial direction, and the ring-ring interface contains 14 equatorial side pores that can be up to 6 A in diameter, depending on the side-chain conformations. The side pores are lined with conserved hydrophobic residues and apparently represent the long-sought exit route for peptide products from the barrel chamber
-
ClpP structures have been solved from five different organisms
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mutant A153P, disruption of handle region resulting in an altered ring-ring dimerization interface. There exists a flexible N-terminal loop in each enzyme subunit that is important for complex formation with ClpXP and ClpAP
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modeling of chaperone ClpC and ClpP3/R subunit structures. The R-ring of ClpP3 is most likely to associate with ClpC
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10
-
inactivation after a short time
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
ATP and nonhydrolyzable analogs of ATP stabilize subunit ClpA
-
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
-
Subunit ClpA is sensitive to freezing
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-15°C, 20% glycerol, 70% loss of activity after 1 month, subunit ClpX
-
-30°C, subunit ClpA, 1 month, 50% loss of activity
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-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
-
-70°C, ClpP stable
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-70°C, stable without substantial loss of activity, subunit ClpX
-
-70°C, subunit ClpP stable
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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
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a subcomplex of ClpP can be produced by dissociation, comprising ClpP1 and ClpR1, 2, 3 and 4, similar to the ClpR-ring described in land plants. Purified ClpP preparation also contains two ClpT subunits, ClpT3 and ClpT4, which like the land plant ClpT1 and ClpT2 show 2 Clp-N domains
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by anion exchange and gel filtration
-
by immunoprecipitation
-
ClpA, ClpAP, Clp-SC
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ClpP-His6, ClpA and ClpPDELTAN
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ClpP3/R complex purified on Ni2+ affinity column and by gel filtration, purification of ClpC and ClpS1
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glutathione-sepharose 4B bead column chromatography
of the recombinant proteins by Ni2+-NTA chromatography and gel filtration, untagged protein by phenyl sepharose and Sephacryl 16/60 S-300 column chromatography
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SSD domains of the ATPase activity
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using Ni-NTA chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
as His tagged fusion protein
-
cloning and mutational analysis of the ATP-binding component ClpA
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Clp sequenced
Clp sequenced, nuclear encoded gene
Clp sequenced, plastid encoded gene
Clp sequenced; Clp sequenced
clpP3 and clpR genes cloned into the pACYC Duet vector for co-expression in Escherichia coli BL21-STAR cells
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Escherichia coli DH5alpha transformed with a pEX18Tc-clpP or a pME6031-clpP plasmid by heat shock transformation
expressed in Escherichia coli
-
expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli using a His-tagged fusion protein
-
expression in Escherichia coli
-
exprression in Escherichia coli
into pET-15b vector and expressed as His-tag recombinant protein in Escherichia coli BL21(DE3)pLysS strain
of the ATP-binding subunit ClpA of the ATP-dependent Clp protease
-
of the subunit ClpP containing the proteolytic active site
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plasmid encoding ClpPDELTAN transformed into Escherichia coli SG1146GaBL21(DE3) cells
-
the entire ClpP gene (amino acid residues 1-196) transferred into pET-22b(+) and expressed in Escherichia coli BL21(DE3)
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
proteolytic subunits ClpP1 and ClpP2 are co-expressed under normal growth conditions with a single promoter region for the clpP1P2 operon; proteolytic subunits ClpP1 and ClpP2 are co-expressed under normal growth conditions with a single promoter region for the clpP1P2 operon
stress conditions enhance the mRNA expression of subunit ClpP2
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
G773R
-
inactive mutant
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
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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
H230A
-
RKH mutant to investigate the role of the RKH sequence loops
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
K229A
-
RAH mutant to investigate the role of the RKH sequence loops
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
R228A
-
AKH mutant to investigate the role of the RKH sequence loops
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
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
S99A
-
does not digest peptide into smaller fragments
E135A
-
mutant shows no activity
E135R
-
mutant shows no activity
G127
-
mutant shows no activity
G128
-
mutant shows no activity
G131
-
mutant shows no activity
L144E
-
mutant shows no activity
L144G
-
mutant shows no activity
L144M
-
Km (N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin): 0.523 mM, kcat: 0.003/sec
L144R
-
mutant shows no activity
S98A
-
mutant shows no activity
A153P
-
crystallization data, disruption of handle region resulting in an altered ring-ring dimerization interface
S101A
-
mutation of the active site Ser-101 in ClpP3 inactivates the entire ClpCP3/R protease, the mutation has no effect on the formation of the recombinant ClpP3/R core complex of 270 kDa. The mutated ClpP3/R complex stimulates the steady-state ATPase activity of ClpC to the same extent as wild-type ClpP3/R. The mutated ClpP3/R core fails to degrade the alpha-casein nor the more sensitive FITC-casein
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
-
synthetic beta-lactones as novel inhibitors for specific and selective targeting of the key virulence regulator ClpP
medicine
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