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- PDB-7p81: Crystal structure of ClpP from Bacillus subtilis in complex with ... -

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Basic information

Entry
Database: PDB / ID: 7p81
TitleCrystal structure of ClpP from Bacillus subtilis in complex with ADEP2 (compact state)
Components
  • ADEP2
  • ATP-dependent Clp protease proteolytic subunit
KeywordsHYDROLASE / ClpP / Acyldepsipeptides / ADEP2 / compressed / antibiotics
Function / homology
Function and homology information


endopeptidase Clp / endopeptidase Clp complex / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / ATPase binding / serine-type endopeptidase activity / identical protein binding / cytoplasm
Similarity search - Function
ClpP, Ser active site / Endopeptidase Clp serine active site. / ClpP, histidine active site / Endopeptidase Clp histidine active site. / ATP-dependent Clp protease proteolytic subunit / Clp protease proteolytic subunit /Translocation-enhancing protein TepA / Clp protease / 2-enoyl-CoA Hydratase; Chain A, domain 1 / 2-enoyl-CoA Hydratase; Chain A, domain 1 / ClpP/crotonase-like domain superfamily ...ClpP, Ser active site / Endopeptidase Clp serine active site. / ClpP, histidine active site / Endopeptidase Clp histidine active site. / ATP-dependent Clp protease proteolytic subunit / Clp protease proteolytic subunit /Translocation-enhancing protein TepA / Clp protease / 2-enoyl-CoA Hydratase; Chain A, domain 1 / 2-enoyl-CoA Hydratase; Chain A, domain 1 / ClpP/crotonase-like domain superfamily / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
ADEP2 / ATP-dependent Clp protease proteolytic subunit
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.79 Å
AuthorsLee, B.-G. / Kim, L. / Kim, M.K. / Kwon, D.H. / Song, H.K.
Funding support Korea, Republic Of, 3items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea)2020R1A2C3008285 Korea, Republic Of
National Research Foundation (NRF, Korea)2020R1A5A1019023 Korea, Republic Of
National Research Foundation (NRF, Korea)2021M3A9I4030068 Korea, Republic Of
CitationJournal: EMBO J / Year: 2022
Title: Structural insights into ClpP protease side exit pore-opening by a pH drop coupled with substrate hydrolysis.
Authors: Leehyeon Kim / Byung-Gil Lee / Minki Kim / Min Kyung Kim / Do Hoon Kwon / Hyunmin Kim / Heike Brötz-Oesterhelt / Soung-Hun Roh / Hyun Kyu Song /
Abstract: The ClpP serine peptidase is a tetradecameric degradation molecular machine involved in many physiological processes. It becomes a competent ATP-dependent protease when coupled with Clp-ATPases. ...The ClpP serine peptidase is a tetradecameric degradation molecular machine involved in many physiological processes. It becomes a competent ATP-dependent protease when coupled with Clp-ATPases. Small chemical compounds, acyldepsipeptides (ADEPs), are known to cause the dysregulation and activation of ClpP without ATPases and have potential as novel antibiotics. Previously, structural studies of ClpP from various species revealed its structural details, conformational changes, and activation mechanism. Although product release through side exit pores has been proposed, the detailed driving force for product release remains elusive. Herein, we report crystal structures of ClpP from Bacillus subtilis (BsClpP) in unforeseen ADEP-bound states. Cryo-electron microscopy structures of BsClpP revealed various conformational states under different pH conditions. To understand the conformational change required for product release, we investigated the relationship between substrate hydrolysis and the pH-lowering process. The production of hydrolyzed peptides from acidic and basic substrates by proteinase K and BsClpP lowered the pH values. Our data, together with those of previous findings, provide insight into the molecular mechanism of product release by the ClpP self-compartmentalizing protease.
History
DepositionJul 21, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 29, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Jul 20, 2022Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.4Nov 13, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ATP-dependent Clp protease proteolytic subunit
B: ATP-dependent Clp protease proteolytic subunit
C: ATP-dependent Clp protease proteolytic subunit
D: ATP-dependent Clp protease proteolytic subunit
E: ATP-dependent Clp protease proteolytic subunit
F: ATP-dependent Clp protease proteolytic subunit
G: ATP-dependent Clp protease proteolytic subunit
H: ATP-dependent Clp protease proteolytic subunit
I: ATP-dependent Clp protease proteolytic subunit
J: ATP-dependent Clp protease proteolytic subunit
K: ATP-dependent Clp protease proteolytic subunit
L: ATP-dependent Clp protease proteolytic subunit
M: ATP-dependent Clp protease proteolytic subunit
N: ATP-dependent Clp protease proteolytic subunit
O: ATP-dependent Clp protease proteolytic subunit
P: ATP-dependent Clp protease proteolytic subunit
Q: ATP-dependent Clp protease proteolytic subunit
R: ATP-dependent Clp protease proteolytic subunit
S: ATP-dependent Clp protease proteolytic subunit
T: ATP-dependent Clp protease proteolytic subunit
U: ATP-dependent Clp protease proteolytic subunit
V: ATP-dependent Clp protease proteolytic subunit
W: ATP-dependent Clp protease proteolytic subunit
X: ATP-dependent Clp protease proteolytic subunit
Y: ATP-dependent Clp protease proteolytic subunit
Z: ATP-dependent Clp protease proteolytic subunit
a: ATP-dependent Clp protease proteolytic subunit
b: ATP-dependent Clp protease proteolytic subunit
c: ADEP2
d: ADEP2
e: ADEP2
f: ADEP2
g: ADEP2
h: ADEP2
i: ADEP2
j: ADEP2
k: ADEP2
l: ADEP2
m: ADEP2
n: ADEP2
o: ADEP2
p: ADEP2
q: ADEP2
r: ADEP2
t: ADEP2
u: ADEP2
v: ADEP2
w: ADEP2


Theoretical massNumber of molelcules
Total (without water)633,55048
Polymers633,55048
Non-polymers00
Water5,386299
1
A: ATP-dependent Clp protease proteolytic subunit
B: ATP-dependent Clp protease proteolytic subunit
C: ATP-dependent Clp protease proteolytic subunit
D: ATP-dependent Clp protease proteolytic subunit
E: ATP-dependent Clp protease proteolytic subunit
F: ATP-dependent Clp protease proteolytic subunit
G: ATP-dependent Clp protease proteolytic subunit
H: ATP-dependent Clp protease proteolytic subunit
I: ATP-dependent Clp protease proteolytic subunit
J: ATP-dependent Clp protease proteolytic subunit
K: ATP-dependent Clp protease proteolytic subunit
L: ATP-dependent Clp protease proteolytic subunit
M: ATP-dependent Clp protease proteolytic subunit
N: ATP-dependent Clp protease proteolytic subunit
c: ADEP2
d: ADEP2
e: ADEP2
f: ADEP2
g: ADEP2
h: ADEP2
i: ADEP2
j: ADEP2
k: ADEP2
l: ADEP2


Theoretical massNumber of molelcules
Total (without water)316,77524
Polymers316,77524
Non-polymers00
Water32418
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
O: ATP-dependent Clp protease proteolytic subunit
P: ATP-dependent Clp protease proteolytic subunit
Q: ATP-dependent Clp protease proteolytic subunit
R: ATP-dependent Clp protease proteolytic subunit
S: ATP-dependent Clp protease proteolytic subunit
T: ATP-dependent Clp protease proteolytic subunit
U: ATP-dependent Clp protease proteolytic subunit
V: ATP-dependent Clp protease proteolytic subunit
W: ATP-dependent Clp protease proteolytic subunit
X: ATP-dependent Clp protease proteolytic subunit
Y: ATP-dependent Clp protease proteolytic subunit
Z: ATP-dependent Clp protease proteolytic subunit
a: ATP-dependent Clp protease proteolytic subunit
b: ATP-dependent Clp protease proteolytic subunit
m: ADEP2
n: ADEP2
o: ADEP2
p: ADEP2
q: ADEP2
r: ADEP2
t: ADEP2
u: ADEP2
v: ADEP2
w: ADEP2


Theoretical massNumber of molelcules
Total (without water)316,77524
Polymers316,77524
Non-polymers00
Water30617
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)112.979, 173.075, 160.001
Angle α, β, γ (deg.)90.000, 91.660, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein ...
ATP-dependent Clp protease proteolytic subunit / Caseinolytic protease / Endopeptidase Clp / Stress protein G7


Mass: 22043.270 Da / Num. of mol.: 28
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (strain 168) (bacteria)
Strain: 168 / Gene: clpP, yvdN, BSU34540 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P80244, endopeptidase Clp
#2: Protein/peptide
ADEP2


Type: Cyclic depsipeptide / Class: Antibiotic / Mass: 816.931 Da / Num. of mol.: 20 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) / References: ADEP2
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 299 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.95 Å3/Da / Density % sol: 58.29 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 100 mM sodium citrate pH 5.6, 100 mM Li2SO4 and 9~11% (w/v) PEG 6,000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Photon Factory / Beamline: AR-NW12A / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 210 / Detector: CCD / Date: Jun 23, 2007
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.79→50 Å / Num. obs: 146652 / % possible obs: 96.03 % / Redundancy: 5.4 % / Biso Wilson estimate: 51.95 Å2 / Rsym value: 0.097 / Net I/σ(I): 19.79
Reflection shellResolution: 2.79→2.9 Å / Num. unique obs: 11129 / Rsym value: 0.466

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
PDB_EXTRACT3.27data extraction
HKL-2000data reduction
HKL-2000data scaling
MOLREPphasing
Coot1.19.2_4158model building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3ktg

3ktg


Resolution: 2.79→45.51 Å / SU ML: 0.4 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 32.74 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.29 2016 1.38 %
Rwork0.2348 144583 -
obs0.2356 146599 95.77 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 116.72 Å2 / Biso mean: 55.3698 Å2 / Biso min: 20.08 Å2
Refinement stepCycle: final / Resolution: 2.79→45.51 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms36809 0 1140 299 38248
Biso mean--56.23 46.83 -
Num. residues----4804
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.79-2.860.3583940.30057226732067
2.86-2.930.39171150.29798952906783
2.93-3.020.34641550.2914101111026694
3.02-3.120.32981450.2823105551070099
3.12-3.230.35171410.27941078410925100
3.23-3.360.36491560.27521064010796100
3.36-3.510.33831500.26591074110891100
3.51-3.70.2821540.24771073910893100
3.7-3.930.32741480.23961075210900100
3.93-4.230.28941550.22211076610921100
4.23-4.660.24511410.19331077110912100
4.66-5.330.26051550.21611081610971100
5.33-6.710.28981490.23571084410993100
6.71-45.510.21361580.1905108861104499

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