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- PDB-8xop: Cryo-EM structure of ClpP1P2 in complex with ADEP1 from Streptomy... -

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Basic information

Entry
Database: PDB / ID: 8xop
TitleCryo-EM structure of ClpP1P2 in complex with ADEP1 from Streptomyces hawaiiensis
Components
  • (ATP-dependent Clp protease proteolytic subunit) x 2
  • ADEP1
KeywordsHYDROLASE / protease / protein degradation / proteostasis / proteolysis
Function / homology
Function and homology information


endopeptidase Clp / ATP-dependent peptidase activity / serine-type endopeptidase activity / proteolysis / 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 / ClpP/crotonase-like domain superfamily
Similarity search - Domain/homology
ADEP1 / ATP-dependent Clp protease proteolytic subunit / ATP-dependent Clp protease proteolytic subunit
Similarity search - Component
Biological speciesStreptomyces hawaiiensis (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsXu, X. / Long, F.
Funding support China, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2021YFA0909500 China
CitationJournal: mBio / Year: 2024
Title: Structural insights into the Clp protein degradation machinery.
Authors: Xiaolong Xu / Yanhui Wang / Wei Huang / Danyang Li / Zixin Deng / Feng Long /
Abstract: The Clp protease system is important for maintaining proteostasis in bacteria. It consists of ClpP serine proteases and an AAA+ Clp-ATPase such as ClpC1. The hexameric ATPase ClpC1 utilizes the ...The Clp protease system is important for maintaining proteostasis in bacteria. It consists of ClpP serine proteases and an AAA+ Clp-ATPase such as ClpC1. The hexameric ATPase ClpC1 utilizes the energy of ATP binding and hydrolysis to engage, unfold, and translocate substrates into the proteolytic chamber of homo- or hetero-tetradecameric ClpP for degradation. The assembly between the hetero-tetradecameric ClpP1P2 chamber and the Clp-ATPases containing tandem ATPase domains from the same species has not been studied in depth. Here, we present cryo-EM structures of the substrate-bound ClpC1:shClpP1P2 from , and shClpP1P2 in complex with ADEP1, a natural compound produced by and known to cause over-activation and dysregulation of the ClpP proteolytic core chamber. Our structures provide detailed information on the shClpP1-shClpP2, shClpP2-ClpC1, and ADEP1-shClpP1/P2 interactions, reveal conformational transition of ClpC1 during the substrate translocation, and capture a rotational ATP hydrolysis mechanism likely dominated by the D1 ATPase activity of chaperones.IMPORTANCEThe Clp-dependent proteolysis plays an important role in bacterial homeostasis and pathogenesis. The ClpP protease system is an effective drug target for antibacterial therapy. can produce a class of potent acyldepsipeptide antibiotics such as ADEP1, which could affect the ClpP protease activity. Although hosts one of the most intricate ClpP systems in nature, very little was known about its Clp protease mechanism and the impact of ADEP molecules on ClpP. The significance of our research is in dissecting the functional mechanism of the assembled Clp degradation machinery, as well as the interaction between ADEP1 and the ClpP proteolytic chamber, by solving high-resolution structures of the substrate-bound Clp system in . The findings shed light on our understanding of the Clp-dependent proteolysis in bacteria, which will enhance the development of antimicrobial drugs targeting the Clp protease system, and help fighting against bacterial multidrug resistance.
History
DepositionJan 2, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 27, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 24, 2024Group: 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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: ADEP1
P: ADEP1
Q: ADEP1
R: ADEP1
S: ADEP1
T: ADEP1
U: ADEP1
V: ADEP1
W: ADEP1
X: ADEP1
Y: ADEP1
Z: ADEP1
a: ADEP1
b: ADEP1


Theoretical massNumber of molelcules
Total (without water)337,15228
Polymers337,15228
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
ATP-dependent Clp protease proteolytic subunit / / ClpP1 / Endopeptidase Clp


Mass: 22632.605 Da / Num. of mol.: 7 / Mutation: S113A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces hawaiiensis (bacteria) / Gene: clpP1, clpP, CEB94_14110 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A5B9BGY8, endopeptidase Clp
#2: Protein
ATP-dependent Clp protease proteolytic subunit / / ClpP2 / Endopeptidase Clp


Mass: 24058.240 Da / Num. of mol.: 7 / Mutation: S131A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces hawaiiensis (bacteria) / Gene: clpP2, clpP, CEB94_14105 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A5B9BIX9, endopeptidase Clp
#3: Protein/peptide
ADEP1


Type: Cyclic depsipeptide / Class: Antibiotic / Mass: 736.855 Da / Num. of mol.: 14 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) / References: ADEP1
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: ClpP1,ClpP2 / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Streptomyces hawaiiensis (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.19.2_4158: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 504306 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 147.2 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003620069
ELECTRON MICROSCOPYf_angle_d0.607427223
ELECTRON MICROSCOPYf_chiral_restr0.04383150
ELECTRON MICROSCOPYf_plane_restr0.00443556
ELECTRON MICROSCOPYf_dihedral_angle_d6.56263276

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