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- EMDB-38536: Cryo-EM structure of the ClpC1:ClpP1P2 degradation complex in Str... -

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Entry
Database: EMDB / ID: EMD-38536
TitleCryo-EM structure of the ClpC1:ClpP1P2 degradation complex in Streptomyces hawaiiensis
Map datamerged focus map
Sample
  • Complex: ClpP1,ClpP2,ClpC1,casein
    • Protein or peptide: ATP-dependent Clp protease proteolytic subunit
    • Protein or peptide: ATP-dependent Clp protease proteolytic subunit
    • Protein or peptide: NDP-hexose 4-ketoreductase
    • Protein or peptide: casein
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
Keywordsprotease / protein degradation / proteostasis / proteolysis / HYDROLASE
Function / homology
Function and homology information


endopeptidase Clp / endopeptidase Clp complex / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / cellular response to heat / ATPase binding / serine-type endopeptidase activity / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
UVR domain / UVR domain profile. / ClpA/B, conserved site 1 / Chaperonins clpA/B signature 1. / ClpP, Ser active site / Endopeptidase Clp serine active site. / ClpA/ClpB, AAA lid domain / AAA lid domain / Clp amino terminal domain, pathogenicity island component / ClpP, histidine active site ...UVR domain / UVR domain profile. / ClpA/B, conserved site 1 / Chaperonins clpA/B signature 1. / ClpP, Ser active site / Endopeptidase Clp serine active site. / ClpA/ClpB, AAA lid domain / AAA lid domain / Clp amino terminal domain, pathogenicity island component / ClpP, histidine active site / Endopeptidase Clp histidine active site. / Clp, repeat (R) domain / Clp repeat (R) domain profile. / : / ATP-dependent Clp protease proteolytic subunit / Clp, N-terminal domain superfamily / Clp protease proteolytic subunit /Translocation-enhancing protein TepA / Clp protease / ClpA/B family / Clp ATPase, C-terminal / AAA domain (Cdc48 subfamily) / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein / ClpP/crotonase-like domain superfamily / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ATP-dependent Clp protease proteolytic subunit / ATP-dependent Clp protease proteolytic subunit / NDP-hexose 4-ketoreductase
Similarity search - Component
Biological speciesStreptomyces hawaiiensis (bacteria) / Bos taurus (domestic cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.84 Å
AuthorsXu X / Long F
Funding support China, 1 items
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 1, 2024-
Header (metadata) releaseMar 27, 2024-
Map releaseMar 27, 2024-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_38536.png

Downloads & links

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Map

FileDownload / File: emd_38536.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmerged focus map
Projections & slices

Image control

Size
Brightness
Contrast
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AxesX (Sec.)Y (Row.)Z (Col.)
0.84 Å/pix.
x 384 pix.
= 322.56 Å		0.84 Å/pix.
x 384 pix.
= 322.56 Å		0.84 Å/pix.
x 384 pix.
= 322.56 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.9
Minimum - Maximum-4.1009536 - 46.977795
Average (Standard dev.)0.032050695 (±1.3010077)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 322.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_38536_msk_1.map
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Additional map: sharpened full map

Fileemd_38536_additional_1.map
Annotationsharpened full map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_38536_half_map_1.map
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Half map: #1

Fileemd_38536_half_map_2.map
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Sample components

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Entire : ClpP1,ClpP2,ClpC1,casein

EntireName: ClpP1,ClpP2,ClpC1,casein
Components
  • Complex: ClpP1,ClpP2,ClpC1,casein
    • Protein or peptide: ATP-dependent Clp protease proteolytic subunit
    • Protein or peptide: ATP-dependent Clp protease proteolytic subunit
    • Protein or peptide: NDP-hexose 4-ketoreductase
    • Protein or peptide: casein
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: ClpP1,ClpP2,ClpC1,casein

SupramoleculeName: ClpP1,ClpP2,ClpC1,casein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Streptomyces hawaiiensis (bacteria)

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Macromolecule #1: ATP-dependent Clp protease proteolytic subunit

MacromoleculeName: ATP-dependent Clp protease proteolytic subunit / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO / EC number: endopeptidase Clp
Source (natural)Organism: Streptomyces hawaiiensis (bacteria)
Molecular weightTheoretical: 24.216336 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MASMTGGQQM GRGSEFGGLG DQVYNRLLNE RIIFLGQPVD DDIANKITAQ LLLLASDPEK DIYLYINSP GGSITAGMAI YDTMQYIKND VVTIAMGLAA AMGQFLLSAG TPGKRFALPN AEILIHQPSA GLAGSASDIK I HAERLLHT ...String:
MGSSHHHHHH SSGLVPRGSH MASMTGGQQM GRGSEFGGLG DQVYNRLLNE RIIFLGQPVD DDIANKITAQ LLLLASDPEK DIYLYINSP GGSITAGMAI YDTMQYIKND VVTIAMGLAA AMGQFLLSAG TPGKRFALPN AEILIHQPSA GLAGSASDIK I HAERLLHT KKRMAELTSQ HTGQTIEQIT RDSDRDRWFD AFEAKEYGLI DDVMTTAAGM PGGGGTGA

UniProtKB: ATP-dependent Clp protease proteolytic subunit

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Macromolecule #2: ATP-dependent Clp protease proteolytic subunit

MacromoleculeName: ATP-dependent Clp protease proteolytic subunit / type: protein_or_peptide / ID: 2 / Number of copies: 7 / Enantiomer: LEVO / EC number: endopeptidase Clp
Source (natural)Organism: Streptomyces hawaiiensis (bacteria)
Molecular weightTheoretical: 22.80785 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MEYDPYAKLF EERVIFLGVQ IDDASANDVM AQLLCLESMD PDRDISVYIN SPGGSFTALT AIYDTMQYV KPDVQTVCMG QAAAAAAVLL AAGTPGKRMA LPNARVLIHQ PYSETGRGQV SDLEIAANEI LRMRSQLEDM L AKHSTTPV ...String:
MGSSHHHHHH SSGLVPRGSH MEYDPYAKLF EERVIFLGVQ IDDASANDVM AQLLCLESMD PDRDISVYIN SPGGSFTALT AIYDTMQYV KPDVQTVCMG QAAAAAAVLL AAGTPGKRMA LPNARVLIHQ PYSETGRGQV SDLEIAANEI LRMRSQLEDM L AKHSTTPV EKIREDIERD KILTAEDALS YGLIDQVIST RKMDNSSLR

UniProtKB: ATP-dependent Clp protease proteolytic subunit

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Macromolecule #3: NDP-hexose 4-ketoreductase

MacromoleculeName: NDP-hexose 4-ketoreductase / type: protein_or_peptide / ID: 3 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Streptomyces hawaiiensis (bacteria)
Molecular weightTheoretical: 77.312055 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MASMTGGQQM GRGSEFAEGT PSTSLVLDQF GRNLTQAARE SKLDPVIGRE KEIERVMQVL SRRTKNNPV LIGEPGVGKT AVVEGLAQAI VKGEVPETLK DKHLYTLDLG ALVAGSRYRG DFEERLKKVL KEIRTRGDII L FIDALHTL ...String:
MGSSHHHHHH SSGLVPRGSH MASMTGGQQM GRGSEFAEGT PSTSLVLDQF GRNLTQAARE SKLDPVIGRE KEIERVMQVL SRRTKNNPV LIGEPGVGKT AVVEGLAQAI VKGEVPETLK DKHLYTLDLG ALVAGSRYRG DFEERLKKVL KEIRTRGDII L FIDALHTL VGAGAAEGAI DAASILKPML ARGELQTIGA TTLDEYRKHL EKDAALERRF QPIQVAEPSL PHTIEILKGL RD RYEAHHR VSITDEALVQ AATLADRYIS DRFLPDKAID LIDEAGSRMR IRRMTAPPDL REFDEKIAGV RRDKESAIDS QDA EKAASL RDKEKQLLAA KAKREKEWKA GDMDVVAEVD GELIAEVLAT ATGIPVFKLT EEESSRLLRM EDELHKRVIG QVDA VKALS KAIRRTRAGL KDPKRPGGSF IFAGPSGVGK TELSKALAEF LFGDEDALIS LDMSEFSEKH TVSRLFGSPP GYVGY EEGG QLTEKVRRKP FSVVLFDAVE KAHPDIFNSL LQILEDGRLT DSQGRVVDFK NTVIIMTTNL GTRDISKGFN LGFAAQ GDT KSNYERMKNK VSDELKQHFR PEFLNRVDDV VVFPQLSQAD ILKIVDLMID KVDERLKDRD MGIELSSSAK ELLSKKG YD PVLGARPLRR TIQREIEDSL SEKILFGELR PGHIVVVDTE GEGETKTFTF RGEE

UniProtKB: NDP-hexose 4-ketoreductase

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Macromolecule #4: casein

MacromoleculeName: casein / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (domestic cattle)
Molecular weightTheoretical: 2.060531 KDa
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 7 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 7 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 5 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 50 sec.
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 1.84 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 183537
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Output model

PDB-8xoo:
Cryo-EM structure of the ClpC1:ClpP1P2 degradation complex in Streptomyces hawaiiensis

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