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- EMDB-46971: Human mitochondrial ClpP in complex with Bortezomib -

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Basic information

Entry
Database: EMDB / ID: EMD-46971
TitleHuman mitochondrial ClpP in complex with Bortezomib
Map dataD7 symmetry map
Sample
  • Complex: Human mitochondrial ClpP in complex with Bortezomib
    • Protein or peptide: ATP-dependent Clp protease proteolytic subunit, mitochondrial
  • Ligand: N-[(1R)-1-(DIHYDROXYBORYL)-3-METHYLBUTYL]-N-(PYRAZIN-2-YLCARBONYL)-L-PHENYLALANINAMIDE
  • Ligand: water
KeywordsProtease / Proteolysis / HYDROLASE
Function / homology
Function and homology information


membrane protein proteolysis / mitochondrial protein catabolic process / endopeptidase Clp / endopeptidase Clp complex / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / Mitochondrial protein degradation / proteolysis involved in protein catabolic process / peptidase activity / ATPase binding ...membrane protein proteolysis / mitochondrial protein catabolic process / endopeptidase Clp / endopeptidase Clp complex / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / Mitochondrial protein degradation / proteolysis involved in protein catabolic process / peptidase activity / ATPase binding / endopeptidase activity / mitochondrial matrix / serine-type endopeptidase activity / mitochondrion / proteolysis / identical protein binding
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
ATP-dependent Clp protease proteolytic subunit, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.49 Å
AuthorsUday AB / Zeytuni N / Goncalves M / Vahidi S
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)PJT-191940 Canada
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Mechanism of allosteric activation in human mitochondrial ClpP protease.
Authors: Monica M Goncalves / Adwaith B Uday / Taylor J B Forrester / S Quinn W Currie / Angelina S Kim / Yue Feng / Yulia Jitkova / Algirdas Velyvis / Robert W Harkness / Matthew S Kimber / Aaron D ...Authors: Monica M Goncalves / Adwaith B Uday / Taylor J B Forrester / S Quinn W Currie / Angelina S Kim / Yue Feng / Yulia Jitkova / Algirdas Velyvis / Robert W Harkness / Matthew S Kimber / Aaron D Schimmer / Natalie Zeytuni / Siavash Vahidi /
Abstract: Human ClpP protease contributes to mitochondrial protein quality control by degrading misfolded proteins. ClpP is overexpressed in cancers such as acute myeloid leukemia (AML), where its inhibition ...Human ClpP protease contributes to mitochondrial protein quality control by degrading misfolded proteins. ClpP is overexpressed in cancers such as acute myeloid leukemia (AML), where its inhibition leads to the accumulation of damaged respiratory chain subunits and cell death. Conversely, hyperactivating ClpP with small-molecule activators, such as the recently discovered ONC201, disrupts mitochondrial protein degradation and impairs respiration in cancer cells. Despite its critical role in human health, the mechanism underlying the structural and functional properties of human ClpP remains elusive. Notably, human ClpP is paradoxically activated by active-site inhibitors. All available structures of human ClpP published to date are in the inactive compact or compressed states, surprisingly even when ClpP is bound to an activator molecule such as ONC201. Here, we present structures of human mitochondrial ClpP in the active extended state, including a pair of structures where ClpP is bound to an active-site inhibitor. We demonstrate that amino acid substitutions in the handle region (A192E and E196R) recreate a conserved salt bridge found in bacterial ClpP, stabilizing the extended active state and significantly enhancing ClpP activity. We elucidate the ClpP activation mechanism, highlighting a hormetic effect where substoichiometric inhibitor binding triggers an allosteric transition that drives ClpP into its active extended state. Our findings link the conformational dynamics of ClpP to its catalytic function and provide high-resolution structures for the rational design of potent and specific ClpP inhibitors, with implications for targeting AML and other disorders with ClpP involvement.
History
DepositionSep 10, 2024-
Header (metadata) releaseApr 23, 2025-
Map releaseApr 23, 2025-
UpdateApr 30, 2025-
Current statusApr 30, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_46971.png

Downloads & links

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Map

FileDownload / File: emd_46971.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationD7 symmetry map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.68 Å/pix.
x 384 pix.
= 259.2 Å		0.68 Å/pix.
x 384 pix.
= 259.2 Å		0.68 Å/pix.
x 384 pix.
= 259.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.675 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0312
Minimum - Maximum-0.036086865 - 0.13058369
Average (Standard dev.)0.0004543209 (±0.0057202526)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 259.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: D7 symmetry half map

Fileemd_46971_half_map_1.map
AnnotationD7 symmetry half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: D7 symmetry half map

Fileemd_46971_half_map_2.map
AnnotationD7 symmetry half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human mitochondrial ClpP in complex with Bortezomib

EntireName: Human mitochondrial ClpP in complex with Bortezomib
Components
  • Complex: Human mitochondrial ClpP in complex with Bortezomib
    • Protein or peptide: ATP-dependent Clp protease proteolytic subunit, mitochondrial
  • Ligand: N-[(1R)-1-(DIHYDROXYBORYL)-3-METHYLBUTYL]-N-(PYRAZIN-2-YLCARBONYL)-L-PHENYLALANINAMIDE
  • Ligand: water

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Supramolecule #1: Human mitochondrial ClpP in complex with Bortezomib

SupramoleculeName: Human mitochondrial ClpP in complex with Bortezomib / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: ATP-dependent Clp protease proteolytic subunit, mitochondrial

MacromoleculeName: ATP-dependent Clp protease proteolytic subunit, mitochondrial
type: protein_or_peptide / ID: 1 / Number of copies: 14 / Enantiomer: LEVO / EC number: endopeptidase Clp
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.179875 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SLIPIVVEQT GRGERAYDIY SRLLRERIVC VMGPIDDSVA SLVIAQLLFL QSESNKKPIH MYINSPGGVV TAGLAIYDTM QYILNPICT WCVGQAASMG SLLLAAGTPG MRHSLPNSRI MIHQPSGGAR GQATDIAIQA EEIMKLKKQL YNIYAKHTKQ S LQVIESAM ...String:
SLIPIVVEQT GRGERAYDIY SRLLRERIVC VMGPIDDSVA SLVIAQLLFL QSESNKKPIH MYINSPGGVV TAGLAIYDTM QYILNPICT WCVGQAASMG SLLLAAGTPG MRHSLPNSRI MIHQPSGGAR GQATDIAIQA EEIMKLKKQL YNIYAKHTKQ S LQVIESAM ERDRYMSPME AQEFGILDKV LVHPPQDGED EPTLVQKEPV EAAPAAEPVP AST

UniProtKB: ATP-dependent Clp protease proteolytic subunit, mitochondrial

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Macromolecule #2: N-[(1R)-1-(DIHYDROXYBORYL)-3-METHYLBUTYL]-N-(PYRAZIN-2-YLCARBONYL...

MacromoleculeName: N-[(1R)-1-(DIHYDROXYBORYL)-3-METHYLBUTYL]-N-(PYRAZIN-2-YLCARBONYL)-L-PHENYLALANINAMIDE
type: ligand / ID: 2 / Number of copies: 14 / Formula: BO2
Molecular weightTheoretical: 384.237 Da
Chemical component information

ChemComp-BO2:
N-[(1R)-1-(DIHYDROXYBORYL)-3-METHYLBUTYL]-N-(PYRAZIN-2-YLCARBONYL)-L-PHENYLALANINAMIDE / medication, anticancer*YM

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 168 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration18 mg/mL
BufferpH: 7.8
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3Tris
100.0 mMKClPottassium chloride
10.0 mMMgCl2Magnesium chloride
1.0 mMC4H10O2S2Dithiothreitol
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 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: NONE / Details: Ab-initio model generated in cryoSPARC
Final reconstructionApplied symmetry - Point group: D7 (2x7 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.49 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4) / Number images used: 408161
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4) / Details: Homogenous refinement in cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4) / Details: Homogenous refinement in cryoSPARC
FSC plot (resolution estimation)

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