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- PDB-6dkf: Caseinolytic protease (ClpP) from Staphylococcus aureus mutant - V7A -

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Basic information

Entry
Database: PDB / ID: 6dkf
TitleCaseinolytic protease (ClpP) from Staphylococcus aureus mutant - V7A
ComponentsATP-dependent Clp protease proteolytic subunit
KeywordsHYDROLASE / Protease
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 / 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
ATP-dependent Clp protease proteolytic subunit
Similarity search - Component
Biological speciesStaphylococcus aureus subsp. aureus str. Newman (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsRipstein, Z.A. / Vahidi, S. / Kay, L.E. / Rubinstein, J.L.
Funding support Canada, 6items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
Natural Sciences and Engineering Research Council (NSERC, Canada) Canada
Canadian Institutes of Health Research (CIHR) Canada
Natural Sciences and Engineering Research Council (NSERC, Canada) Canada
Canadian Institutes of Health Research (CIHR) Canada
Natural Sciences and Engineering Research Council (NSERC, Canada) Canada
CitationJournal: Proc Natl Acad Sci U S A / Year: 2018
Title: Reversible inhibition of the ClpP protease via an N-terminal conformational switch.
Authors: Siavash Vahidi / Zev A Ripstein / Massimiliano Bonomi / Tairan Yuwen / Mark F Mabanglo / Jordan B Juravsky / Kamran Rizzolo / Algirdas Velyvis / Walid A Houry / Michele Vendruscolo / John L ...Authors: Siavash Vahidi / Zev A Ripstein / Massimiliano Bonomi / Tairan Yuwen / Mark F Mabanglo / Jordan B Juravsky / Kamran Rizzolo / Algirdas Velyvis / Walid A Houry / Michele Vendruscolo / John L Rubinstein / Lewis E Kay /
Abstract: Protein homeostasis is critically important for cell viability. Key to this process is the refolding of misfolded or aggregated proteins by molecular chaperones or, alternatively, their degradation ...Protein homeostasis is critically important for cell viability. Key to this process is the refolding of misfolded or aggregated proteins by molecular chaperones or, alternatively, their degradation by proteases. In most prokaryotes and in chloroplasts and mitochondria, protein degradation is performed by the caseinolytic protease ClpP, a tetradecamer barrel-like proteolytic complex. Dysregulating ClpP function has shown promise in fighting antibiotic resistance and as a potential therapy for acute myeloid leukemia. Here we use methyl-transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, biochemical assays, and molecular dynamics simulations to characterize the structural dynamics of ClpP from (SaClpP) in wild-type and mutant forms in an effort to discover conformational hotspots that regulate its function. Wild-type SaClpP was found exclusively in the active extended form, with the N-terminal domains of its component protomers in predominantly β-hairpin conformations that are less well-defined than other regions of the protein. A hydrophobic site was identified that, upon mutation, leads to unfolding of the N-terminal domains, loss of SaClpP activity, and formation of a previously unobserved split-ring conformation with a pair of 20-Å-wide pores in the side of the complex. The extended form of the structure and partial activity can be restored via binding of ADEP small-molecule activators. The observed structural plasticity of the N-terminal gates is shown to be a conserved feature through studies of and ClpP, suggesting a potential avenue for the development of molecules to allosterically modulate the function of ClpP.
History
DepositionMay 29, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 27, 2018Provider: repository / Type: Initial release
Revision 1.1Jul 11, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.pdbx_database_id_PubMed ..._citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Jul 25, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jan 15, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Mar 13, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
B: ATP-dependent Clp protease proteolytic subunit
D: ATP-dependent Clp protease proteolytic subunit
C: ATP-dependent Clp protease proteolytic subunit
A: 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
I: ATP-dependent Clp protease proteolytic subunit
K: ATP-dependent Clp protease proteolytic subunit
J: ATP-dependent Clp protease proteolytic subunit
H: 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


Theoretical massNumber of molelcules
Total (without water)301,11914
Polymers301,11914
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 21508.479 Da / Num. of mol.: 14 / Mutation: V7A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus subsp. aureus str. Newman (bacteria)
Strain: Newman / Gene: clpP, NWMN_0736 / Plasmid: pET24 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): Bl21(DE3) CodonPlus / References: UniProt: A6QF76, endopeptidase Clp

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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: Caseinolytic protease from Staphylococcus aureus (V7A)
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.301 MDa / Experimental value: YES
Source (natural)Organism: Staphylococcus aureus (strain Newman) (bacteria) / Strain: Newman
Source (recombinant)Organism: Escherichia coli Bl21(DE3) (bacteria) / Strain: BL21(DE3) CodonPlus / Plasmid: pET24
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMpotassium chlorideKCl1
21 mMethylenediaminetetraacetic acidEDTA1
35 mMmagnesium chlorideMgCl21
45 %Igepal CA-6301
550 mMimidazole1
SpecimenConc.: 30 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Modified Vitrobot

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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 FIELD / Nominal magnification: 75000 X / Calibrated defocus min: 1000 nm / Calibrated defocus max: 3200 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 60 sec. / Electron dose: 43 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 1837 / Details: movies were collected with 44 fractions
Image scansWidth: 4096 / Height: 4096 / Movie frames/image: 30 / Used frames/image: 1-30

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4CTF correction
7Coot0.8.2model fitting
9cryoSPARC0.5initial Euler assignment
10cryoSPARC0.5final Euler assignment
11cryoSPARCclassification
12cryoSPARC0.53D reconstruction
13PHENIX1.10.1-2155model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 878240
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 324522 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model building

3D fitting-ID: 1 / Pdb chain-ID: A / Pdb chain residue range: 20-193 / Source name: PDB / Type: experimental model

IDPDB-IDAccession codeInitial refinement model-ID
13V5E

3v5e

3V5E1
23QWD

3qwd

3QWD2

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