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- PDB-6vgq: ClpP1P2 complex from M. tuberculosis with GLF-CMK bound to ClpP1 -

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Basic information

Entry
Database: PDB / ID: 6vgq
TitleClpP1P2 complex from M. tuberculosis with GLF-CMK bound to ClpP1
Components
  • ATP-dependent Clp protease proteolytic subunit 1
  • ATP-dependent Clp protease proteolytic subunit
  • Z-Gly-leu-phe-CH2Cl
KeywordsHYDROLASE / Complex / protease / ClpP / tuberculosis
Function / homology
Function and homology information


endopeptidase Clp complex / endopeptidase Clp / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / peptidoglycan-based cell wall / ATPase binding / serine-type endopeptidase activity / proteolysis / plasma membrane / cytosol / 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 / ATP-dependent Clp protease proteolytic subunit 2 / ATP-dependent Clp protease proteolytic subunit 1
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsRipstein, Z.A. / Vahidi, S. / Rubinstein, J.L. / Kay, L.E.
Funding support Canada, 2items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)FDN-503573 Canada
Canadian Institutes of Health Research (CIHR)PJT-162186 Canada
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: An allosteric switch regulates ClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR.
Authors: Siavash Vahidi / Zev A Ripstein / Jordan B Juravsky / Enrico Rennella / Alfred L Goldberg / Anthony K Mittermaier / John L Rubinstein / Lewis E Kay /
Abstract: The 300-kDa ClpP1P2 protease from collaborates with the AAA+ (ATPases associated with a variety of cellular activities) unfoldases, ClpC1 and ClpX, to degrade substrate proteins. Unlike in other ...The 300-kDa ClpP1P2 protease from collaborates with the AAA+ (ATPases associated with a variety of cellular activities) unfoldases, ClpC1 and ClpX, to degrade substrate proteins. Unlike in other bacteria, all of the components of the Clp system are essential for growth and virulence of mycobacteria, and their inhibitors show promise as antibiotics. MtClpP1P2 is unique in that it contains a pair of distinct ClpP1 and ClpP2 rings and also requires the presence of activator peptides, such as benzoyl-leucyl-leucine (Bz-LL), for function. Understanding the structural basis for this requirement has been elusive but is critical for the rational design and improvement of antituberculosis (anti-TB) therapeutics that target the Clp system. Here, we present a combined biophysical and biochemical study to explore the structure-dynamics-function relationship in MtClpP1P2. Electron cryomicroscopy (cryo-EM) structures of apo and acyldepsipeptide-bound MtClpP1P2 explain their lack of activity by showing loss of a key β-sheet in a sequence known as the handle region that is critical for the proper formation of the catalytic triad. Methyl transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, and biochemical assays show that, on binding Bz-LL or covalent inhibitors, MtClpP1P2 undergoes a conformational change from an inactive compact state to an active extended structure that can be explained by a modified Monod-Wyman-Changeux model. Our study establishes a critical role for the handle region as an on/off switch for function and shows extensive allosteric interactions involving both intra- and interring communication that regulate MtClpP1P2 activity and that can potentially be exploited by small molecules to target .
History
DepositionJan 8, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 18, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 1, 2020Group: 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

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Assembly

Deposited unit
N: ATP-dependent Clp protease proteolytic subunit 1
H: ATP-dependent Clp protease proteolytic subunit 1
I: ATP-dependent Clp protease proteolytic subunit 1
J: ATP-dependent Clp protease proteolytic subunit 1
K: ATP-dependent Clp protease proteolytic subunit 1
L: ATP-dependent Clp protease proteolytic subunit 1
M: ATP-dependent Clp protease proteolytic subunit 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
U: Z-Gly-leu-phe-CH2Cl
O: Z-Gly-leu-phe-CH2Cl
P: Z-Gly-leu-phe-CH2Cl
Q: Z-Gly-leu-phe-CH2Cl
R: Z-Gly-leu-phe-CH2Cl
S: Z-Gly-leu-phe-CH2Cl
T: Z-Gly-leu-phe-CH2Cl


Theoretical massNumber of molelcules
Total (without water)304,50921
Polymers304,50921
Non-polymers00
Water0
1


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

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Components

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


Mass: 21065.934 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: clpP1, clpP, Rv2461c, MTV008.17c / Plasmid: pet24a+
Details (production host): Cleavable N-terminal His 6 -SUMO tag
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WPC5, endopeptidase Clp
#2: Protein
ATP-dependent Clp protease proteolytic subunit / / Endopeptidase Clp


Mass: 21914.957 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: clpP2, clpP, ERS007703_00186, ERS023446_00571, EZX46_04555, FDK60_08755, FDK62_16525, SAMEA2682864_03098, SAMEA2683035_00557
Plasmid: pet24a+ / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A045HBE0, UniProt: P9WPC3*PLUS, endopeptidase Clp
#3: Protein/peptide
Z-Gly-leu-phe-CH2Cl


Mass: 520.449 Da / Num. of mol.: 7 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
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: ClpP1P2 complex bound to GLF-CMK / Type: COMPLEX
Details: Complex formed between P1 and P2 heptameric rings with inhibitor GLF-CMK
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Bl21(DE3) / Plasmid: pet24a+
Buffer solutionpH: 7
Details: IGEPAL-CA630 was added shortly prior to vitrification
Buffer component
IDConc.NameBuffer-ID
150 mMImidazole1
2100 mMPotassium Chloride1
35 mMDithiothreitol1
40.025 %IGEPAL-CA6301
SpecimenConc.: 20 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Mono-disperse complexes
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Blotted for 4.5 seconds at an offset of -5 mm

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 70 K
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: 1 / Num. of real images: 1645
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1cryoSPARC2.1particle selection
2EPUimage acquisition
4cryoSPARC2.1CTF correction
7UCSF Chimeramodel fitting
9RosettaEMmodel refinement
10PHENIXmodel refinement
11cryoSPARC2.1initial Euler assignment
12cryoSPARC2.1final Euler assignment
13cryoSPARC2.1classification
14cryoSPARC2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 257060
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 143748 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 5DZK

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